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https://openalex.org/W4382138286	https://bmcgenomics.biomedcentral.com/counter/pdf/10.1186/s12864-023-09456-5	English	null	Homoeologous evolution of the allotetraploid genome of Poa annua L.	BMC genomics	2,023	cc-by	17,886	Abstract Background Poa annua (annual bluegrass) is an allotetraploid turfgrass, an agronomically significant weed, and one of the most widely dispersed plant species on earth. Here, we report the chromosome-scale genome assemblies of P. annua’s diploid progenitors, P. infirma and P. supina, and use multi-omic analyses spanning all three species to better understand P. annua’s evolutionary novelty. Results We find that the diploids diverged from their common ancestor 5.5 – 6.3 million years ago and hybridized to form P. annua ≤ 50,000 years ago. The diploid genomes are similar in chromosome structure and most notably distinguished by the divergent evolutionary histories of their transposable elements, leading to a 1.7 × difference in genome size. In allotetraploid P. annua, we find biased movement of retrotransposons from the larger (A) sub- genome to the smaller (B) subgenome. We show that P. annua’s B subgenome is preferentially accumulating genes and that its genes are more highly expressed. Whole-genome resequencing of several additional P. annua accessions revealed large-scale chromosomal rearrangements characterized by extensive TE-downsizing and evidence to sup- port the Genome Balance Hypothesis. Conclusions The divergent evolutions of the diploid progenitors played a central role in conferring onto P. annua its remarkable phenotypic plasticity. We find that plant genes (guided by selection and drift) and transposable elements (mostly guided by host immunity) each respond to polyploidy in unique ways and that P. annua uses whole-genome duplication to purge highly parasitized heterochromatic sequences. The findings and genomic resources presented here will enable the development of homoeolog-specific markers for accelerated weed science and turfgrass breeding. Keywords Chromosomal rearrangements, Transposable elements (TEs), Retrotransposons, Phenotypic plasticity, Allopolyploid, Whole-genome duplication (WGD), Genome evolution, Genome sequencing, Weed, Turfgrass 4 USDA ARS, Forage and Range Research, Logan, UT, USA 5 Department of Plant, Soil, and Microbial Sciences, Michigan State University, East Lansing, MI, USA 4 USDA ARS, Forage and Range Research, Logan, UT, USA 5 Department of Plant, Soil, and Microbial Sciences, Michigan State University, East Lansing, MI, USA Homoeologous evolution of the allotetraploid genome of Poa annua L. Christopher W. Benson1,2, Matthew R. Sheltra1,2, Peter J. Maughan3, Eric N. Jellen3, Matthew D. Robbins4, B. Shaun Bushman4, Eric L. Patterson5, Nathan D. Hall5 and David R. Huff1 BMC Genomics BMC Genomics Benson et al. BMC Genomics (2023) 24:350 https://doi.org/10.1186/s12864-023-09456-5 Open Access © The Author(s) 2023, corrected publication 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/. 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. Background dominance because silencing TEs can involve methyla- tion spillover to nearby genes, which can reduce their expression relative to the less TE dense homoeolog [10, 15]. Studies focused on WGD are challenging due to the high sequence similarity between homoeologs but will be instrumental to better understanding the cis–trans regu- latory relationships that govern phenotypic plasticity in allopolyploid crops. g Polyploidy, or whole-genome duplication (WGD), is a repeated phenomenon in the evolution of plants and fre- quently associated with the emergence of novel traits, elevated stress tolerance, and niche expansion. In addi- tion to the abundance of young and recently formed polyploids, it is now evident that all angiosperms have remnants of ancient WGD [1, 2]. Polyploidy can influ- ence cellular processes, including transposable element (TE) activity, gene expression changes, epigenetic modifi- cations, and chromosomal restructuring [3, 4]. Allopoly- ploidy is a type of WGD that arises when two or more distinct species hybridize through an interspecific cross. The merged genomes of an allopolyploid are referred to as subgenomes and are ancestrally related but have sepa- rate evolutionary histories. Ancestrally related chromo- somes between the subgenomes are called homoeologs and share similar structure and gene orientation. Allopol- yploids predominantly use bivalent chromosome pairing during meiosis, but when bivalence fails, homoeologs can recombine and homoeologous exchanges (HEs) can occur [5, 6]. After many generations (or possibly only a few) [7], most allopolyploids eventually establish a ‘domi- nant’ subgenome, with higher expression of homoeologs and fewer lost genes (less fractionated) as the species returns to a diploid-like state (diploidization) [8–12]. Interestingly, recent work suggests that certain features of the parental genomes might help predispose subgenomes for dominance after allopolyploidy [13, 14]. For exam- ple, TE density may be a useful predictor of subgenome One of the most ubiquitous allopolyploids on earth is the grass species, Poa annua L. (2n = 4x = 28). Poa annua is an allotetraploid that originated from an inter- specific cross between diploid species, Poa infirma Kunth and Poa supina Schrader (Fig. 1a) [16–19]. The parental diploids of P. annua are restricted to their niches where P. infirma thrives in arid Mediterranean climates and P. supina prefers the boreal and alpine regions of cen- tral Europe. In contrast to its progenitors, P. annua has remarkable phenotypic variability that has allowed it to establish seeding populations on all seven continents and 96% of cities around the world (Fig. . Open Access T d d Benson et al. BMC Genomics (2023) 24:350 Benson et al. BMC Genomics (2023) 24:350 Page 2 of 20 Background 1b) [20–22]. It is a problematic weed in urban, agricultural, and turf- grass ecosystems, partially due to its evolved resistance to more than 10 different herbicide modes of action [23]. Despite its unfavorable reputation, P. annua has devel- oped an agronomic niche on golf course putting greens where it often invades and outcompetes turfgrass species that were bred to thrive under the intensive management conditions of 2-3 mm mowing height [24]. Some golf course superintendents come to view P. annua as an elite putting surface and allow it to slowly envelope the entire Fig. 1 The evolutionary origin of allotetraploid Poa annua. a Images of parental diploids, P. infirma and P. supina, and derived allotetraploid, P. annua. Two biotypes of P. annua are shown; a wild-type plant with annual lifespan and upright growth and a dwarf-type plant with perennial lifespan and prostrate growth. Grey arrows indicate the parental relationship between species. b The present-day geographic ranges of diploid and allotetraploid Poa species shows transgressive versatility and niche expansion of P. annua. Coordinate data downloaded from the Global Biodiversity Information Facility on 9/19/2021 with Antarctic additions according to Chwedorzewska et al., [22] Fig. 1 The evolutionary origin of allotetraploid Poa annua. a Images of parental diploids, P. infirma and P. supina, and derived allotetraploid, P. annua. Two biotypes of P. annua are shown; a wild-type plant with annual lifespan and upright growth and a dwarf-type plant with perennial lifespan and prostrate growth. Grey arrows indicate the parental relationship between species. b The present-day geographic ranges of diploid and allotetraploid Poa species shows transgressive versatility and niche expansion of P. annua. Coordinate data downloaded from the Global Biodiversity Information Facility on 9/19/2021 with Antarctic additions according to Chwedorzewska et al., [22] Benson et al. BMC Genomics (2023) 24:350 Page 3 of 20 contained approximately the additive number of lncR- NAs as its diploid parents with fewer lncRNAs in the A (infirma) subgenome (14,394) and more lncRNAs in the B (supina) subgenome (15,057). putting green. In fact, seven of the top ten golf courses in the United States utilize P. annua putting greens (top100golfcourses.com). p g The parental diploids of P. annua can hybridize at low frequencies (0.20%) and the offspring are amphihap- loid (polyhaploid; i.e., plants that contain a single set of unpaired chromosomes for each subgenome) [25]. Genome assembly and annotationhi Genome assembly and annotation The P. infirma (2n = 2x = 14) and P. supina (2n = 2x = 14) genomes each assembled into seven pseudomolecules that represented 96% of the estimated genome sizes by k-mer analysis and contained > 97% of the 1,614-core conserved orthologs in the Embryophyta OrthoDB (v10), supporting high-quality chromosome-level genome assemblies for both species (see methods; Supplementary Table 1; Supplementary Fig. 1). The chromosome-level assemblies represent the collapsed haploid (unphased) genomes for each species (n = 7). Chromosomes were named according to a pre-established nomenclature pre- sented by Robbins et al. [30], where P. infirma contributes the ‘A’ subgenome to P. annua and P. supina contrib- utes the ‘B’ subgenome. A prefix designates the species of origin, such that P. infirma chromosomes are ‘PiA’, P. supina’s are ‘PsB’, and P. annua’s are either ‘PaA’ or ‘PaB’ (Supplementary Fig. 2a). Poa infirma and P. supina chromosomes were 81% and 65% repetitive, respectively. These percentages amount to 489 Mb (1.77 ×) more repetitive DNA in P. infirma than P. supina, suggesting that TEs have played an out- sized role in the disparate genome sizes between the two diploids, particularly on orthologous chromosomes 1 and 2. The majority of annotated repetitive sequences were classified as Gypsy and Copia long terminal repeat (LTR) retrotransposons (598 Mb (53%) of the P. infirma genome and 241 Mb (38%) of the P. supina genome). The sequence length of the non-repetitive portions in each diploid is very similar, totaling 211 Mb in the P. infirma genome and 225 Mb in the P. supina genome. The subge- nomes of P. annua have slightly less repetitive DNA than their corresponding diploid progenitor genomes, with 7% less repetitive DNA in the A (infirma) subgenome and 2% less in the B (supina) subgenome. Repetitive DNA and TEs were annotated using custom built repeat libraries and included class I retrotranspo- sons as well as class II DNA transposons. Genes were predicted using the BRAKER2 pipeline on the repeat- masked genome assemblies [31]. Full-length Iso-Seq transcripts from each species was incorporated with protein evidence from Arabidopsis and related grasses for ab initio gene prediction. In addition, we identified 14,743 long noncoding RNAs (lncRNAs) in the P. infirma genome and 13,963 in the P. supina genome. Poa annua Genome characteristics and syntenyhi Genome characteristics and synteny The P. infirma genome is 1,125 Mb in length, which makes it 489 Mb (1.77 ×) larger than the P. supina genome (636 Mb), despite being closely related species and sister taxa within the section Micrantherae (syn. Ochlopoa). Most (76%) of the excess in genome size is due to orthologous chromosomes 1 and 2 being a com- bined 374 Mb larger in the P. infirma genome (Fig. 2ab; Supplementary Fig. 3). The subgenomes of P. annua are similar in composition to the genomes of the diploid progenitors, with the A subgenome (1,116 Mb) being 1% shorter than the P. infirma genome and the B subgenome (662 Mb) being 4% larger than the P. supina genome (Supplementary Fig. 2c). At the gene level, the A (infirma) subgenome had 6% fewer genes than P. infirma (37,123 and 39,420, respectively), and the B (supina) subgenome had 4% more genes than P. supina (39,536 and 37,935 respectively). Overall, the P. annua reference genome is 99% of the length of its progenitor genomes and contains 99% of its parental genes, most of which (95%) are rep- resented as colinear syntenic blocks (Fig. 2c; Supplemen- tary Fig. 2c; Supplementary Fig. 4).i Background Amphihaploid plants (2n = 14) are sterile at first but have been observed to spontaneously transition to fertile allo- tetraploids (2n = 28) [26], suggesting that P. annua’s path to polyploidy may have involved mitotic (somatic dou- bling) rather than meiotic error (unreduced gametes). Interestingly, amphihaploids are frequently found on golf course putting greens [27], suggesting that polyploid P. annua can return to amphihaploidy [28, 29] in certain environmental conditions and may oscillate between the two cytotypes. Here, we leverage the genomes of the diploid progenitors to accurately assign P. annua homoe- ologs to their appropriate parental origin. Using this methodology, we unravel P. annua’s polyploid evolution- ary history with the goal to better understand its pheno- typic plasticity and provide a valuable genetic resource for turfgrass breeders and weed scientists. Nucleotide divergence, molecular dating, and bursts of LTRs supina) was very close to zero and used to estimate the date that the two progenitor diploids hybridized to form P. annua. With a Poaceae mutational rate of 5.76174 × 10–9 substitutions per synonymous site per year [32], our Ks values suggest that the diploids diverged from their common ancestor 5.5 – 6.3 million years ago (Mya) and hybridized to form polyploid P. annua 0 – 600,000 years ago (Supplementary Fig. 5). The most recent of the ancestral WGD events in the Poaceae is rho (ρ) and pre-dates the divergence of the BOP (C3) and PACMAD (C4) grasses [33]. Syntenic gene pairs from rho have a Ks = 1 in our Poa species and corresponds to a date of 87 Mya, which largely overlaps with the reported rho WGD date of 85–97 Mya and helps to corroborate our methodology (Supplementary Fig. 6) [34]. Furthermore, our estimated date of diploid diver- gence is in agreement with a recent analysis based on plastid markers [35]. corresponding parental sequences was 98% (i.e., PaA to PiA alignments and PaB to PsB; Supplementary Fig. 2b). To estimate divergence and hybridization times, we cal- culated the synonymous substitutions rate (Ks) between homologous and homoeologous gene pairs. Gene pairs between P. infirma (A) and P. supina (B) have a peak Ks = 0.065 and was used to estimate the date that the two species diverged from their common ancestor. Ks between P. annua’s A subgenome and P. infirma (and also P. annua’s B subgenome and P. supina) was very close to zero and used to estimate the date that the two progenitor diploids hybridized to form P. annua. With a Poaceae mutational rate of 5.76174 × 10–9 substitutions per synonymous site per year [32], our Ks values suggest that the diploids diverged from their common ancestor 5.5 – 6.3 million years ago (Mya) and hybridized to form polyploid P. annua 0 – 600,000 years ago (Supplementary Fig. 5). The most recent of the ancestral WGD events in the Poaceae is rho (ρ) and pre-dates the divergence of the BOP (C3) and PACMAD (C4) grasses [33]. Nucleotide divergence, molecular dating, and bursts of LTRs Genomic similarity can be assessed at the nucleotide level using measures of average nucleotide identity (ANI) and is a useful indicator of genetic divergence between sequence alignments. The ANI between P. infirma (A) and P. supina (B) orthologous chromosomes is 95%. The ANI when comparing P. annua chromosomes to their Page 4 of 20 Benson et al. BMC Genomics (2023) 24:350 Benson et al. BMC Genomics i h l l h f h Fig. 2 The comparative colinear relationship of three Poa genomes. a Macrosyntenic comparison of the P. annua genome (PaA & PaB; x-axis) to the combined P. infirma (PiA) and P. supina (PsB) genomes (y-axis). PiA to PaA and PsB to PaB comparisons are orange. Breaks in the contiguity of the orange line illustrate recent structural modifications occurring after the hybridization of the tetraploid (post-polyploidy). PiA to PaB and PsB to PaA comparisons are purple and illustrate structural modifications that occurred after the parental diploid species diverged from their common ancestor. The ‘S’ curve in some syntenic comparisons illustrates differences in chromosome size. Colors denote synonymous substitution rate (Ks). The Ks values in the scale bar indicates three important events: polyploid hybridization (Ks = 0), speciation of the parents (Ks = 0.065), and the rho (ρ) WGD event (Ks = 1). b The photomicrograph and syntenic ribbon plot depict the relative chromosome size, structure, and collinear relationship between the genomes of the P. infirma, P. supina, and allotetraploid P. annua. Scale bar in the bottom corner of the photomicrograph indicates the relative chromosome sizes. c The ratio of syntenic depth between genes of the diploid parents and genes of P. annua indicate a 1:1 relationship Fig. 2 The comparative colinear relationship of three Poa genomes. a Macrosyntenic comparison of the P. annua genome (PaA & PaB; x-axis) to the combined P. infirma (PiA) and P. supina (PsB) genomes (y-axis). PiA to PaA and PsB to PaB comparisons are orange. Breaks in the contiguity of the orange line illustrate recent structural modifications occurring after the hybridization of the tetraploid (post-polyploidy). PiA to PaB and PsB to PaA comparisons are purple and illustrate structural modifications that occurred after the parental diploid species diverged from their common ancestor. The ‘S’ curve in some syntenic comparisons illustrates differences in chromosome size. Colors denote synonymous substitution rate (Ks). Nucleotide divergence, molecular dating, and bursts of LTRs The Ks values in the scale bar indicates three important events: polyploid hybridization (Ks = 0), speciation of the parents (Ks = 0.065), and the rho (ρ) WGD event (Ks = 1). b The photomicrograph and syntenic ribbon plot depict the relative chromosome size, structure, and collinear relationship between the genomes of the P. infirma, P. supina, and allotetraploid P. annua. Scale bar in the bottom corner of the photomicrograph indicates the relative chromosome sizes. c The ratio of syntenic depth between genes of the diploid parents and genes of P. annua indicate a 1:1 relationship Fig. 2 The comparative colinear relationship of three Poa genomes. a Macrosyntenic comparison of the P. annua genome (PaA & PaB; x-axis) to the combined P. infirma (PiA) and P. supina (PsB) genomes (y-axis). PiA to PaA and PsB to PaB comparisons are orange. Breaks in the contiguity of the orange line illustrate recent structural modifications occurring after the hybridization of the tetraploid (post-polyploidy). PiA to PaB and PsB to PaA comparisons are purple and illustrate structural modifications that occurred after the parental diploid species diverged from their common ancestor. The ‘S’ curve in some syntenic comparisons illustrates differences in chromosome size. Colors denote synonymous substitution rate (Ks). The Ks values in the scale bar indicates three important events: polyploid hybridization (Ks = 0), speciation of the parents (Ks = 0.065), and the rho (ρ) WGD event (Ks = 1). b The photomicrograph and syntenic ribbon plot depict the relative chromosome size, structure, and collinear relationship between the genomes of the P. infirma, P. supina, and allotetraploid P. annua. Scale bar in the bottom corner of the photomicrograph indicates the relative chromosome sizes. c The ratio of syntenic depth between genes of the diploid parents and genes of P. annua indicate a 1:1 relationship corresponding parental sequences was 98% (i.e., PaA to PiA alignments and PaB to PsB; Supplementary Fig. 2b). To estimate divergence and hybridization times, we cal- culated the synonymous substitutions rate (Ks) between homologous and homoeologous gene pairs. Gene pairs between P. infirma (A) and P. supina (B) have a peak Ks = 0.065 and was used to estimate the date that the two species diverged from their common ancestor. Ks between P. annua’s A subgenome and P. infirma (and also P. annua’s B subgenome and P. Nucleotide divergence, molecular dating, and bursts of LTRs Syntenic gene pairs from rho have a Ks = 1 in our Poa species and corresponds to a date of 87 Mya, which largely overlaps To further evaluate the date of hybridization and explore the 1.7-fold difference in genome size between A and B, we examined the mutation rates between pairs of LTRs. LTRs multiply by escaping host silencing and ‘burst’ into activity for a short time before being re- silenced [36, 37]. Repeats of an LTR are identical when inserted, owing to their copy-and-paste mode of trans- position [38]. Mutations between an ancestral LTR and its transposed derivative are a reflection of its evo- lutionary divergence. Our analysis suggests that the A genome experienced a burst in proliferation of LTRs that climaxed ~ 340,000 years ago, while bursts of LTRs in the B genome occurred more recently, with peak rate of transposition dating back to ~ 50,000 years ago (Fig. 3a). Because the density of LTR insertion times in P. infirma and P. supina closely mirror that of P. annua’s A and B subgenomes, it is likely that those bursts occurred during Page 5 of 20 Benson et al. BMC Genomics (2023) 24:350 Benson et al. BMC Genomics Fig. 3 Retrotransposon mobility in Pa annua. a Dating the insertion times of LTRs shows varying bursts of mobility in the subgenomes of P. annua (PaA & PaB) and its diploid progenitors, P. infirma (PiA) and P. supina (PsB). b Transposed gene duplications that mobilized post-polyploidy (0 – 50,000 years ago) show biased movement from the A subgenome (left) to the B subgenome (right). Light grey are ancestral copies that translocated to the opposite subgenome (inter-subgenome), while dark grey are ancestral copies that translocated and stayed within their parental subgenome (intra-subgenome). Blue is the location of the novel (transposed) copy. Transposed gene duplications are heavily enriched for LTR-associated activity Fig. 3 Retrotransposon mobility in Pa annua. a Dating the insertion times of LTRs shows varying bursts of mobility in the subgenomes of P. annua (PaA & PaB) and its diploid progenitors, P. infirma (PiA) and P. supina (PsB). b Transposed gene duplications that mobilized post-polyploidy (0 – 50,000 years ago) show biased movement from the A subgenome (left) to the B subgenome (right). Light grey are ancestral copies that translocated to the opposite subgenome (inter-subgenome), while dark grey are ancestral copies that translocated and stayed within their parental subgenome (intra-subgenome). Single‑gene duplications and retrotransposon activity g g p p y In addition to the WGD that formed P. annua, smaller scale duplications can also accompany polyploidy and are collectively referred to as single-gene duplications [39]. We identified 2,008 tandemly duplicated and 1,815 proxi- mally duplicated genes in the P. infirma genome. These numbers are similar to P. supina with 1,940 tandem and 1,914 proximal duplications. As compared to its progeni- tor genomes, allotetraploid P. annua has slightly fewer single-gene duplications in the A (infirma) subgenome (1,806 tandem and 1,736 proximal duplicated genes), and slightly more in the B (supina) subgenome (1,999 tan- dem and 2,160 proximal). Transposed duplications are another type of single-gene duplication and are thought to occur extensively after polyploidy [40–42]. We used the progenitor P. infirma and P. supina genomes as out- groups to identify pairs of transposed genes that were mobilized after the diploids hybridized to form P. annua (post-polyploidy). We found 63% more transposed dupli- cations in P. annua’s B subgenome than in P. annua’s A subgenome (5,917 and 3,438 transposed genes, respec- tively). This result is similar to the pattern observed with Nucleotide divergence, molecular dating, and bursts of LTRs Blue is the location of the novel (transposed) copy. Transposed gene duplications are heavily enriched for LTR-associated activity Fig. 3 Retrotransposon mobility in Pa annua. a Dating the insertion times of LTRs shows varying bursts of mobility in the subgenomes of P. annua (PaA & PaB) and its diploid progenitors, P. infirma (PiA) and P. supina (PsB). b Transposed gene duplications that mobilized post-polyploidy (0 – 50,000 years ago) show biased movement from the A subgenome (left) to the B subgenome (right). Light grey are ancestral copies that translocated to the opposite subgenome (inter-subgenome), while dark grey are ancestral copies that translocated and stayed within their parental subgenome (intra-subgenome). Blue is the location of the novel (transposed) copy. Transposed gene duplications are heavily enriched for LTR-associated activity the speciation of the diploids and prior to the hybridi- zation event that formed P. annua. Thus, we suggest a narrower timeframe for P. annua hybridization at 0 – 50,000 years ago. We expect that the 489 Mb difference in TE content and genome size between P. infirma and P. supina is greatly impacted by the two species varying abilities to silence retrotransposons. proximal and tandem duplications and may point to a post-polyploidy expansion of the B subgenome and con- traction of the A subgenome within P. annua. Interestingly, 74% of transposed duplications in the B subgenome remained within B, while 46% of A duplica- tions remained within the A subgenome, suggesting that inter-subgenomic duplications preferentially move from the A (infirma) subgenome and integrate into B (supina; Fig. 3b; χ2 test, P < 0.0001). Inter-subgenomic transposed duplications are enriched for functions associated with Gypsy and Copia-type LTRs, suggesting that they are heavily involved with retrotransposon activity. Taken together with our molecular dating of LTRs, we expect that the observed bias in inter-subgenome transpositions is a reflection of the two subgenomes uneven abilities to inhibit retrotransposons and is a continuation of the TE momentum that was established during the independent evolutions of the diploids. The observed bias in inter-sub- genome transpositions may point to a trans relationship, where retrotransposons ‘diffuse’ from the subgenome with higher TE content to the subgenome with lower TE content. Fractionation bias Gene loss (fractionation) occurs via intrachromosomal recombination resulting in short deletions and is a typical behavior of ancient allopolyploids [45]. We compared the A and B subgenomes of P. annua to the A and B genomes of its progenitors and identified consistent gene retention (97%) across all chromosomes, likely reflecting the recent timescale of the P. annua WGD event (Supplementary Fig. 7). Although this result seems to clash with our observations at the single-gene and HE levels, it is impor- tant to note the distinction between these methodolo- gies. The fractionation analysis used here [46] calculates the number of genes retained in P. annua with respect to the syntenic sequences in the progenitor genomes. Con- sequently, single-gene duplications would only impact our fractionation analysis if they had duplicated in the progenitor genome but not in P. annua. The impact of HEs on our fractionation analysis is relatively small, since there are only 1,208 genes within HEs and most (~ 61%) have an ancestrally syntenic ortholog in the homoeolo- gous subgenome and therefore would not impact frac- tionation values. Fig. 4 Homoeologous exchanges in Poa annua. a A karyotypic view of P. annua’s allotetraploid evolution illustrated by the haplotig-level genome assemblies. Chromosome lengths are scaled according to their relative size. Karyotype of the common ancestor is unknown, and a theoretical karyotype is depicted. b A bimodal distribution of genes within HEs across 15 re-sequenced P. annua genotypes shows biased reshuffling favoring gene movement to the B subgenome. On the left are genes found in rare HEs that occur in one or a few genotypes. On the right, genes commonly found within HEs occurring in most or all genotypes. c A graphical depiction of the largest HE in the P. annua genome. Circled in the syntenic dotplot, the disjunction highlights that the 2.2 Mb HE is an unbalanced Pa7A to Pa7B translocation. d An IGV alignment window shows a HE in the P. annua genome. Poa annua reads are tagged by their parental origin and mapped to the P. annua reference genome (see methods). Reads with P. supina origin are shown in the top half of the image, while reads with P. infirma origin are on the bottom half. Because this is a B subgenome chromosome (Pa7B), regions with P. infirma origin are putative HEs Fig. 4 Homoeologous exchanges in Poa annua. a A karyotypic view of P. Homoeologous exchanges Crossing over between ancestrally related chromo- somes is a common occurrence in newly formed allopolyploids and are referred to as HEs [43, 44]. We assessed HEs in P. annua (i.e., A segments in the B sub- genome and B segments in the A subgenome) using the parental sequences as a guide to assign P. annua reads Page 6 of 20 Page 6 of 20 Benson et al. BMC Genomics (2023) 24:350 Benson et al. BMC Genomics (2023) 24:350 as either being derived from P. infirma or P. supina. We detected 1,299 HEs in the P. annua genome (Fig. 4; 657 A segments in the B subgenome and 642 B segments in the A subgenome). Almost 2% of P. annua’s gene anno- tations are within HEs. Of those, 68% are A to B, sug- gesting that there may be an asymmetric exchange of genic sequences between the two subgenomes (823 A genes in the B subgenome vs 385 B genes in the A sub- genome; χ2 test, P < 0.0001). The average length of an HE was 16 kb for A to B subgenome HEs and 13 kb for B to A subgenome HEs. Interestingly, 1.6% of the B sub- genome consists of A sequences (10.4 Mb), while 0.7% of the A subgenome is B sequences (8.3 Mb). A to B HEs were most enriched for genes involved in gibberel- lin 3-beta-dioxygenase activity, while B to A HEs were enriched in genes involved in telomere maintenance. The largest HE is a 2.2 Mb Pa7A to Pa7B exchange containing 103 genes (Fig. 4c). Three of P. annua’s 26 annotated histone H3-K4 methylation genes reside in this 2.2 Mb HE. The differences in HEs between sub- genomes points to a visible but tenuous bias accumula- tion of genes in the B subgenome. Homoeolog expression bias and polyploid plasticity stress (unmowed). We vegetatively propagated dwarf- and wild-type P. annua plants and subjected one clone to mowing stress for three months, while leaving the other clone unmowed for three months. We observed no corre- lation in the expression profiles between biotypes (dwarf or wild) across our biological replicates (Supplementary Fig. 8; Supplementary Fig. 9), indicating that dwarf-types and wild-types exhibit similar transcriptional behav- ior under both mowed and unmowed conditions. After removing biotypes as a variable, we identified 5,505 and 6,400 differentially expressed pairs of homoeologs in our unmowed and mowed comparisons, respectively. We found that both mowed and unmowed plants showed a homoeolog expression bias favoring the B subgenome (Wilcoxon test: p = 0.001 and p = 0.0008, respectively), indicating that P. annua preferentially utilizes B (supina) genes regardless of mowing stress (Fig. 5). Although P. annua’s B subgenome expression bias is statistically significant in both treatment comparisons, the bias is not as evident as reported in other neo-allopolyploids [7, 51–53], likely reflecting the recent timescale of the hybridization but perhaps also pointing to a more equi- table relationship between P. annua’s subgenomes where primary metabolic function is partitioned across pairs of homoeologs (Supplementary Fig. 10). Only chromosomes omoeolog expression bias and polyploid plasticity P. annua is typically described as having two distinct biotypes; plants with wild-type morphology and plants with dwarf-type morphology (Fig. 1a; sometimes referred to as annual- and perennial-types, respectively) [47]. Plants with wild-type habit resemble P. infirma, while the dwarf-types more closely resemble P. supina. Genetic factors contribute to P. annua’s morphology, but broad phenotypic plasticity has also been reported where envi- ronmental stressors such as animal disturbance, intense wind, soil properties, temperature, elevation, and even golf course-style management can influence plants to preferentially favor one biotype over the other [48]. The two contrasting morphologies likely play an important role in P. annua’s ability to infiltrate and persist across a spectrum of climactic conditions [49]. Shimizu-Inatsugi et al. [50] introduced the Polyploid Plasticity Hypothesis stating that an allopolyploid spe- cies might differentially utilize the expression profiles of its progenitor genomes depending on the environment. With agronomic and turfgrass breeding in mind, we aimed to test the hypothesis that P. annua might pref- erentially express genes from the B (supina) subgenome when exposed to mowing stress and the A (infirma) sub- genome when allowed to grow in the absence of mowing Fig. Fractionation bias annua’s allotetraploid evolution illustrated by the haplotig-level genome assemblies. Chromosome lengths are scaled according to their relative size. Karyotype of the common ancestor is unknown, and a theoretical karyotype is depicted. b A bimodal distribution of genes within HEs across 15 re-sequenced P. annua genotypes shows biased reshuffling favoring gene movement to the B subgenome. On the left are genes found in rare HEs that occur in one or a few genotypes. On the right, genes commonly found within HEs occurring in most or all genotypes. c A graphical depiction of the largest HE in the P. annua genome. Circled in the syntenic dotplot, the disjunction highlights that the 2.2 Mb HE is an unbalanced Pa7A to Pa7B translocation. d An IGV alignment window shows a HE in the P. annua genome. Poa annua reads are tagged by their parental origin and mapped to the P. annua reference genome (see methods). Reads with P. supina origin are shown in the top half of the image, while reads with P. infirma origin are on the bottom half. Because this is a B subgenome chromosome (Pa7B), regions with P. infirma origin are putative HEs Page 7 of 20 Benson et al. BMC Genomics (2023) 24:350 Benson et al. BMC Genomics (2023) 24:350 Whole‑genome resequencing and large‑scale chromosomal modifications i Homoeologous exchanges and bursts of activity in trans- posable elements contribute to genomic instability in polyploids but do not provide a satisfying explanation for the reported 80% variation in DNA content between P. annua genotypes [54, 55]. To explore intraspecific vari- ation in P. annua at the whole-chromosome and DNA sequence level, we re-sequenced 13 geographically dis- tinct accessions and two additional elite breeding lines. Together, the 15 samples represent nine countries and four continents (Supplementary Fig. 11). The Illumina reads were aligned to the P. annua reference genome with a depth of coverage ranging between 13–26 ×. More than 99% of all reads mapped to the P. annua reference genome. SNP density across a 1 Mb sliding window showed large variability in sequence divergence within subgenomes, suggesting that there may have been multi- ple hybrid origins (Supplementary Fig. 12). Of the 76,541 gene annotations in the reference genome, we found that 7,808 were absent (dispensable) from at least one of the 15 samples, leaving 68,733 ‘core’ genes approximately evenly split between subgenomes (Supplementary Fig. 13; 52% of core genes were from the B subgenome). Dispen- sable genes were enriched for function in RNA-mediated transposon integration, suggesting that retrotransposons are actively proliferating in the species in a genotype-spe- cific manner. In addition to core and dispensable genes, we used the diploid genomes to identify HEs and deter- mine the parental origin for P. annua homoeologs across all 15 samples. There were 5,217 genes within HEs in at least one sample. A to B HEs were enriched for func- tions associated with primary metabolism, while B to A HEs were enriched for functions associated with tel- omere maintenance. Most (60%) genes within HEs were transferred from the A to the B subgenome, continuing Perhaps the most parsimonious path to this karyotype involves meiotic error, where 1A and 1B form a quad- rivalent and adjacent disjunction leads to two 1A’s going to one pole and two 1B’s going to the other. When ferti- lized by a normal nucleus, the resulting offspring would be 1A1B1B1B (or 1A1A1A1B). Subsequent generations would lead to introgression of 1A at recombination sites, which would cause most of the genic regions of the dis- placing 1B chromosome to return to a 1A-like state. Homoeolog expression bias and polyploid plasticity 5 Homoeolog expression bias tests the polyploid plasticity hypothesis under golf course-style mowing stress. Clonally propagated plants were exposed to mowing stress (bottom), or not exposed to mowing stress (top). In the histograms, solid bars are homoeologous gene pairs with a FDR ≤ 0.05. Open bars include all testable gene pairs. Pink are significantly biased toward the A (infirma) subgenome and grey are significantly biased toward the B (supina) subgenome. The bar plots adjacent to the histograms show differentially expressed genes across all seven homoeologous pairs of chromosomes Fig. 5 Homoeolog expression bias tests the polyploid plasticity hypothesis under golf course-style mowing stress. Clonally propagated plants were exposed to mowing stress (bottom), or not exposed to mowing stress (top). In the histograms, solid bars are homoeologous gene pairs with a FDR ≤ 0.05. Open bars include all testable gene pairs. Pink are significantly biased toward the A (infirma) subgenome and grey are significantly biased toward the B (supina) subgenome. The bar plots adjacent to the histograms show differentially expressed genes across all seven homoeologous pairs of chromosomes Page 8 of 20 Benson et al. BMC Genomics (2023) 24:350 to point toward a biased accumulation of genes in B (supina) homoeologs (Fig. 4b; χ2 test, P < 0.0001). one, four, and six showed consistent expression bias toward B homoeologs, suggesting that these three chro- mosomes contribute disproportionally to homoeolog expression bias at the whole-genome level (Fig. 5). Thus, we conclude that counter to the polyploid plasticity hypothesis, P. annua utilizes genes from both subge- nomes with modest homoeolog expression bias favoring B (supina) genes irrespective of mowing treatments. one, four, and six showed consistent expression bias toward B homoeologs, suggesting that these three chro- mosomes contribute disproportionally to homoeolog expression bias at the whole-genome level (Fig. 5). Thus, we conclude that counter to the polyploid plasticity hypothesis, P. annua utilizes genes from both subge- nomes with modest homoeolog expression bias favoring B (supina) genes irrespective of mowing treatments. Reads mapped to the P. annua reference genome (and diploid progenitor genomes) provide a view of structural modifications at the whole-chromosome level. Using this approach, we identified remarkable variation in chromo- some structure post-polyploidization. The largest is a 224 Mb deletion in the centromeric and pericentromeric region of chromosome 1A in some samples that amounts to 70% of the length of the reference chromosome (Fig. 6; Fig. 7; Supplementary Fig. 14). Homoeolog expression bias and polyploid plasticity Coinciding with the dele- tion at 1A is a 32 Mb duplication at chromosome 1B. Split reads and improperly paired reads at the deletion and duplication breakpoints suggest that the duplicated region at 1B resides within the deleted region of 1A, and indeed, capillary electrophoresis using homoeolog-spe- cific markers across the chromosomal breakpoint con- firms this to be the case (Supplementary Fig. 15). The 1B duplication contains the highest density of LTRs across the chromosome, suggesting that the rearrangement most likely spans the centromere (Fig. 7b). There are 1,996 annotated genes and 133 functional enrichments (mostly transposon-associated categories) in the 224 Mb centromeric deletion. The 32 Mb homoeologous cen- tromere brings back 1,321 of the 1,996 deleted genes and all but four of the functionally enriched categories. In addition to homoeolog expression analysis, we also used our transcriptional data to compare gene expression between pairs of recently transposed gene duplications that were identified during our analysis of single-gene duplications. We identified, 973 pairs of transposed genes as being differentially regulated between their novel and ancestral copies. Of those, 847 (87%) were upregulated in the novel copy and most were A to B transpositions. Whole‑genome resequencing and large‑scale chromosomal modifications 6 Depth of coverage plotted along the Poa annua reference genome shows large-scale variation in chromosome structure. Regions of reduced coverage indicate deletions relative to the reference genome, while regions with elevated coverage show duplications. The P. annua reference genome is unphased (haplotypes are collapsed), so regions with 1/2 × coverage indicate chromosomes with haplotype-specific duplications and deletions (heterozygotes). Samples ‘Germany’ and ‘Arizona’ were imaged 125 days after germination. ‘Ohio’ was imaged 56 days after germination. The two breeding lines (‘Pa-14’ dwarf and wild) were collected from a field experiment and allowed to grow unmowed for 56 days prior to imaging. Mean and median map coverage are indicated with green and red lines, respectively Fig. 6 Depth of coverage plotted along the Poa annua reference genome shows large-scale variation in chromosome structure. Regions of reduced coverage indicate deletions relative to the reference genome, while regions with elevated coverage show duplications. The P. annua reference genome is unphased (haplotypes are collapsed), so regions with 1/2 × coverage indicate chromosomes with haplotype-specific duplications and deletions (heterozygotes). Samples ‘Germany’ and ‘Arizona’ were imaged 125 days after germination. ‘Ohio’ was imaged 56 days after germination. The two breeding lines (‘Pa-14’ dwarf and wild) were collected from a field experiment and allowed to grow unmowed for 56 days prior to imaging. Mean and median map coverage are indicated with green and red lines, respectively synthase (EPSPS) in the shikimate pathway. Resistant P. annua’s have been reported to have increased EPSPS copy number variation and missense mutations [63]. The A subgenome EPSPS gene is 3,891 bp in length, while the B subgenome copy is 9,092 bp. We identify large geno- typic variation in the structure of EPSPS homoeologs, particularly in the B subgenome, where nearly 6 kb is deleted from the two largest introns in some genotypes (Supplementary Fig. 18). We do not see evidence of copy number variation in our resequencing data, but that is likely because none of our accessions originated from a population with known herbicide resistance. individual genotypes display some variability in the exact nucleotide coordinates of the breakpoint but are often within several kilobases of each other (Supplementary Fig. 16; Supplementary Fig. 17). Some individuals appear to contain large-scale variability between their paren- tal haplotypes (heterozygotes). For example, sample ‘Ohio’ has a copy of 1A that resembles the P. The plant cell’s response to WGD Comparative genomics between the P. annua reference genome and the genomes of its diploid parents sug- gests that some tetraploid genotypes are remarkably unchanged since polyploidy. In contrast to paleo-allopol- yploids where biased fractionation is a hallmark of dip- loidization, it appears that neo-allotetraploid P. annua is more accurately characterized by biased gene reshuf- fling, where the B (supina) subgenome has preferentially acquired genes from the A (infirma) subgenome in the Whole‑genome resequencing and large‑scale chromosomal modifications Alternatively, it is possible that dysploidy and Robert- sonian rearrangements (fusion-fission) played an inter- mediate role, where again, introgression back to the population resulted in the observed karyotype [56]. To our knowledge, cytological studies have not recorded any evidence of dysploidy in P. annua. y p y Chromosome 1A has more repetitive DNA (90%) than any other chromosome in the P. annua reference genome, which likely plays a role in the observed restructuring in some genotypes [57]. Most (99%) of the 224 Mb deleted region is low-complexity repetitive sequence, indicating that it would likely be wound into pericentromeric het- erochromatin and suppressed from meiotic recombina- tion [58–60]. Fittingly, rearrangements at 1A appear to reside at the periphery of heterochromatic sequences (Fig. 7b). Large-scale chromosomal rearrangements in P. annua occur in intragenic recombination ‘hotspots’ (resulting in a gene fusion between homoeologs), where Benson et al. BMC Genomics (2023) 24:350 Page 9 of 20 Benson et al. BMC Genomics Fig. 6 Depth of coverage plotted along the Poa annua reference genome shows large-scale variation in chromosome structure. Regions of reduced coverage indicate deletions relative to the reference genome, while regions with elevated coverage show duplications. The P. annua reference genome is unphased (haplotypes are collapsed), so regions with 1/2 × coverage indicate chromosomes with haplotype-specific duplications and deletions (heterozygotes). Samples ‘Germany’ and ‘Arizona’ were imaged 125 days after germination. ‘Ohio’ was imaged 56 days after germination. The two breeding lines (‘Pa-14’ dwarf and wild) were collected from a field experiment and allowed to grow unmowed for 56 days prior to imaging. Mean and median map coverage are indicated with green and red lines, respectively Fig. 6 Depth of coverage plotted along the Poa annua reference genome shows large-scale variation in chromosome structure. Regions of reduced coverage indicate deletions relative to the reference genome, while regions with elevated coverage show duplications. The P. annua reference genome is unphased (haplotypes are collapsed), so regions with 1/2 × coverage indicate chromosomes with haplotype-specific duplications and deletions (heterozygotes). Samples ‘Germany’ and ‘Arizona’ were imaged 125 days after germination. ‘Ohio’ was imaged 56 days after germination. The two breeding lines (‘Pa-14’ dwarf and wild) were collected from a field experiment and allowed to grow unmowed for 56 days prior to imaging. Mean and median map coverage are indicated with green and red lines, respectively Fig. Whole‑genome resequencing and large‑scale chromosomal modifications annua ref- erence genome, while the other haplotype contains the 224/32 Mb centromeric displacement discussed above (Fig. 8; Supplementary Fig. 15). Such variability between haplotypes is surprising given that homologous chro- mosomes recognize each other by sequence similarity and incorrect pairing could lead to multivalents, which are associated with improper segregation and reduced fertility. Variation of EPSPS P. annua is most commonly known as a noxious weed. It can be managed with both pre- and post-emergent her- bicides, but repeated application has resulted in the evo- lution of multiple herbicide resistance pathways [61, 62]. Glyphosate resistance has been particularly problematic for managers of P. annua. Glyphosate works by inhibit- ing the enzyme 5-enolpyruvylshikimate-3-phosphate Benson et al. BMC Genomics (2023) 24:350 Page 10 of 20 Benson et al. BMC Genomics (2 Fig. 7 Sample ‘Arizona’ mapped to the Poa annua reference genome illustrates intraspecific variation in chromosome structure. a Depth of coverage is plotted in pastel alongside a cartoon representation of the P. annua reference genome. Sample ‘Arizona’ reads that preferentially map to the A and B subgenome parents are blue and orange, respectively. Orange segments within blue chromosomes and blue segments within orange chromosomes are putative HEs. Regions of the reference that have no reads mapped are white and indicate deletions. b The chromosome structures of the P. annua reference genome and sample ‘Arizona’ at chromosomes 1A and 1B. The 2 × increase in coverage at chromosome 1B signifies the presence of a large insertion, while the reduction in coverage at 1A signifies a deletion. Split reads at the deletion and duplication breakpoints show that the 1B duplication resides within the deleted region of the 1A chromosome. Gene and LTR density across the chromosomes indicate that the structural modification likely spans centromeric and pericentromeric sequences. The scale bar below chromosomes show hashes that are 10 Mb in length properties of the A and B subgenomes, such as chroma- tin type (heterochromatin or euchromatin) and DNA methylation, play a role in susceptibility. We expect that newly formed allopolyploids with broadly divergent TE immunities will approach retrotransposon-equilibrium as the subgenome with fewer TE inhibitory mechanisms will be preferentially bloated by TEs. absence of measurable loss. It is possible that reshuffling precedes fractionation, and if homoeolog expression bias and TE content are accurate early predictors of subge- nome dominance, as is the case in other allopolyploids [52, 64, 65], the bias accumulator of genes (i.e., the B sub- genome) will be preferentially retained (dominant). The plant cell’s response to retrotransposons in light of WGD The lifecycle of LTRs begins in the nucleus, gets trans- ferred to the cytoplasm as mRNA, and re-enters the nucleus where it integrates into the genome (for review see Sabot and Schulman [66]). The location of LTR inte- gration is at least partially stochastic and dictated by spa- tial nearness of susceptible host DNA upon re-entry into the nucleus. Our data suggests that the subgenomes of P. annua have varying ability to inhibit LTRs, both within and between subgenomes. Because inter-subgenome defense likely involves silencing LTRs after they re-enter the nucleus, we hypothesize that the observed bias in transposon movement from the A (infirma) subgenome to the B (supina) subgenome is partially driven by differ- ences in the subgenome’s ability to repress retrotranspo- sons post-transcriptionally. It is also likely that intrinsic Although the P. annua reference genome closely resem- bles the parental genomes, this is not the case for all P. annua individuals, with some genotypes appearing heavily restructured relative to the genomes of the dip- loid progenitors. It is likely not a coincidence that the observed chromosomal rearrangements result in the substitution of a heavily TE-parasitized region with a less parasitized homoeologous segment. It appears that WGD has provided P. annua with the homoeologous ‘spare parts’ to purge highly parasitized sequences. This result supports the Genome Balance Hypothesis, which pre- dicts that differences in the amount of pericentric hetero- chromatin between subgenomes (as observed between Page 11 of 20 Benson et al. BMC Genomics (2023) 24:350 Benson et al. BMC Genomics Fig. 8 Sample reads mapped to the Poa annua reference genome depict intraspecific variation in chromosome structure. Depth of coverage is plotted along the chromosome. Regions where duplications and deletions occur are highlighted in pink and grey, respectively, and evident by depth of coverage that deviates from the mean. The pairs of cartoon chromosomes depict the two parental haplotypes of 1A (blue) and 1B (orange). The inferred chromosome structure of each sample is based on map coverage and coordinates of split-reads at breakpoint junctions. Sample ‘Ohio’ shows a haplotype-specific modification (heterozygote), likely originating from a cross between a Germany-like sample and an Arizona-like sample. The scale bar is in Mb Fig. 8 Sample reads mapped to the Poa annua reference genome depict intraspecific variation in chromosome structure. Depth of coverage is plotted along the chromosome. The plant cell’s response to retrotransposons in light of WGD Regions where duplications and deletions occur are highlighted in pink and grey, respectively, and evident by depth of coverage that deviates from the mean. The pairs of cartoon chromosomes depict the two parental haplotypes of 1A (blue) and 1B (orange). The inferred chromosome structure of each sample is based on map coverage and coordinates of split-reads at breakpoint junctions. Sample ‘Ohio’ shows a haplotype-specific modification (heterozygote), likely originating from a cross between a Germany-like sample and an Arizona-like sample. The scale bar is in Mb homoeologs of P. annua have already begun to intermin- gle in meaningful ways. Purifying selection might slow the diploidization process but ultimately, we expect that P. annua will be exposed to the ecological consequences of subgenome homogenization and the inevitable func- tional divergence (neofunctionalization and subfunction- alization) and pseudogenization of duplicated alleles. It will be interesting to continue to unravel the aspects of the parental genomes that seem to have helped equip P. annua for success as an allopolyploid and to see if it can maintain its environmental foothold as one of the most 1A and 1B) will cause chromosomes to move to the poles at uncoordinated times, and that the centromere of one of the parents will be retained to overcome those seg- regation issues [67]. We expect that our genome rese- quencing provides a snapshot of an organism caught in that act of positive selection for a balanced genome. 1A and 1B) will cause chromosomes to move to the poles at uncoordinated times, and that the centromere of one of the parents will be retained to overcome those seg- regation issues [67]. We expect that our genome rese- quencing provides a snapshot of an organism caught in that act of positive selection for a balanced genome. Conclusions Poa annua is a globally distributed neo-allotetraploid grass that benefits from the heterotic effects of having two distinct subgenomes, but our work shows that the Page 12 of 20 Page 12 of 20 Benson et al. BMC Genomics (2023) 24:350 sequencing yielded 72 Gb of Q20 reads for P. infirma (29 × fold coverage) and 45 Gb of Q20 reads for P. supina (30 × fold coverage). For Omni-C proximity ligation (Dovetail Genomics), genomic DNA was re-extracted from the same genotypes after 72-h of dark treatment. One proximity ligation library was prepared for each spe- cies and sequenced using the Illumina HiSeq platform to obtain 464 million reads (28 × coverage) for P. infirma and 466 million reads (49 × coverage) for P. supina using 75 × 75 bp paired-end sequencing. We pooled a variety of tissues and treatment types for full-length RNA-sequenc- ing with Iso-Seq (PacBio) to help facilitate high qual- ity gene annotations. Tissue types included germinating seedlings, fresh leaves and root, and juvenile and mature inflorescences. Treatments included clonally propagated individuals that were exposed to 8-h light, 16-h light, cold (4 °C) treatment for two weeks, treated to 1″ simulated mowing stress for one week (five total cuts), and expo- sure to 100 mM NaCl for two weeks. Meristematic crown tissue was collected for each of the described treatments. All RNA samples were extracted using the Qiagen RNe- asy Plant Mini Kit. RNAs for Iso-Seq were pooled and libraries were constructed using the PacBio express kit (v2.0). Each of the two Iso-Seq libraries per species ran for 24 h on an 8 M SMRT cells with a Sequel II instru- ment and yielded 4,026,288 million P. supina transcripts and 3,689,421 P. infirma full-length Iso-Seq transcripts. prolific plants on earth over the next tens and hundreds of millennia as its genome continues to be modified. i Here, we present the chromosome-scale genome sequences of P. infirma and P. supina, the diploid pro- genitor species of allotetraploid P. annua. The genomic resources generated here, and in Robbins et al. [30], com- prise one of the first reports detailing an allopolyploid and its progenitors sequenced to chromosome level. The insights into polyploid evolution that were generated as a result of this work have expanded our understanding of the relationship between plant homoeologs and TEs. Few species have the environmental versatility that P. Collecting genomic and transcriptomic resourcesi Collecting genomic and transcriptomic resourcesi Seeds of P. infirma were obtained from the turfgrass breeding collection at the Pennsylvania State University and represent the only publicly available source of this species. The P. infirma accession used here was origi- nally collected in Spain and acquired by Dr. Shui-zhang Fei at Iowa State University. The ‘Supranova’ cultivar was selected to represent P. supina as it is the most widely used cultivar on the market with agronomic application as a turfgrass, primarily known for its shade tolerance. Seeds were germinated on moist filter paper in petri dishes before being transferred to potting soil in a growth chamber at 20 °C and 8-h day lengths. A single genotype was selected for each species and clonally propagated by manually splitting plants at the basal meristem. Conclusions annua has, and as such, the species serves as an appro- priate model for studying biotic and abiotic stress toler- ance in cereal crops and other agronomically significant polyploids. The genomic resources detailed in this work should serve as a powerful tool for turfgrass breeders and herbicide biologists to solve emerging agricultural challenges by facilitating better targeting of P. annua’s homoeologs and enabling the development of genetic markers that span chromosomal breakpoints for cost- effective surveys of chromosome architecture. Nuclear genome assembly The purged haplo- type assemblies and raw Omni-C reads were input into the HiRise pipeline (Dovetail Genomics) to scaffold con- tigs, identify chimeric scaffolds, and build a final genome assembly based on proximity ligation (Supplementary Fig. 1). Taxonomic classification with Kraken2 (v2.1.1) [74] was used to filter out potential contaminants from the final assemblies and verify that the chromosomes did not contain non-plant DNA, which may indicate a chi- meric assembly. The P. infirma genome assembled into seven pseudomolecules and 873 supplementary scaf- folds. The P. supina nuclear genome contained seven pseudomolecules and 357 supplementary scaffolds. Poa supina pseudomolecules ranged between 73 and 115 Mb, while P. infirma ranged between 90 and 331 Mb in length. The seven chromosomes of each species were re-oriented, if necessary, to reflect identical strand ori- entation across all pairs of orthologous chromosomes. Chromosomes were renamed according to pre-estab- lished chromosomal nomenclature and large structural modifications between each diploid and the allotetra- ploid P. annua were verified by sequence alignment using minimap2 with parameters ‘–secondary = no -cx asm10’ (v.2.24) [75]. centromeric and telomeric repeats using ‘bedtools nuc’ and a 1 Mb sliding window to count the occurrences of common repetitive sequences found in the centromeric and telomeric sequences of Poaceae. The purged haplo- type assemblies and raw Omni-C reads were input into the HiRise pipeline (Dovetail Genomics) to scaffold con- tigs, identify chimeric scaffolds, and build a final genome assembly based on proximity ligation (Supplementary Fig. 1). Taxonomic classification with Kraken2 (v2.1.1) [74] was used to filter out potential contaminants from the final assemblies and verify that the chromosomes did not contain non-plant DNA, which may indicate a chi- meric assembly. The P. infirma genome assembled into seven pseudomolecules and 873 supplementary scaf- folds. The P. supina nuclear genome contained seven pseudomolecules and 357 supplementary scaffolds. Poa supina pseudomolecules ranged between 73 and 115 Mb, while P. infirma ranged between 90 and 331 Mb in length. The seven chromosomes of each species were re-oriented, if necessary, to reflect identical strand ori- entation across all pairs of orthologous chromosomes. Chromosomes were renamed according to pre-estab- lished chromosomal nomenclature and large structural modifications between each diploid and the allotetra- ploid P. annua were verified by sequence alignment using minimap2 with parameters ‘–secondary = no -cx asm10’ (v.2.24) [75]. Genome annotation RNA-sequencing runs SRR1634026 and SRR1634028 were downloaded from NCBI’s Sequence Read archive database representing P. supina and P. infirma, respec- tively. Poa annua sequences from experiments SRR1634028, SAMD00020897, and SAMD00020898 were also acquired. All NCBI sequencing experiments were then trimmed for adapter content and low qual- ity using bbduk with ‘tbo tpe ktrim = r k = 23 mink = 11 hdist = 1’. Cleaned reads from NCBI could be larger than 20 gigabytes, so we randomly subset each experimen- tal run into a single 400-megabyte file. Each fastq file was aligned to the respective genome using the splice- aware algorithm, HISAT2 (v2.2.1) [83]. Iso-Seq tran- scripts for each species were aligned using minimap2 with ‘-ax splice:hq -uf’. NCBI and Iso-Seq alignment files were sorted by name and converted to bam format. The OrthoDB plant protein database (v10) was downloaded and expanded to include amino acid sequence annota- tions from the Poales available through NCBI refseq and Uniprot TrEMBL. BRAKER2 (v2.1.5) was run in ETP mode to incorporate both the enhanced OrthoDB pro- tein data and the RNA alignment data from NCBI and Iso-Seq to train GeneMark-ETP with proteins processed by ProtHint. Augustus was trained based on the Gene- Mark-ETP predictions and the resulting protein predic- tions were hints from both sources. BRAKER2 added 5’ and 3’ UTRs using ‘–addUTR = on’ to call GUSHR. Annotations were filtered using sequence similarity to orthologous groups and phylogenies in the eggNOG [84] database (v2.0.5) using diamond alignments to retain only those annotations with fine-grained orthologous relationships. BUSCO (v3.0.2) was used in transcriptome mode to identify the majority (96% and 91%) of the 1,614 conserved embryophyta_odb10 orthologs were present in our P. infirma and P. supina chromosome annotations, respectively, supporting high-quality genome annota- tions. Long noncoding RNAs were identified using RNA- plonc (v1.1) [85] that uses a classifier approach developed specifically for plants. The chloroplast genome assem- blies were functionally annotated using GeSeq [86]. Nuclear genome assembly resulting TE consensus classification libraries were used as input into RepeatMasker (v4.1.2) to softmask each of the genome assemblies using the wublast engine. LTR_ FINDER_parallel (v1.1; with parameter ‘-harvest_out’) [81] and LTR_retriever were run separately on all three species to calculate the insertion times for intact LTR ele- ments. A rice mutational rate of 1.3 × 10–8 substitutions per year was used to calculate insertion times using the formula T = K/2µ, where K is the divergence rate calcu- lated based on LTR sequence identity and µ is the neutral mutational rate in mutations per bp per year [82]. Chloroplast genome assembly Raw whole-genome sequenced HiFi reads were mapped to the P. annua chloroplast reference genome (GenBank acc: NC_036973.1) using minimap2 (v2.24) using ‘map- hifi’. Samtools (v1.9) [76] was used to identify mapped reads with a minimum query length (mlen) > 8000, query value (qval) > 60 and GC content between 32 – 52%. Reads with a length > 20,000 bp were then included in a final de novo assembly of the chloroplast genome with HiCanu (v2.1) using default parameters. A circu- lar genome was predicted by HiCanu, which was sub- sequently trimmed as projected by HiCanu at the same starting point as the reference chloroplast genome. Sequence alignment of the circular chloroplast genomes for each species verified that P. infirma is the maternal parent to P. annua (Supplementary Fig. 20). Nuclear genome assembly K-mers were extracted from long-read (HiFi) sequenc- ing data using Jellyfish (v2.2.10) [68] with 21-mers and a hash with 100 M elements (parameters ‘-m 21 -s 100 M’). GenomeScope (v.1) [69] was used to plot k-mers and esti- mate genome size, level of heterozygosity, and amount of repetitive sequence using 15,000 bp read lengths (Supplementary Fig. 19). K-mer analysis confirmed that P. supina was highly heterozygous and P. infirma was highly homozygous [47]. As a result, we selected differ- ent assembly pipelines for each species that best accom- modated its unique biology. The genome of the highly heterozygous and obligate outcrosser, P. supina, was assembled with HiCanu (v2.1) [70] and purged to hap- lotig level using the Purge_Dups (v1.0.1) pipeline [71] with manual cutoffs adjusted according to its heterozy- gosity (calcuts parameters ‘-l 7 -m 40 -u 160’; minimum alignment score (-a) to 80). Poa infirma is self-pollinated and highly homozygous. As a result, we assembled the P. infirma genome using HiFiasm (v0.3) [72] with its built- in haplotype purging algorithm that is better suited for homozygous genome assemblies’. The Benchmarking Universal Single-Copy Orthologs (BUSCO; v3.0.2) soft- ware was used to estimate assembly completeness and their quality [73]. We also scanned for incorrectly placed Genomic DNA was extracted from fresh leaf tissue using the cetyltrimethylammonium bromide (CTAB) method as outlined by OPS Diagnostics protocols with minimal vortexing and cut pipet tips to promote high molecular weight DNA extractions. Sample integrity was verified using pulsed-field electrophoresis and indi- cated an average size range between 50–70 kb. DNA was sheared to 20 kb length using a Megaruptor (PacBio). HiFi libraries were constructed using the PacBio Express kit, v2.0, and size selection was performed on a SageELF (Sage Science) to obtain narrow 15–20 kb libraries for sequencing using a PacBio Sequel II (Brigham Young University, DNA Sequencing Center). Three 8 M SMRT cells with 30-h movies were used for each diploid. PacBio Page 13 of 20 Benson et al. BMC Genomics (2023) 24:350 Benson et al. BMC Genomics (2023) 24:350 centromeric and telomeric repeats using ‘bedtools nuc’ and a 1 Mb sliding window to count the occurrences of common repetitive sequences found in the centromeric and telomeric sequences of Poaceae. Repeat masking and LTR insertion times De novo repeat libraries were created for each diploid assembly using the Dfam (v3.1) [77] database to classify transposable DNA sequences. RepeatModeler (v2.0.3) [78] with the parameter ‘-LTRStruct’ was used to model TE family relationships and identify repetitive ele- ments by employing programs RECON, RepeatScout, LTRHarvest [79] and LTR_retriever (v2.8.7) [80]. The Page 14 of 20 Benson et al. BMC Genomics (2023) 24:350 Cytology separately using STAR (v2.7.8a) [91]. The EAGLE model subsequently evaluated the likelihood of each reads sub- genome origin based on genotypic variants and assigns a likelihood score. Alignments with SNP evidence to support subgenome origin are dubbed homoeolog- specific and quantified with featureCounts (v2.0.2) [92]. The resulting counts matrix was filtered to retain only the genes that had at least one read per sample. The ‘run_DE_analysis.pl’ script from the trinityrnaseq toolkit (v2.13.0) [93] was used to run DESeq2 (v1.38) [94] on the counts matrix for subgenome-specific differential expres- sion analysis. Because plant biotypes (dwarf or wild) were nested within treatments (mowed or unmowed), biotype as a variable was removed to prevent erroneous interpre- tation in our mowed vs unmowed and A vs B subgenome comparisons (Supplementary Fig. 9). C-banded chromosome preparations were made from root-tip meristematic cells according to the protocol described by Mitchell et al. [87] except that 0.02% colchi- cine, not trifluralin, was used to arrest microtubule for- mation for 2–4 h at room temperature. RNA‑seq expression analysis and homoeolog expression bias Plants were collected from an ongoing field trial from the turfgrass breeding program at the Pennsylvania State University. For each P. annua breeding line, at least one typical dwarf-type and one aberrant wild-type plant were collected from a genetically pure unmowed stand. Dwarf-types were defined as any genotype with diam- eter ≤ 1.5 cm, while aberrant wild-types had a diam- eter ≥ 6 cm. Plants were transplanted to a greenhouse (27 °C high and 17 °C low) and clonally propagated over two months. To simulate mowing treatment, one clone of each genotype was trimmed three times per week and maintained at 1.5 cm height and the other clone was left untrimmed. The experiment was conducted on 30 plants representing 15 unique genotypes (six dwarf-types and nine wild-types). Spacing on the bench was randomly assigned. Treatments were applied between May 10th and August 16th, 2020. All plants were allowed to grow unmowed for an additional three weeks prior to tissue collection to reduce the influence of wounding stress in our data analysis. Tissue was collected from the grass’s basal meristem. Gene ontology enrichment analysis gy y Gene ontologies and functional enrichments of differ- entially expressed genes were classified using the Trino- tate pipeline. Blastp and Blastx (v2.12) [95] were used to align P. annua amino acids and coding sequence files against the Uniprot Swissprot database with parameters ‘-max_target_seqs 1 -outfmt 6 -evalue 1e-3’. Hmmscan (v3.3.2) was used to incorporate protein domain identi- fication based on query against the pfam database. An id2go formatted file was then generated using the blastx, blastp, and hmmscan results to incorporate the Swis- sprot and pfam alignments using go-basic and pfam2go annotations from geneontology.org. The id2go formatted file was incorporated into ‘analyze_diff_expr.pl’ (trinityr- naseq toolkit) with the ‘–examine_GO_enrichment’ flag to call the R package Goseq to scan for enriched gene ontologies in our subgenome-specific differential expres- sion matrix. The id2go formatted file was also used as input into Goatools script ‘find_enrichments.py’ [96] to identify enriched ontologies in various subsets of genes of interest. Candidates from enriched subsets were fur- ther analyzed using EggNOG-mapper and BLAST for functional annotation at the single-gene level. Unique libraries for each sample were created using the Lexogen SENSE mRNA-seq library kit with the goal of producing long insert sizes of ~ 485 bp for simplified and accurate inference of parental origin across homoe- ologous pairs [88]. A pilot study was conducted using a MiSeq with Nano kit reagents (v2) to obtain 500 Mb of 250 × 250 bp paired-end sequencing. The pilot analy- sis revealed that insert sizes were generally shorter than anticipated with 75% of inserts being ≤ 260 bp. Adjusting for shorter library lengths, we sequenced the RNAs using an S1 flow cell on an Illumina NovaSeq 6000 (Pennsylva- nia State University, Genomics Core Facilities) to obtain 150 × 150 bp paired-end reads with ~ 48 million reads/ sample. Resequencing P. annua Resequencing P. annua 15 geographically distinct P. annua accessions were sequenced to survey genotypic variation across the spe- cies. Poa annua is a heavily admixed species, and as such, it is difficult to know the degree to which accessions are genetically isolated from each other. Our goal was to choose 15 accessions across distinct locations to maxi- mize our chances of capturing a wide range of genetic diversity. Samples ‘Germany’ (W6 28,152), ‘Nunavut’ (PI 236900), ‘India’ (PI 217625), and ‘Belgium’ (PI 442543) were acquired from the Germplasm Resources Informa- tion Network (GRIN) through the US Department of Agriculture. Samples ‘Washington’ (Tacoma), ‘Scotland’ (Galloway), ‘New Zealand’ (Manawata), ‘Arizona’, ‘Que- bec’, ‘Wales’ (Aberystwyth), ‘Sweden’ (Särö), ‘New York’ (Pa-33) and ‘Ohio’ (Columbus) were acquired from a breeding collection maintained at the Pennsylvania State University. Seeds were germinated on moist filter paper. A single genotype of each of the thirteen samples was transferred to potting soil (Promix) and grown in green- house. In addition to the thirteen geographically distinct accessions, two breeding lines were included. ‘Pa-14 dwarf’ and ‘Pa-14 wild-type’ are derived from the same breeding pedigree of an unstable line (Pa-14), where ‘wild’ describes an aberrant wild-type plant and ‘dwarf’ describes an agronomically desirable dwarf individual. DNA was extracted from all 15 samples using fresh leaf tissue and the CTAB method as described above. Plants were genotyped to confirm that they were authentic P. annua’s using the Trx2 nuclear gene with PCR parame- ters described in Mao and Huff [18], and Patterson et al. [105]. Genomic DNA (300 ng) from each sample was input into the Illumina DNA PCR-Free Prep kit to create uniquely indexed libraries. The samples were pooled and an equimolar concentration was verified using a MiSeq Nano 150 × 150 bp. The pooled sample was sequenced on a NovaSeq S1 (Pennsylvania State University, Genomics Core Facilities) with 150 × 150 bp paired-end sequenc- ing to generate a target of 1.3 – 1.6 billion pairs and Comparative genomics Comparative genomics P. annua (PaA & PaB) and a concatenated file contain- ing the diploid parents (PiA & PsB) were uploaded into CoGe SynMap tool [97] with DAGChainer options ‘-D 20 -A 5’ and tandem duplication distance set to 10. Syn- onymous mutation (Ks) was calculated on the syntenic CDS pairs using CodeML of the PAML package. Ks val- ues were plotted on a density plot to visualize Ks peaks associated with parental divergence and hybridization. For CoGe’s fractionation bias calculation, syntenic blocks were merged using the ‘Quota Align Merge’ algorithm with a maximum distance between two genes (-Dm) set We used Eagle-RC (v1.1.2) [89] to classify RNA-seq reads to their appropriate subgenome using explicit genotypic differences between them to calculate the like- lihood that an RNA read came from a particular subge- nome. Briefly, variant candidates for statistical inference were generated using reciprocal LAST (v1387) [90] to identify homoeologous genes and an Eagle-RC python script (homeolog_genotypes.py) to create the variant file (VCF). Reads were mapped to the parental genomes Page 15 of 20 Page 15 of 20 Benson et al. BMC Genomics (2023) 24:350 Benson et al. BMC Genomics (2023) 24:350 to 40. Syntenic depth was calculated with the ‘Quota Align’ algorithm and ratio of coverage depth set to 1-to- 2. The window size for fractionation bias was adjusted to 100 genes and set to only use syntenic genes in the tar- get genome. MCScanX [98] was used to detect syntenic blocks of genes between P. annua and the diploid progen- itors. The collinear file was input into SynVisio [99], an interactive multiscale synteny visualization tool to depict regions of shared homology. Syntenic pairs and mac- rosynteny in monocots was calculated using the MCscan (python version) with Ananas comosus and Brachypo- dium distachyon coding sequences and genomes down- loaded from Phyotozome (v12) [100]. A C-score of 0.99 was used to select only 1:1 orthologous blocks and is stringent enough to filter out syntenic blocks that were not LAST reciprocal best hit. Translated transcriptomes of model grasses were acquired through Phytozome and were asigned into orthogroups using Orthofinder with the Diamond algorithm for similarity searches. Average nucleotide identity (ANI) was calculated using the gap- compressed per-base sequence divergence output (de tag) of a PAF formatted full genome assembly alignment using minimap2. Comparative genomics DupGen_finder [42] was used to iden- tify single-gene duplicate pairs and classify them as either WGD, tandem, proximal, transposed, or dispersed. A concatenated fasta file containing both parental diploids (PiA & PsB) was used as an outgroup so that the trans- posed classification included only those genes that were duplicated after the hybridization of P. annua. mapped to the PiA genome and reads that mapped to the PsB genome. Both pools of P. annua reads assigned a custom tag based on their parental mapping and were re-mapped to P. annua. If a P. annua read mapped best to the P. infirma (PiA) parent but subsequently mapped to P. annua’s B (supina) subgenome, it was a candidate for HE. All four HE methods were compared and it was deter- mined that the primary mapping approach was superior as it was visually verifiable in the Integrative Genomics Viewer (IGV) [104] and produced HE statistics that were most intermediate to the other methods. The JCVI chro- mosomal painting tool (jvci.graphics.chromosome) was used to visualize P. annua’s HEs. Identification of HEs Primer pair 1AF (5′- GGCGGACACCTTTGACACC) and 1AR (5′- GGATACTCAGACAATGATAG) amplify using stand- ard PCR settings with a 53 °C annealing temperature and 1:00 extension time. Primer pair 1AF (5′- GGCGGACAC CTTTGACACC) and 1BR (5′- GGGTGACAGAGTTCC CAGTG) amplify using standard PCR settings with a 65 °C annealing temperature and 1:20 extension time. 1AF to 1AR spans a chromosomal breakpoint and only amplifies in the absence of the 32/224 Mb structural modification. 1AF to 1BR spans the same chromosomal breakpoint but only amplifies in the presence of a rearrangement (Supplemen- tary Fig. 15).i g Raw Illumina reads were trimmed for adapter sequences using bbduk as described above and aligned to the P. annua reference genome using bwa-mem2. Scaffolds corresponding to the chloroplast and mito- chondrial genomes were included in the P. annua refer- ence genome to prevent erroneous alignment of plastid sequences to the genome. Coverage across chromosomes and scaffolds were plotted using WGSCoveragePlotter. jar (jvarkit). Putative HEs were annotated similarly as described above. Briefly, raw reads were mapped to a file containing the parental P. infirma (A) and P. supina (B) genomes. Primary alignments were tagged according to their parental origin and re-mapped to the P. annua ref- erence genome. Reads that mapped to a different paren- tal genome than P. annua subgenome were potentially a HE. We then classified each coordinate in the P. annua reference as either derived from P. supina, derived from P. infirma, or novel (not derived from either parent). In contrast to the HE pipeline used above that used HiFi (ccs) data, novel regions were annotated independently as opposed to being unincluded in the bed file. This adjust- ment allowed more accurate visualization of short-reads with jcvi.graphics.chromosome. SNPs were identified from each of the 15 sam- ples using their corresponding P. annua align- ment file. Picard MarkDuplicates was used to tag duplicated reads and reduce the frequency of incor- rect SNP calls. The duplicate-marked bam files were used to generate genotype likelihood calls across all samples and chromosomes using parameters ‘-q 40 –ff UNMAP,SECONDARY,QCFAIL,DUP’ with bcftools mpileup and subsequently input into bcftools call with default parameters. Variants were further filtered with vcftools using parameters ‘–remove-indels –maf 0.1 – max-missing 0.9 –minQ 30 –min-meanDP 10 –max- meanDP 80 –minDP 10 –maxDP 80’. Presence-absence variants were analyzed using an SGSGeneloss-based protocol, described in Fernandez et al. [106]. Identification of HEs Illumina reads were mapped to the paren- tal genomes and subsequently the P. annua genome to identify HE regions as described above. The alignment file for each sample was converted to bed format using bamToBed from bedtools (v2) [107]. The alignment bed was merged with the gene annotation file using bedtools intersect to identify regions of overlap. If a gene’s coor- dinates contained < 20% coverage in the sample, it was deemed lost (dispensible) in that sample. If it had > 90% coverage in the opposite parent, it was deemed a gene within an HE. Identification of HEs HEs regions were characterized using several different methods to assure accurate identification. First, CNVkit (v0.9) [101] was used to identify and visualize copy num- ber variants by mapping HiFi (ccs) reads from P. annua onto the parental diploid genomes (PiA & PsB). Second, minimpa2 with parameter ‘-x map-hifi’ was used to map P. annua HiFi reads to the concatenated fasta containing the parental diploid genomes (PiA & PsB). The resulting bam file was input into SVIM (v1.0.2) [102] and used to detect structural variants from our long-read sequenc- ing data and extract split-reads with translocation break- points, called BNDs by SVIM. Split-reads were extracted from the bam file and used to detect beginning and endpoint of a HE block. Thirdly, we used mmseqs [103] with parameters ‘easy-rbh -s 7.5’ to identify P. annua coding sequences with reciprocal best hits correspond- ing to the other subgenome (PaA genes with RBHs on PsB or PaB genes with RBHs on PiA). Finally, we used a primary mapping approach where P. annua HiFi reads are aligned to a fasta file containing both parental dip- loid genome (PiA & PsB). Reads with primary mapping flag were retained and sorted into two pools, reads that Page 16 of 20 Page 16 of 20 Benson et al. BMC Genomics (2023) 24:350 Benson et al. BMC Genomics (2023) 24:350 15–20 × coverage per genotype across the haploid (1.78 Gb in size [30]) genome. 15–20 × coverage per genotype across the haploid (1.78 Gb in size [30]) genome. as Delly2, Manta, and Lumpy are not equipped to identify insertions larger than several kilobases in length. Large- scale structural rearrangements were verified using homoe- olog-specific primers and Sanger sequencing. Primer pair 1AF (5′- GGCGGACACCTTTGACACC) and 1AR (5′- GGATACTCAGACAATGATAG) amplify using stand- ard PCR settings with a 53 °C annealing temperature and 1:00 extension time. Primer pair 1AF (5′- GGCGGACAC CTTTGACACC) and 1BR (5′- GGGTGACAGAGTTCC CAGTG) amplify using standard PCR settings with a 65 °C annealing temperature and 1:20 extension time. 1AF to 1AR spans a chromosomal breakpoint and only amplifies in the absence of the 32/224 Mb structural modification. 1AF to 1BR spans the same chromosomal breakpoint but only amplifies in the presence of a rearrangement (Supplemen- tary Fig. 15).i as Delly2, Manta, and Lumpy are not equipped to identify insertions larger than several kilobases in length. Large- scale structural rearrangements were verified using homoe- olog-specific primers and Sanger sequencing. Supplementary Information Geo- graphic distribution of 13 re-sequenced Poa annua accessions. Two additional plants (PA-14 dwarf and Pa-14 WT) were also sequenced but not depicted on the map. Supplementary Fig. 12. SNP density across a 1 Mb sliding window demonstrates variability in sequence identity between Poa annua accessions and across chromosomes. Pink is sample ‘Arizona’ and blue is sample ‘Wales’. Coverage plots of both sam- ples are included for reference. The scale bar is 320 Mb in length with each hash representing 10 Mb. Supplementary Fig. 13. The core Poa annua genome. Of the 76,541 gene annotationsin the P. annua reference genome, 68,733 are present in all 15 re-sequenced genotypes. 7,808 genes are dispensable and absent in at least one genotype. Supplemen‑ tary Fig. 14. Depth of coverage plotted along the Poa annua reference genome suggests large-scale structural modification in P. annua chromosomes. Supplementary Fig. 15. Homoeolog-specific markers and Sanger sequencing verifies the composition of a large-scale chromosomal rearrangement in Poa annua. Alignment of the homoeolo- gous sequences of Pa1A and Pa1B span the breakpoint of a large-scale chromosomal rearrangement in re-sequenced genotype ‘Arizona’ but not ‘Germany’. Sample ‘Ohio’ has both a rearranged and non-rearranged chro- mosome, verifying the haplotype specificity (heterozygotes) of chromosome rearrangements in some individuals. Black arrows highlight homoeolog distinguishing SNPs on either side of the breakpoint. Supplementary Fig. 16. Sequence alignment of three samples to the Poa annua reference genome illustrates recombination hotspots. boxes). Arrows that are perpendicular to genes are gene fusion events. Supplementary Fig. 17. Sequence alignments at chromosome 1A illustrates local variability at crossover ‘hotspots’. Black arrows indicate positions where both pairs of homologous chromosomes break at the same location and red arrows point to haplotype-variable breakpoints. Blue boxes at the top of the alignment window show genes with exons (boxes) and introns (lines connecting boxes). Arrows that are perpendicu- lar to genes are gene fusion events. Supplementary Fig. 18. Sequence alignment of four Poa annua accessions shows structural variation at its two EPSPS homoeologs. ajg15317 and ajg73723 are EPSP synthases on chromosomes Pa5A and Pa5B, respectively. Blue boxes at the top depicts the genes exons (boxes) and introns (lines connecting boxes). The ajg15317 transcript is 3,891 bp in length, while ajg73723 is 9,092 bp. Grey boxes are reads that aligned to the reference genome as proper pairs. Open boxes are reads that mapped equally well to five or more locations in the genome. Funding This research was supported by the United States Golf Association under the Turfgrass and Environmental Research Program; the Pennsylvania Turfgrass Coun- cil (PTC); the Huck Institute of Life Sciences, Pennsylvania State University; the College of Agricultural Sciences, Pennsylvania State University; and Hatch Project PA 4592. This research was also supported by the USDA National Institute of Food and Agriculture, Hatch project 1023293 and SCRI project 2020-02585. The funding bodies did not contribute to the design of the study, or the collection, analysis, and interpretation of data. Supplementary Information BMC Genomics (2023) 24:350 Benson et al. BMC Genomics annua and the genomes of the diploid parents (PiA and PsB). Arrows and corresponding values highlight the peak density of synonymous substitutions. Supplementary Fig. 6. Sequence alignment of five model monocots spanning two whole-genome duplications. (a) A phylogenetic tree shows the species relationships. Pineapple (Ananas comosus) serves as outgroup because its speciation from the BOP and PACMAD clades of grasses predates the most recent of the ancestral Poaceae WGD events, rho (r). (b) Genomic alignment between monocots Ananas comosus, Brachypodium distachyon, Poa infirma, P. supina, and P. annua. Percentages in red show the ratio of 1:1 orthogroups relative to A. comosus. The syntenic block highlighted in green shows the colinear evolution of a cluster of genes. Supplementary Fig. 7. Retention of Poa annua genes across the parental chromosomes of P. infirma and P. supina. PiA to PaA and PsB to PaB have elevated gene retention (~97%) across chromosomes and reflects minimal gene loss (fractionation) since polyploidization. PiA to PaB and PsB to PaA have lower gene retention (~61%) and reflects the quantity of genes retained since the two parental lineages diverged from their common ancestor. The red arrow highlights the largest homoeologous exchange in the P. annua genome. Supple‑ mentary Fig. 8. Principal component analysis compares the gene expression profiles of 30 Poa annua samples. On the right, a PCA including all samples, with variables being subgenome (A or B), treatment (mowed or unmowed), and biotype (dwarf-type or wild-type). On the left, a PCA of the A subgenome illustrates that samples cluster by mowing treatment but biotypes are nested. Supplementary Fig. 9. Differential gene expression analysis across the A and B subgenomes of Poa annua. (a) Principal component analysis of the differential gene expression profiles of P. annua samples with biotypes (dwarf-type or wild-type) removed from the analysis. (b) A heatmap of the DEGs across subgenome and treatment. Blue genes are upregulated and orange are downregulated. (c) Clusters of genes with similar expression profiles. On the left, a cluster of genes upregulated in the B subgenome. On the right, a cluster upregulated in unmowed plants. In the DEG hierarchal cluster and dendrogram, red=B_mowed, green=B_unmowed, purple=A_unmowed, blue=A_mowed. Supplementary Fig. 10. Treemaps cluster enriched gene ontologies in the subgenome (A vs B) and treatment (mowed vs unmowed) comparisons based on semantic similarity of enriched terminologies. Supplementary Fig. 11. Supplementary Information Red pairs have longer than anticipated insert lengths and depict putative indels at ajg73723’s longest introns. Sample ‘Sweden’ is heterozygous for a 2,738 deletion at the second intron, while ‘Wales’ and breeding line ‘Pa-14 dwarf’ are homozygous for the deletion. Only the breeding line sample, ‘Pa-14 dwarf’ contained a 2,954 deletion at the 7th intron. Purple alignments in ajg15317 show reads with mates that map to the other subgenome homoeolog (ajg73723), within the indel at the second intron. Supplementary Fig. 19. Linear K-mer profiles and fitted models of the Poa infirma and P. supina genomes. Black lines show the fit of the model to the distribution of K-mer frequencies (blue). Sequencing errors are identified by low coverage k-mers shown in orange. The P. infirma and P. supina models follow a diploid distribution with low and high heterozygosity, respectively. Supplementary Fig. 20. The chloroplast sequences of Poa infirma and P. supina. (a) Chloroplast maps for P. supina and P. infirma. (b) Sequence alignment of chloroplasts show that P. annua’s maternal parent is P. infirma.(PPTX 45713 KB) Authors’ contributions CWB and DRH conceived and designed the project and all research activities. CWB, DRH, MDR, and BSB collected the samples, extracted DNA and RNA, and directed the Illumina and PacBio sequencing. CWB and MDR assembled and annotated the genomes. CWB performed the comparative genomics with contributions from MRS, ELP, NDH, and PJM. The chloroplast genome was assembled and annotated by PJM, and ENJ performed the cytology. CWB performed the gene expression analysis and analysis of EPSPS alleles. CWB designed and implemented the homoeolog-specific markers. CWB, PJM, ELP, and MDR coordinated data submission. CWB wrote the manuscript with review and revisions from all other authors. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12864-023-09456-5. Additional file 1: Supplementary Table 1. Features of the genome assemblies and annotations. Parental species, Poa infirma (PiA) and P. supina (PsB), relative to the subgenomes of P. annua (PaA and PaB). Values correspond to the seven pseudomolecules. BUSCOs are n=1,614. Supplementary Fig. 1. Linked density histograms of the Poa infirma and P. supina genomes. The Omni-C plot represents long-range cis information using proximity ligation and mapping position of paired-end data. Red indicates the number of read pair interactions within each bin. Supplementary Fig. 2. The homoeologous and orthologous sequences of Poa annua and its diploid parents, P. infirma and P. supina. (a) The evolutionary pathway and chromosomal relationships within and between the homologous sequences of P. annua and its diploid progenitors. (b) The distribution of sequence identity measured by the gap-compressed sequence identity of full-genome alignments. Percentages above violin plots indicate the median. (c) Chromosome lengths of the genome assemblies of all three species. Supplementary Fig. 3. Whole-genome sequence alignment depicts the primary mapping of parental chromosomes (PiA & PsB) to allotetraploid, Poa annua (PaA & PaB). The black arrow over Pa2B highlights 30 Mb of novel sequence in the tetraploid genome assembly, mostly composed of repetitive DNA (22.7Mb). Supplementary Fig. 4. The distribution of shared orthologous clusters (gene families) between the A and B genomes of Poa infirma (PiA), P. supina (PsB), and P. annua (PaA & PaB). Single-copy gene clusters are not depicted. Supplementary Fig. 5. Estimated molecular divergence of the PaA and PaB subgenomes of P. Large-scale structural variants were annotated manu- ally by analyzing the depth of coverage of each sample alignment to both progenitor and allotetraploid genomes. Duplicated and deleted sequences will cause deviation from the mean and median coverage. Duplicated sequences align to the next most homologous coordinates in the reference genome and are visible by elevated coverage at that site. Deleted sequences are detectible by reduced coverage at the missing region. Transposed sequences are represented in equal proportion in the reference and in the sample, therefore they do not cause deviations from the mean cov- erage. Split reads and improperly paired reads at the junc- tions of duplicated and deletion breakpoints can be used to further specify the exact coordinated of the exchanges. Commonly used tools that identify structural variants such Page 17 of 20 Benson et al. References Xiong Z, Gaeta RT, Edger PP, Cao Y, Zhao K, Zhang S, et al. Chromosome inheritance and meiotic stability in allopolyploid Brassica napus. G3 Genes\|Genomes\|Genetics. 2021;11:jkaa011. 29. Dunwell JM. Haploids in flowering plants: origins and exploitation. Plant Biotechnol J. 2010;8:377–424. 29. Dunwell JM. 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https://openalex.org/W4225424848	https://www.frontiersin.org/articles/10.3389/fenvs.2022.902915/pdf	English	null	Application of Invasive Plants as Biochar Precursors in the Field of Environment and Energy Storage	Frontiers in environmental science	2,022	cc-by	8,670	MINI REVIEW published: 04 May 2022 doi: 10.3389/fenvs.2022.902915 Application of Invasive Plants as Biochar Precursors in the Field of Environment and Energy Storage Lei Yang, Yuanyuan Deng, Zihan Shu, Qiang Chen, Hailan Yang and Xiaofei Tan* College of Environmental Science and Engineering, Hunan University and Key Laboratory of Environmental Biology and Control (Hunan University), Ministry of Education, Changsha, China Plant invasion caused due to various human activities has become a serious problem affecting ecosystem diversity and imposes a burden on the economy. In recent years, there have been increasing studies on the application of biochar (BC) in the ﬁeld of environmental protection. Invasive plants, which are considered as a kind of hazardous waste biomass, can be used as feedstocks to prepare BC. Consumption of invasive plants for BC preparation can achieve a win-win situation in ecology and resources. This can solve a series of ecological problems caused by invasive plants to a certain extent while also realizing the resource utilization of wastes and bringing considerable economic beneﬁts. Based on previous studies, this paper summarizes the progress of preparing and using invasive plant biochar (IPB). This includes the production, modiﬁcation, merit and demerit of IPB, its application in improving soil quality, the adsorption of pollutants, application in energy storage, and climate change mitigation potential. It provides a basis for further study of IPB based on the currently existing problems and proposes a direction for future development. Edited by: Edited by: Xiaodong Xin, Dongguan University of Technology, China Reviewed by: Hang Yu, Dalian Maritime University, China Luxin Zhang, Xi’an University of Architecture and Technology, China Keywords: biochar, invasive plants, resource utilization, environmental remediation, energy storage INTRODUCTION Correspondence: Xiaofei Tan tanxf@hnu.edu.cnmailto Correspondence: Xiaofei Tan tanxf@hnu.edu.cnmailto Invasive plants are one of the important factors that affect the global ecological environment (Simberloff et al., 2013). Due to the strong adaptability and rapid spreading ability of invasive plants, their negative impact on the local ecosystem cannot be ignored. Many effective methods to limit the invasion of alien plants have been derived, including chemical, biological, and alternative controls (Terry et al., 2021). These treatment methods have certain effects in long-term practice but still require considerable manpower and material resources. Hence, we need to ﬁnd a more economical and effective way to deal with invasive plants. As an emerging material for environmental protection, biochar (BC) is characterized by its economy and efﬁciency (Beesley et al., 2011). Studies show that biochar production from invasive plants is suitable for mitigating ecological damage caused by biological invasion (Feng et al., 2021). Biochar production using invasive plants as raw materials realizes the effective utilization of waste and controls the expansion of invasive plants to a certain extent. Thus, this method can potentially alleviate the issue of invasive plants. Specialty section: This article was submitted to Toxicology, Pollution and the Environment, a section of the journal Frontiers in Environmental Science Received: 23 March 2022 Accepted: 18 April 2022 Published: 04 May 2022 Specialty section: This article was submitted to Toxicology, Pollution and the Environment, Specialty section: This article was submitted to Toxicology, Pollution and the Environment, a section of the journal Frontiers in Environmental Science Specialty section: This article was submitted to Toxicology, Pollution and the Environment, a section of the journal Frontiers in Environmental Science Received: 23 March 2022 Accepted: 18 April 2022 Published: 04 May 2022 Citation: Yang L, Deng Y, Shu Z, Chen Q, Yang H and Tan X (2022) Application of Invasive Plants as Biochar Precursors in the Field of Environment and Energy Storage. Front. Environ. Sci. 10:902915. doi: 10.3389/fenvs.2022.902915 Biochar possesses excellent physical and chemical properties. It can be prepared by pyrolysis of waste biomass (Manya, 2012; Tan et al., 2015; Feng et al., 2021). Presently, biochar is often used to improve the soil environment and remove various pollutants in the environment. Hence, it is an important material that aids in environment recovery (Zhang et al., 2013; Ahmad et al., 2014b; Mohan et al., 2014). However, the physical and chemical properties of biochar are greatly affected by raw materials and preparation methods. Hence, ﬁnding suitable raw materials and corresponding preparation methods is particularly important. May 2022 \| Volume 10 \| Article 902915 1 Frontiers in Environmental Science \| www.frontiersin.org Application of Invasive Plants Biochar Yang et al. growth, invasive plants can cause signiﬁcant harm to the local ecological environment (Vila et al., 2011; Eviner et al., 2017). Invasive plants increase the risk of extinction of native species by disrupting biogeographical areas and encroaching on their living space and resources. Many invasive plants also alter ecosystem functions by altering nutrient cycling, habitat structure, and disturbance mechanisms (Pysek et al., 2020). Concurrently, some invasive plants release toxic substances while consuming survival resources, which critically threatens the health and safety of humans and livestock (Weller et al., 2015; Jank and Rath, 2021; Simmons et al., 2021). The economic cost of plant invasion is increasing worldwide, which brings great ﬁnancial burden to the local economy (Leung et al., 2002; Diagne et al., 2021). Invasive plants are extremely diverse and renewable; therefore, using these plants as raw materials for biochar will be more economical and easier to obtain than other biomass wastes (Wang et al., 2021). Some researchers have used invasive plants as raw materials to produce biochar which is mainly used in agricultural soil improvement and pollutant adsorption (Cheng et al., 2017; Beckinghausen et al., 2020). However, the advantages and disadvantages of invasive plant biochar (IPB) and their preparation methods need to be systematically analyzed. Most studies about IPB are focused on its application in environmental protection, while there is little information about its application in the ﬁeld of energy. There are still many challenges in the implementation of IPB that need further analysis and understanding. Deﬁnition Invasive plants are those that are introduced into a non-native or alien environment and are capable of adept multiplication and can cause damage to the environment, economy, or human health (Prabakaran et al., 2019). Invasive plants usually have the following characteristics: 1) Strong ecological adaptability and high genetic diversity; 2) Strong and rapid reproductive ability, i.e., they can produce offspring in adverse environments; 3) Strong transmission capacity, which is suitable for the transmission of seeds or propagators through media, and a high transmission rate (Wan et al., 2010; Meng et al., 2020). Currently, the invasive plants most commonly used as raw materials for BC are water hyacinth, Eupatorium Adenophorum, Spartina Alterniﬂora, Alternanthera Philoxeroides, Solidago Canadensis L, etc. (Nguyen D. T. C. et al., 2021; Cui et al., 2022). These invasive plants are mostly herbaceous, abundant and easily accessible. It is beneﬁcial to collect a large amount of raw materials to prepare BC. The stems of herbaceous plants are densely covered with relatively small vascular bundles, between which are a large number of thin-walled cells (Buranov and Mazza, 2008), which is more conducive to the preparation of BC with large speciﬁc surface area and well-developed porosity. g g Some of the most adaptable plants appear in non-native environments due to intentional, unintentional, or accidental human activity (Leprieur et al., 2008; Blackburn et al., 2011; Besnard and Cuneo, 2016). Generally, there are three main pathways of plant invasion: 1) natural invasion; 2) initiative taken to introduce; 3) passive spread (Rahel and Olden, 2008; Hulme, 2009). After successfully entering a new habitat, invasive plants often gain a great competitive advantage over native plants due to the lack of natural enemies and a suitable living environment. Simultaneously, invasive plants may produce various organic acids, allelopathic substances, and hormones, which can destroy the structure of rhizosphere soil microbial community and interrupt the interaction between the soil community and native plants. This results in successful invasion (Simberloff et al., 2013; Jabran et al., 2015; Zhang et al., 2019). Citation: Therefore, based on the characteristics of invasive plants and the preparation method of IPB, this paper focuses on the preparation and modiﬁcation methods of biochar using invasive plants as raw materials, as well as on the application and potential of IPB in the environmental and energy ﬁeld. Finally, this paper elucidates the current problems facing IPB and the possible future developmental direction of IPB research. Commonly used methods for the processing of invasive plants are mechanical control (removal by using artiﬁcial or mechanical cleaning, shading, and other ways to remove invasive plants), chemical control (through the use of chemicals to kill or control invasive plants), and biological control (introducing other biological agents to control the growth of invasive plants) (Hussner et al., 2017; Weihua Li et al., 2015). Using invasive plants in different ways is a desirable method to reduce the damage caused by them. As shown in Figure 1, common utilization methods of invasive plants include composting, feed, activated carbon, biochar, etc. (Reaser et al., 2007; Atyosi et al., 2019; Feng et al., 2021). Since traditional methods to deal with invasive plants consume signiﬁcant manpower and material resources, we urgently need more economical and efﬁcient ways to deal with invasive plants. PREPARATION AND MODIFICATION OF IPB The most common way to prepare biochar is by pyrolysis (Jahirul et al., 2012). Pyrolysis conditions (temperature, heating rate, etc.) have a signiﬁcant effect on the physicochemical properties of biochar (Mašek et al., 2013). During thermal decomposition, hemicellulose, cellulose, and lignin (components of biomass) are crosslinked, depolymerized, and cleaved at their respective temperatures to produce solid, liquid, and gaseous products (Cha et al., 2016). Biochar preparation by pyrolysis has the advantages of simplicity and low cost. Hazards and Handling Methods The inherent ability of plants to undergo rapid adaptive evolution on genotypes or exhibit phenotypic plasticity makes them formidable invaders (Prabakaran et al., 2019). Due to the characteristics of strong adaptability and rapid At present, there are numerous relevant studies on the preparation of biochar using invasive plants as raw materials May 2022 \| Volume 10 \| Article 902915 Frontiers in Environmental Science \| www.frontiersin.org 2 Yang et al. Application of Invasive Plants Biochar FIGURE 1 \| Characteristics of invasive plants, IPB preparation and utilization. FIGURE 1 \| Characteristics of invasive plants, IPB preparation and utilization. prepared at high temperature has good stability, and the removal effect of heavy metals is better (Fan et al., 2019). Pyrolysis temperature also affects the functional groups on biochar (Xiaoling Dong et al., 2013). At lower temperatures, the degree of carbonization of IPB is not high, so there are more oxygen-containing functional groups on the surface. The speciﬁc surface area and pore volume of IPB can be changed by acid-base modiﬁcation, and some surface functional groups can be introduced to improve the adsorption performance of biochar (Sizmur et al., 2017). The modiﬁcation of supported metal and its oxides mainly uses the binding force between supported metal elements and biochar to improve adsorption. Metal element oxides attached to the surface of biochar can provide more adsorption sites and can also endow magnetic characteristics to biochar, which is convenient for separation, recovery, and regeneration. Water hyacinth biochar was used as the carrier of Fe3O4 particles to achieve effective separation and recovery of materials (Zhuang et al., 2020). Citric acid was applied for biochar modiﬁcation (raw material: Anemonis crematis), which introduced extensive carboxyl groups on the surface of biochar and generated numerous active sites (Xu et al., 2016). (GuhaRay et al., 2019; Velez et al., 2018). In addition to the direct preparation of biochar by pyrolysis, modiﬁed biochar can also be prepared by pre-treatment of raw materials to improve the relevant properties of IPB (Saravanakumar et al., 2019; Zeng et al., 2019). This can aid in enhancing its applicability in various scenarios. Using invasive plants as the carrier, biochar was modiﬁed by acid and base, supported metal and its oxides, oxidizer, etc., and had better performance than the original biochar (Figure 1). Frontiers in Environmental Science \| www.frontiersin.org Hazards and Handling Methods (2021) Large pore structure, rich functional groups, graphene structure Adsorption of phthalate esters in water Hazards and Handling Methods Through acid modiﬁcation, the surface area of BC can be changed to a certain extent while also removing metal and other impurities on the surface of biochar and introducing oxygen-containing functional groups (Shen et al., 2010). Alkali modiﬁcation can increase the speciﬁc surface area (BET) and oxygen- containing functional groups of BC (Ahmed et al., 2016). The modiﬁcation of BC by metal or metal oxide can enhance the adsorption and catalytic performance and endow it with magnetic properties, which is conducive to recycling (Li et al., 2021). Oxidant modiﬁcation can increase oxygen-containing functional groups on BC, which helps improve its performance (Uchimiya et al., 2012) (Table 1). Invasive plants used to prepare biochar often have porous structures (such as water hyacinth). This enables the resulting biochar to have a larger surface area and provide more active sites, which greatly improves the ability of IPB to adsorb pollutants (Zhuang et al., 2020). Pyrolysis temperature also affects the properties of IPB. As high temperatures can remove some impurities in IPB, the porosity of IPB increases with increasing temperature. Simultaneously, high temperatures can increase the carbonization degree of biochar and carbon content of biochar. Eupatorium adenophorum biochar The preparation of BC from invasive plants has obvious advantages. Invasive plants are easy to obtain and provide a large number of high quality and cheap raw materials for BC preparation. Consumption of invasive plants for BC preparation can achieve a win-win situation in ecology and resources. Invasive plants have inherent structural advantages, such as the hollow structure of water hyacinth, which can achieve excellent structural characteristics in the initial BC. Most invasive plants May 2022 \| Volume 10 \| Article 902915 Frontiers in Environmental Science \| www.frontiersin.org 3 Application of Invasive Plants Biochar Yang et al. Yang et al. TABLE 1 \| Preparation, modiﬁcation, characteristic, and use of biochar from invasive plants. Invasive plants Production method Modiﬁcation Feature Application References Water hyacinth Pyrolysis/550°C/ 60 min With PCM impregnated High carbon content, high porosity, good thermal conductivity Energy storage Das et al. (2020) Water hyacinth Pyrolysis/500°C/ 180 min Modiﬁcation with alkali (KOH) and hyperthermy Developed carbon nanonetwork and macropore structure Energy storage Mo et al. (2020) Prosopis juliﬂora Pyrolysis/400°C/ 60 min Modiﬁcation with alkali (KOH) Large BET, large number of heteroatoms Energy storage Raj et al. Hazards and Handling Methods (2022) Water hyacinth Pyrolysis/250°C/ 60 min Modiﬁcation with metal oxides (Fe) Large BET, more surface active sites, magnetic Adsorption of As (Ⅴ) in water Zhang et al. (2016) Water hyacinth Pyrolysis/400°C/ 180 min Modiﬁcation with metal oxides (Fe) It has more -OH groups, magnetic Adsorption of Cr (Ⅵ) in water Chen et al. (2019) Water hyacinth Pyrolysis/<700°C Modiﬁcation with metal oxides (Mn) BET and pore volume increased, rich in Mn-OH groups Adsorption of heavy metals in water Zhang et al. (2020) Water hyacinth Pyrolysis/700°C/ 120 min Modiﬁcation with metal oxides (Fe) Large aperture, Fe3O4 nanoparticles aggregate to form larger clusters Degradation of 2,4,6-trichlorophenol and coal gasiﬁcation wastewater Zhuang et al. (2020) Eupatorium adenophorum HTC/220°C/60 min Modiﬁcation with acid (HNO3) Large BET, more pores, more functional groups Adsorption of Pb(Ⅱ) in water Liu et al. (2021) Eupatorium adenophorum Pyrolysis/600°C/ 120 min Modiﬁcation with metal (Fe/Ni) Surface functional groups increased Removal of 2,4, 6-trichlorophenol from water Guo Liu et al. (2019) Spartina alterniﬂora Pyrolysis/350°C/ 120 min — Many oxygen functional groups Adsorption of Cd in soil Cai et al. (2020) Sicyos angulatus Linn Pyrolysis/700°C/ 120 min Steam-activated (45 min) BET and pore volume increased Adsorption of sulfamethoazine from water (SMT) Rajapaksha et al. (2015) Alternanthera philoxeroides Pyrolysis/450°C/ 120 min Modiﬁcation with oxidant (H2O2) Oxygen-containing functional groups and BET increased Adsorption of metformin hydrochloride in water Huang et al. (2016) Alternanthera philoxeroides Pyrolysis — Rich microporous structure Adsorption of rhodamine B in water Du et al. (2018) Solidago canadensis L Pyrolysis/ 400–600°C/ 120 min — The contents of carboxylic acid, phenol and amine are higher Improvement of soil quality in saline- alkali land Tang et al. (2020) Solidago canadensis L Pyrolysis/700°C/ 240 min Ca/Al hydrotalcite or hydroxyapatite modiﬁcation Rich in P-, C-, O- functional groups Adsorption of Eu (Ⅲ) in water Dong et al. (2021) Mesquite Pyrolysis — Large pore Improvement of soil quality Hussain et al. (2021) Ambrosia triﬁda L Pyrolysis/700°C/ 180 min — High aromaticity, low polarity Adsorption of trichloroethylene in water Ahmad et al. (2014a) Lantana camara Pyrolysis/500°C/ 240 min — High ash content Reduction of soil acidity Berihun et al. (2017) Acacia auriculiformis Pyrolysis/500°C/ 120 min — Large BET Removal of dyes in water Nguyen et al. (2021b) Pistia stratiotes Pyrolysis/ 400–700°C/ 180 min Nitrogen doped Large pore structure, rich functional groups, graphene structure Adsorption of phthalate esters in water Zhang et al. ENVIRONMENTAL APPLICATION OF IPB are herbaceous and have high cellulose content. In the pyrolysis process, cellulose is easily volatilized by combustion, so BC produced by invasive plants is characterized by abundant pores. The use of invasive plants to prepare BC is still limited. Invasive plant species are abundant, but only a few of them are used to prepare BC. Meanwhile, due to the high content of cellulose and hemicellulose, the yield of BC produced from invasive plants was relatively low. In the actual production process, invasive plants can be selected according to needs. To generate BC with large speciﬁc surface area and abundant pores, invasive herbaceous plants with high cellulose content can be used. For those with high yield requirements, the selection range can be expanded to adopt invasive plants with high lignin content. are herbaceous and have high cellulose content. In the pyrolysis process, cellulose is easily volatilized by combustion, so BC produced by invasive plants is characterized by abundant pores. The use of invasive plants to prepare BC is still limited. Invasive plant species are abundant, but only a few of them are used to prepare BC. Meanwhile, due to the high content of cellulose and hemicellulose, the yield of BC produced from invasive plants was relatively low. In the actual production process, invasive plants can be selected according to needs. To generate BC with large speciﬁc surface area and abundant pores, invasive herbaceous plants with high cellulose content can be used. For those with high yield requirements, the selection range can be expanded to adopt invasive plants with high lignin content. Soil Quality Improvement (Soil Amendment) Biochar is an excellent soil conditioner and can be used for improving soil quality. It ameliorates the soil environment by 1) affecting soil physical and chemical properties and 2) affecting microbial activity. It can increase soil moisture and nutrient retention capacity, promote growth and biological activity of soil microﬂora, reduce nutrient leaching, increase circulation of soil nutrients, and increase soil organic carbon, which promotes the growth of plants. Biochar is generally alkaline and can be used as a soil modiﬁer to neutralize soil acidity and raise soil pH (Yuan et al., 2011). Similar to other kinds of biochars, when IPB is used to improve soil quality, it can also May 2022 \| Volume 10 \| Article 902915 Frontiers in Environmental Science \| www.frontiersin.org 4 Yang et al. ENVIRONMENTAL APPLICATION OF IPB Application of Invasive Plants Biochar The maximum adsorption capacity for Pb(II) of the Alternanthera philoxeroides biochar was 257.12 mg g−1, which was 5.3 times that of common activated carbon (Yang et al., 2014). The adsorption capacity (Kd value) of water hyacinth biochar for heavy metals (Cd, Cu, Pb, and Zn) is greater than 104 L kg−1, and the removal rate is up to 99.9% in the range of low metal concentration. Thus, it shows high adsorption performance and low adsorption reversibility (Doumer et al., 2016). Biochar prepared with Prosopis juliﬂora as raw material can be effectively separated by a magnetic ﬁeld from the treated water after modiﬁcation with doped magnetic NiO, and the adsorption rate of Pb(II) increased (Saravanakumar et al., 2019). Nitrogen-doped biochar prepared from Pistia stratiotes has a large pore structure and abundant surface functional groups. Through hydrogen bonding, Lewis acid-base interaction, and functional group interaction, the adsorption capacity of diethyl phthalate can reach 161.7 mg g−1 (Zhang et al., 2021). Water hyacinth biochar was modiﬁed with citric acid, and carboxyl group was added to the surface of the biochar under esteriﬁcation. The modiﬁed water hyacinth biochar had good regeneration adsorption performance and the maximum adsorption capacity for methylene blue was improved (up to 395 mg g−1) (Xu et al., 2016). The removal effect of 2,4,6-trichlorophenol (2,4,6-TCP) in water by nano-iron/nickel bimetal loaded with Eupatorium adenophorum biochar is signiﬁcant and the degradation rate of 2,4,6-TCP can be increased by 39.7–71.6% under different conditions (Guo Liu et al., 2019). improve soil fertility, reduce the damage of pollutants on soil quality, and improve the quality and yield of agricultural products, as well as it can reduce the persecution of local species by invasive plants. This IPB application method has good developmental prospects and can be widely used. It was reported that ﬁne-grained water hyacinth biochar reduced soil macropores and contributed to water retention by capillary action, and the hydrophilic surface bond (OH) of biochar further improved water retention capacity (Bordoloi et al., 2019). The pores of IPB can also provide a certain water retention capacity, which can offer resistance to the development of tensile force of soil cracks (Li et al., 2009). Water hyacinth biochar has higher aromaticity and carbon stability than the initial biomass at 300–350°C. ENVIRONMENTAL APPLICATION OF IPB After experimental analysis, it was found that water hyacinth biochar signiﬁcantly improved soil biological activity, with acid phosphatase and alkaline phosphatase activity increasing by 32 and 22.8%, respectively, thereby increasing soil activity by 3 times (Masto et al., 2013). The abundance of pores in IPB can also provide important habitats for microbes. Through ﬁeld trials, Lantana camara biochar signiﬁcantly reduced soil bulk density and exchangeable acidity, while signiﬁcantly increasing soil total porosity, pH, total nitrogen, organic carbon, and available phosphorus and potassium (Berihun et al., 2017). Organic anions can quickly neutralize soil acidity in acidic soil, so the improvement effect of carbonates in biochar on acidic soil can be long-lasting. The effects of Spartina alterniﬂora biochar application on the germination and growth of Salinaria salinosa were investigated under three conditions: non-ﬂooding, intermittent ﬂooding, and continuous ﬂooding. IPB had positive effects on the growth of Salinaria and improved the rhizosphere soil quality, which indicated that biochar is feasible for soil remediation in coastal wetlands (Cai et al., 2021). Additionally, biomass and grain yield of maize treated by the Eupatorium adenophorum biochar were signiﬁcantly improved. This was partly due to changes in soil physical properties, which included improved drainage and air permeability of heavy clay upon the addition of biochar (Obia et al., 2018). IPB can alleviate water, nutrient, and acid stress in soil. Further, the addition of Eupatorium adenophorum biochar improved soil nutrient availability and promoted plant growth by increasing soil pH, Ca/Al ratio, and available phosphorus to keep soil pH stable (Pandit et al., 2018). Contaminants in soil tend to exist in more complex forms than in water and are harder to remove. IPB can complete the adsorption of soil pollutants to some extent. At 600–700°C and pH 6, the Cu(II) removal rates of Spartina alterniﬂora biochar and water hyacinth biochar were 29.4 and 28.2 mg g−1, respectively. The process of Cu(II) ﬁxation had no signiﬁcant effect on Na/K/Mg leaching, nutrient cycling (especially K), and heavy metal retention could be achieved simultaneously (Mi Li et al., 2015). Spartina alterniﬂora biochar prepared under low temperature pyrolysis conditions (350 and 450°C) is beneﬁcial to the passivation of Cd in soil, and the effective Cd content decreases by up to 26.9%, while the increase of soil salinity is not signiﬁcant. Therefore, Spartina alterniﬂora biochar can be used to treat Cd pollution in coastal saline-alkali soil (Cai et al., 2020). Energy Storage and Climate Impact Energy Storage and Climate Impact Biochar is a promising energy storage material with easily regulated surface chemical properties, multi-purpose porous structure, and abundant surface functional groups. Biochar can play an important role in various energy storage conversion reactions and processes (Wu-Jun Liu et al., 2019). It can be used to produce supercapacitors and batteries (Li-ion, Na-ion, Li- S, and metal-Air) (Saning et al., 2019; Senthil and Lee, 2021). In the recent study, a novel and low cost biochar-phase change materials (PCM) hybrid latent heat energy storage material was developed by the addition of water hyacinth biochar as a supporting matrix. It was found that water hyacinth biochar had a 13.82-fold increased coefﬁcient of thermal conductivity of PCM, and possessed enhanced stability due to the high carbon Removal of Pollutants in Water/Soil Removal of Pollutants in Water/Soil With the rapid development of various industries, heavy metals and organic pollutants have increasing inﬂuence on the environment and are one of the main challenges to be overcome in the ﬁeld of environmental sciences and conservation/sustainability (Yang et al., 2018; Hu et al., 2020). At present, IPB is mainly used in environmental remediation to adsorb pollutants in water or soil and reduce the persistence of pollutants in the ecological environment. The IPB has good adsorption and removal effect for some pollutants. Simultaneously, the use of IPB can reduce the ecological harm caused by invasive plants, realize the resource utilization of waste, and reduce the production cost of adsorbents. May 2022 \| Volume 10 \| Article 902915 Frontiers in Environmental Science \| www.frontiersin.org 5 Application of Invasive Plants Biochar Yang et al. content and porosity of biochar (Das et al., 2020). Water hyacinth biochar could absorb metal ions, which helped to improve thermal conductivity in the production of PCM and solved the problem of low thermal conductivity in traditional PCM (Muigai et al., 2020). Hierarchical porous carbon based on water hyacinth biochar could realize rapid ion transfer simultaneously by coupling its layered porous structure with high speciﬁc surface area, and provide rich active sites for energy storage (Mo et al., 2020). The biochar produced by water hyacinth under high temperature conditions had the characteristics of porous and high graphitization, which contributed to overcome the water degradation of ygroscopic perovskite layer and had better air stability. It could be used to realize the continuous manufacture of perovskite solar cells (Pitchaiya et al., 2020). Water hyacinth absorbed Ni2+ in water through phytoremediation technology and could be used as feedstock to prepare biochar. Metal ions could be introduced naturally, and the electrochemical performance of water hyacinth biochar could be signiﬁcantly improved (Shell et al., 2021). IPB has great development potential in energy storage, more researches about the application of IPB in energy storage are needed in the future. raw materials for biochar preparation. Furthermore, the current usage of IPB and modiﬁed biochar elucidates that its performance can be enhanced and meet more environmental protection requirements. In this paper, the common methods of biochar preparation by invasive plants and their application in the environmental and energy storage ﬁeld were discussed based on previous studies. This can provide a basis for the future management of invasive plants. Removal of Pollutants in Water/Soil Biochar preparation from invasive plants also has great potential in energy storage, but research on this aspect is rare. 5) Fully utilizing invasive plants to prepare biochar can further achieve carbon sequestration. However, there is still some controversy over whether biochar can effectively mitigate climate change, and further research should be carried out in this regard in the future. AUTHOR CONTRIBUTIONS LY and XT participate in the outline planning, the data collection, ﬁgures design, writing and editing. YD and ZS participated in the writing and editing. QC, HY, and XT participated in the editing and review. XT participated in project administration and funding acquisition. All authors contributed to the article and approved the submitted version. CONCLUSION The ecological damage caused by invasive plants worldwide cannot be ignored, and efﬁcient mitigation policies are necessary to tackle the problems caused by invasive plants. Using invasive plants to prepare biochar can turn problematic weeds into valuable products, thereby taking advantage of them. This method has become an effective way to control invasive plants by reducing the control cost and realizing the sustainable utilization of resources. Invasive plants, especially water hyacinth, Solidago canadas and Alternanthera philoxeroides are excellent Removal of Pollutants in Water/Soil Biochar preparation from invasive plants can solve ecological problems caused by invasive plants to a certain extent while also providing economic value, realizing resource utilization of waste, and meeting the requirements of sustainable utilization. However, there are still some challenges and obstacles in the application of IPB: 1) There are many kinds of invasive plants, but the existed invasive plants are not fully utilized. Therefore, various invasive plants should be considered while selecting raw materials of biochar in the future. 2) The ability of the original IPB to mitigate environmental problems may be insufﬁcient to meet future environmental conservation requirements. Therefore, various modiﬁcation methods must be considered to signiﬁcantly improve the performance of IPB. gy g Biochar has received increasing attention as a method of almost permanently locking atmospheric carbon in the soil through carbon-negative processes. The pyrolysis of biochar from invasive plants can improve carbon stability and form persistent carbon storage in soil (Gaurav et al., 2020). Applying biochar to agricultural soils can increase soil carbon sequestration, stabilize soil organic carbon pool, inhibit soil CO2 ﬂux, and reduce greenhouse gas emissions to mitigate climate change (Da Dong et al., 2013; Wang et al., 2014). Studies have shown that IBP has a variable impact on greenhouse gas emissions, which is reﬂected by the signiﬁcant decrease in soil N2O emissions, increase in soil CH4 uptake, and complex changes (negative, positive, or negligible) in soil CO2 emissions (Li et al., 2017). Meta-analyses from laboratory and potted and ﬁeld studies found that biochar application signiﬁcantly affected soil greenhouse gas ﬂuxes and their global warming undercurrent values and reduced soil N2O ﬂuxes by 30.92% (He et al., 2017). IPB could be applied to the soil to reduce nitrous oxide emissions by reducing the use of fertilizers and lime (Simmons et al., 2021). The extent and process of climate change mitigation through IPB application requires further in-depth research. 3) Some biochars produced from invasive plants (such as water hyacinth) showed superior properties and performance in the speciﬁc applications. The advantages of invasive plants compared to other raw materials of biochar need further veriﬁcation. 4) Many invasive plants have desirable structures that can meet the requirements of energy storage materials. Biochar preparation from invasive plants also has great potential in energy storage, but research on this aspect is rare. 4) Many invasive plants have desirable structures that can meet the requirements of energy storage materials. Frontiers in Environmental Science \| www.frontiersin.org REFERENCES A Review of Biochars’ Potential Role in the Remediation, Revegetation and Restoration of Contaminated Soils. Environ. Pollut. 159 (12), 3269–3282. doi:10.1016/j.envpol.2011.07.023 Fan, L., Zhou, X., Liu, Q., Wan, Y., Cai, J., Chen, W., et al. (2019). Properties of Eupatorium Adenophora Spreng (Crofton Weed) Biochar Produced at Different Pyrolysis Temperatures. Environ. Eng. Sci. 36 (8), 937–946. doi:10. 1089/ees.2019.0028 Feng, Q., Wang, B., Chen, M., Wu, P., Lee, X., and Xing, Y. 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Xu, Y., Liu, Y., Liu, S., Tan, X., Zeng, G., Zeng, W., et al. (2016). Enhanced Adsorption of Methylene Blue by Citric Acid Modiﬁcation of Biochar Derived from Water Hyacinth (Eichornia Crassipes). Environ. Sci. Pollut. Res. 23 (23), 23606–23618. doi:10.1007/s11356-016-7572-6 Copyright © 2022 Yang, Deng, Shu, Chen, Yang and Tan. 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. Yang, Y., Wei, Z., Zhang, X., Chen, X., Yue, D., Yin, Q., et al. (2014). Biochar from Alternanthera Philoxeroides Could Remove Pb(II) Efﬁciently. Bioresour. 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https://openalex.org/W4247469845	http://jme.bmj.com/content/43/4/226.full.pdf	English	null	Rationing conscience	Journal of medical ethics	2,016	cc-by	5,082	Received 7 July 2016 Revised 1 September 2016 Accepted 8 September 2016 Published Online First 12 October 2016 iiThis is an abbreviated version of a real published case.9 iiiThis case is based on a real one.10 Note that in the real case treatment was provided by a private clinic rather than within the public health system. ivIn this case, the fertility specialist might object to non-provision of treatment either because it is discriminatory (against obese patients) or because it is based on mistaken assessment of the cost/beneﬁt, or both. For the purposes of this paper, either might form the basis of a conscientious objection. vThirty-four per cent of US physicians were prepared to lie on an insurance claim form in a survey version of this case.11 viT li d H d f d ‘ i i ’ d i Paper Paper J Med Ethics: first published as 10.1136/medethics-2016-103795 on 12 Octo on October 23, 2024 by guest. Prote http://jme.bmj.com/ J Med Ethics: first published as 10.1136/medethics-2016-103795 on 12 October 2016. Downloaded from ABSTRACT 1Faculty of Philosophy, Oxford Uehiro Centre for Practical Ethics, University of Oxford, Oxford, UK 2Newborn Care Unit, John Radcliffe Hospital, Oxford, UK Decisions about allocation of limited healthcare resources are frequently controversial. These decisions are usually based on careful analysis of medical, scientiﬁc and health economic evidence. Yet, decisions are also necessarily based on value judgements. There may be differing views among health professionals about how to allocate resources or how to evaluate existing evidence. In speciﬁc cases, professionals may have strong personal views (contrary to professional or societal norms) that treatment should or should not be provided. Could these disagreements rise to the level of a conscientious objection? If so, should conscientious objections to existing allocation decisions be accommodated? In the ﬁrst part of this paper, I assess whether resource allocation could be a matter of conscience. I analyse conceptual and normative models of conscientious objection and argue that rationing could be a matter for conscience. I distinguish between negative and positive forms: conscientious non-treatment and conscientious treatment. In the second part of the paper, I identify distinctive challenges for conscientious objections to resource allocation. Such objections are almost always inappropriate. PAPER Dominic Wilkinson1,2 limited medical resources. Dr A does not discuss the option of cardiac surgery with the parents. 1Faculty of Philosophy, Oxford Uehiro Centre for Practical Ethics, University of Oxford, Oxford, UK Case 2ii 2Newborn Care Unit, John Radcliffe Hospital, Oxford, UK An elderly patient in a persistent vegetative state and multi-organ failure has been in intensive care for a prolonged period. Medical staff believe that continued intensive care would be futile, and that treatment should be withdrawn. However, his family wishes treatment to continue and obtains a court order for treatment to continue pending review. Several doctors in the intensive care unit refuse to be involved in treatment of the patient because they regarded it as unethical. Correspondence to Dr Dominic Wilkinson, Oxford Uehiro Centre for Practical Ethics, Suite 8, Littlegate House, St Ebbes St, Oxford OX1 1PT, UK; dominic.wilkinson@ philosophy.ox.ac.uk INTRODUCTION Whil ll d While controlled allocation of scarce resources (‘rationing’) appears to be inevitable in health systems,1 2, (pp 14–19) health professionals do not always agree about the right way to allocate those resources.3–5 Those disagreements can take differ- ent forms. Consider the following cases: Case 4v Mrs L presents seeking a mammogram. She is con- cerned about breast cancer as her younger sister was recently diagnosed with this condition. Physical examination is normal. From review of the medical literature, Mrs L’s doctor believes that an annual mammogram would be indicated; however, Mrs L’s insurer will not cover the test. Mrs L is of limited means, and will not be able to pay for the mammogram. Her doctor regards the insurance limitations as unfair. She documents ‘suspicious breast lump’ on Mrs L’s insurance claim form (though this is not accurate).vi on October 23, 2024 by guest. Protected by copyright. http://jme.bmj.com/ m Case 3iii A couple, Jane and Peter, is having trouble conceiv- ing. Regional guidelines specify that publicly funded fertility treatment will only be available to women with a body mass index (BMI) in the normal range. Jane is 36 and has a BMI of 39. She has tried to lose weight over a prolonged period without success. Other fertility treatment centres have declined to provide in vitro fertilisation (IVF); however, her new fertility specialist believes that the restrictions on fertility treatment for obese women are unjust.iv She provides fertility treatment to Jane and Peter. on October 23, 2024 by guest. Prote http://jme.bmj.com/ 12 October 2016. Downloaded from Case 1i Dr A is counselling parents following antenatal diagnosis of a major chromosomal disorder. The fetus also has evidence of congenital heart disease. Babies with this chromosomal disorder have a high chance of dying in the newborn period, and will be severely disabled if they survive. There is no ofﬁcial policy not to provide cardiac surgery for infants with this chromosomal disorder. Some infants with this disorder receive cardiac surgery and long-term survival following surgery has been described. However, in Dr A’s view, cardiac surgery would be unethical in this situation; even if it were poten- tially beneﬁcial, it would constitute an unjust use of r 23, 2024 by guest. Protected by copyright. guest. Protected by copyright. To cite: Wilkinson D. J Med Ethics 2017;43: 226–229. Wilkinson D. J Med Ethics 2017;43:226–229. doi:10.1136/medethics-2016-103795 iThis is a composite version of real cases encountered. For examples of the disagreement caused by cases like this, see Gali6 and Janvier.7 As another example of this type of objection, Savulescu imagines a doctor who is convinced by the ‘fair innings’ argument and decides not to admit patients over the age of 70 to the intensive care unit.8 viTavaglione and Hurst defend ‘conscientious’ deceit on behalf of the patient in non-ideal settings like this case.12 Note that individuals who are dishonest might not be 226 Wilkinson D. J Med Ethics 2017;43:226–229. doi:10.1136/medethics-2016-103795 Paper Paper on October 23, 202 http://jme.bmj.com/ J Med Ethics: first published as 10.1136/medethics-2016-103795 on 12 October 2016. Downloaded from In the cases above, doctors did not use the language of ‘con- scientious objection’ (CO). Nevertheless, the nature of the con- cerns and the action taken by the professionals might be thought to at least share a family resemblance with standard cases of COs in medicine.13, (p 8) If professional disagreements about rationing can count as CO, this would be signiﬁcant, since such objections are often granted special status and regarded as deserving of respect and accommodation by the law and profes- sional bodies.13 represent core beliefs. For example, in case 2, one professional’s resignation letter described continued treatment as ‘an abomin- ation’, and wrote ‘I can’t do it’, appearing to indicate the depth of feeling associated with objections to treatment.17 However, some have argued that judgements about the futility of medical treatment represent professional judgements, and should not count as CO.13, (p 6) A recent professional guideline on COs set aside the source or quality of beliefs to focus instead on actions that would be contrary to either personal or professional beliefs: For the sake of this paper, I will set aside substantive ques- tions about how resources should be allocated. I will assume that some form of rationing is permissible. I will also set aside the wider questions about COs, and assume that COs in health- care should, at least sometimes, be accommodated. I will focus particularly on resource allocation in public healthcare systems. CONSCIENTIOUS RATIONING Conceptual questions on October 23, 2024 by guest. Prot http://jme.bmj.com/ 12 October 2016. Downloaded from There are a series of questions that might be asked. First, are objections to resource allocation consistent with the concept of COs? There are different accounts of what it means to have a CO. Some are relatively restrictive. For example, Conscientious Resource-based Non-treatment (Conscientious Non-treatment): A considered decision not to provide a legally and professionally accepted medical treatment on the basis of a personal belief that this would be an unjust use of limited health- care resources CO(narrow deﬁnition): an objection to provide a good or service based on a sincerely held set of moral convictions arising from belief in and relation to a Supreme Being or arising from a belief that has a parallel place in the individual’s life to that ﬁlled by God amongst religious adherents.13, (p 3) Conscientious Resource-based Treatment (Conscientious Treatment): A considered decision to provide a legally available medical treatment on the basis of a personal belief that this would be justiﬁed, despite a professional norm that because of limited healthcare resources the treatment should not be provided This narrow deﬁnition of CO may exclude cases of objection to resource allocation since such objections would not usually relate to religious or quasi-religious beliefs.viii However, objec- tions to abortion or physician-assisted dying do not necessarily relate to beliefs of that kind either, so this deﬁnition may be too restrictive. An alternative account identiﬁes CO as refusals to provide a good or service on the basis that this would be incom- patible with the agent’s core moral beliefs.13, (p 4–5) It is plausi- ble that objections to particular allocation decisions could Conscientious non-treatment or treatment might take place in the absence of a clear guideline or policy about treatment (case 1); however, they are most likely to be identiﬁed where they are in contravention of an existing guideline or policy (eg, cases 3 and 4). regarded as conscientious objectors. For example, people who lie about their health to avoid being drafted for war are viewed differently from those who publicly refuse to serve based on core personal or religious values. However, it depends on whether it is possible to avoid military service by telling the truth. As case 4 highlights, however, similar issues may apply to pro- fessional objections to allocation decisions by insurers or managed care organisations.14 Finally, while acknowledging that in real cases clinicians might have several different reasons for objecting to a particular treatment option, I will concentrate on those concerns that relate to resource allocation per se.vii CO (broad deﬁnition): Objections to providing legal, profession- ally accepted, and otherwise available medical services based on a clinician’s judgment that to do what is requested would be morally wrong.18 All of the above cases involved physicians’ moral disagree- ment with allocation decisions. Some of them, though (cases 3 and 4), led to positive actions rather than to withholding treat- ment. Should these also count as forms of CO? It is beyond the scope of this paper to address differences between negative and positive duties. However, Mark Wicclair has argued persuasively that there are no good reasons to selectively protect negative claims of conscience.19 If positive claims of conscience are included too, it may allow CO to be claimed in cases 3 and 4. (Wicclair leaves open the question of whether positive and negative conscience claims are completely symmetric and should be treated equally. In the context of resource allocation, there are reasons to treat these differently (see below).) For clarity, here are two different forms of CO to resource allocation: CONSCIENTIOUS RATIONING Conceptual questions In countries where there is no recognised mechanism for registering a CO and opting out of military service, someone might dissemble because that is the only way of acting in accordance with their conscience. Analogously, Mrs L’s doctor knows that if he tells the truth on the claim form that she will not have the investigation. His decision to lie is motivated by his conscience-based objection to the insurer’s allocation decision. I am grateful to a reviewer for this point. ii regarded as conscientious objectors. For example, people who lie about their health to avoid being drafted for war are viewed differently from those who publicly refuse to serve based on core personal or religious values. However, it depends on whether it is possible to avoid military service by telling the truth. In countries where there is no recognised mechanism for registering a CO and opting out of military service, someone might dissemble because that is the only way of acting in accordance with their conscience. Analogously, Mrs L’s doctor knows that if he tells the truth on the claim form that she will not have the investigation. His decision to lie is motivated by his conscience-based objection to the insurer’s allocation decision. I am grateful to a reviewer for this point. ii Normative questions There are several reasons that are commonly given for accom- modating COs. Do these apply to cases of disagreement about rationing? 23, 2024 by guest. Protected by copyright. Four commonly cited reasons in favour of accommodating COs include protecting clinicians’ moral integrity, respecting their autonomy, improving the quality of medical care (particu- larly through allowing diversity) and identifying needed changes in professional norms.18 All of these could straightforwardly be applied to instances of professional disagreement about resource allocation. For example, the clinicians who resigned in case 2 clearly felt that their personal moral integrity was threatened by continuing to provide treatment. In cases 1 and 4, permitting the doctors to make a determination about admission to inten- sive care, or the appropriateness of screening mammography would respect their personal and professional freedom to make decisions about medical treatment. It might be argued that uest. Protected by copyright. viiIn cases of ostensibly futile treatment, such as case 1 or 2, clinicians might have concerns about harm to patients, or about limited resources.15 I will concentrate on the latter. We could imagine in the cases cited that the clinicians involved are not necessarily opposed to self-funded treatment, or treatment in a private intensive care unit, but have a particular objection to the use of limited public healthcare resources. iii viiiSome objections to resource-based treatment limitations are linked to speciﬁc religious beliefs (eg, some opposition to Obamacare).16 227 Wilkinson D. J Med Ethics 2017;43:226–229. doi:10.1136/medethics-2016-103795 Paper on October 23, 202 http://jme.bmj.com/ J Med Ethics: first published as 10.1136/medethics-2016-103795 on 12 October 2016. Downloaded from decisions about the appropriateness of different treatment options are a fundamental example of professional autonomy. which resource allocation is an accepted (if regretted) feature of medical practice.) Furthermore, allowing doctors to object to resource allocation decisions would potentially encourage them to take engage with resource allocation questions and determine how best to manage limited resources. It would arguably promote sensitivity among professionals to the claims of their patients, and avoid a sense that they are merely enforcing rules laid down by others. Finally, accommodating COs to resource allocation might help to identify where existing allocation schemes should be modi- ﬁed. For example, if a large number of professionals feel that screening mammography is appropriate and manipulate claim forms to ensure access, perhaps the insurer would be compelled to change its policy. Normative questions If doctors elect to provide fertility treat- ment for a group of patients previously excluded, it may gener- ate important data about the beneﬁt (or non-beneﬁt) of such treatment. What does seem concerning about the decisions in cases 1 and 2 is the apparent variation between clinicians in their responses to the case. It seems worryingly arbitrary that whether or not a patient is offered cardiac surgery, or intensive care is continued depends on which doctor happens to be on call. There appears to be a ‘roster lottery’ affecting the provision of potentially life-saving medical treatment.21 Concern about variation in decision making and in access to treatment might apply to other cases of CO. For example, it is likely to be of concern that some pharmacies will provide emer- gency contraception, while others will not. However, lack of consistency poses a particular problem for resource allocation22 since it seems to be contrary to all major theories of justly dis- tributing scarce resources. Whether resources are allotted on the basis of greatest beneﬁt, greatest clinical need or equal access, patients should only be treated differently if there are ethically relevant differences between them. The identity of the clinician does not appear to be ethically relevant. To sum up the arguments thus far, professional objections to extant resource allocation could be consistent with existing broad concepts of CO in healthcare—either in situations where professionals conscientiously ration treatment or provide it con- trary to rationing policies. The ethical arguments that support accommodation of COs also potentially apply to cases of objec- tion to allocation decisions. This suggests that doctors could conscientiously object to allocation decisions. Could a fair process of allocation yield variable decisions? Norm Daniels in ‘Just Health’ considers the hypothetical example of Jack and Jill, who have identical conditions and require an expensive cancer treatment.23, (pp 135–7) They apply to their respective health authority or health insurer for access to the treatment. One health authority (or health plan) approves the treatment for Jack, while the other declines Jill’s request. Daniels argues that it is not necessarily unjust for the outcome of allocation to be different, as long as the process is consistent; fair decision-making processes may reach different conclusions in the setting of moral uncertainty. Moreover, there may be reasons to allow regional authorities (or insurers) to weigh up their priorities and allocate to different treatments as they see ﬁt. Against conscientious treatment The positive form of CO to resource allocation might raise dif- ferent concerns. The patients of IVF doctors who provide treat- ment outside conventional limits are unlikely to complain of inconsistency or unfairness. However, other women (whose doctors adhere to the guidelines) could complain. The often cited ‘publicity’ condition of fair allocation24 may require those other women to be informed, for example, that some doctors will provide IVF above the standard BMI limit and may lead them to seek that treatment. That would potentially defeat the purpose of making community-level decisions about allocation and imposing limits on treatment.ix Since the ethical considerations are different, we need to con- sider the two forms separately. er 23, 2024 by guest. Protected by copyright. Normative questions However, such defences of regional variation in allocation decisions arguably do not apply to individual decision makers. Idiosyncratic determinations about available resources do not represent a fair decision-making process. Kristin Baeroe con- tends that some variation in microallocation decisions is inevit- able, but proposes that physicians should strive towards common ground and a common basis for distinguishing between cases.22, (p 98) This aim for consistent decision making appears to preclude conscientious non-treatment. I have not argued that resource-based COs are identical to more traditional forms of CO in medicine nor that they are necessarily equally weighty. For example, Christopher Cowley claims that resource-based COs differ from other forms of CO (eg, abortion) in the type and directness of the harm caused if objections are not accommodated.20 This raises the possibility that CO to resource allocation would be treated differently from other forms of CO. on October 23, 2024 by guest. Protected http://jme.bmj.com/ ober 2016. Downloaded from Are there particular features of resource allocation that would count against CO? RATIONING CONSCIENCE Standard instances of CO in medicine represent a conﬂict between the wishes of the professional and the wishes of the patient. Cases of conscientious non-treatment might also have this character. However, conscientious treatment gives rise to a different conﬂict. In cases like 3 and 4 above, the professional’s wishes coincide with those of the patient. Instead, such cases appear to represent a conﬂict between the wishes of the profes- sional and those of wider society (and potentially of other patients). ixWeinstock argues that COs that jeopardise the function of a healthcare system are unreasonable, and should not be accommodated.25 Wilkinson D. J Med Ethics 2017;43:226–229. doi:10.1136/medethics-2016-103795 ixWeinstock argues that COs that jeopardise the function of a healthcare system are unreasonable, and should not be accommodated.25 CONCLUSIONS 11 Freeman VG, Rathore SS, Weinfurt KP, et al. Lying for patients: physician deception of third-party payers. Arch Intern Med 1999;159:2263–70. 11 Freeman VG, Rathore SS, Weinfurt KP, et al. Lying for patients: physician deception of third-party payers. Arch Intern Med 1999;159:2263–70. COs manifest when individuals face a conﬂict between their own values and what they are being asked or required to do. In healthcare, it is often felt to be important to respect the differ- ent ethical viewpoints of professionals and therefore to accom- modate COs to morally controversial treatment options. Since resource allocation decisions are frequently contentious, and involve value judgements, it might be anticipated that these could give rise to COs. In this paper, I have argued that COs to allocation decisions are consistent with broad concepts of CO, and that the arguments in favour of accommodating CO would also apply to conscientious non-treatment or conscientious treat- ment. However, I have also outlined substantial arguments against accommodating CO to allocation; such accommodation would almost always be inappropriate. Conscientious non- treatment or treatment run counter to fundamental principles of allocation including consistency, and the need to impose limits on available treatment. 12 Tavaglione N, Hurst SA. Why physicians ought to lie for their patients. Am J Bioeth 2012;12:4–12. 12 Tavaglione N, Hurst SA. Why physicians ought to lie for their patients. Am J Bioeth 2012;12:4–12. 13 Wicclair MR. Conscientious objection in health care: an ethical analysis. Cambridge: Cambridge University Press, 2011. 14 Childress JF. Conscience and conscientious actions in the context of MCOs. Kennedy Inst Ethics J 1997;7:403–11. 15 Wilkinson DJC, Savulescu J. Knowing when to stop: futility in the ICU. Curr Opin Anaesthesiol 2011;24:160–5. 16 7 things the Bible says about Obamacare. http://www.thefamilylea 7-things-the-bible-says-about-obamacare/ (accessed 6/7/2016). 16 7 things the Bible says about Obamacare. http://www.thefamilyl 7-things-the-bible-says-about-obamacare/ (accessed 6/7/2016). 7-things-the-bible-says-about-obamacare/ (accessed 6/7/2016). 17 CBC News. Doctors offer to treat dying Winnipeg man after colleagues refuse. CBC News 2008 Jun 18 2008. 17 CBC News. Doctors offer to treat dying Winnipeg man after colleagues refuse. CBC News 2008 Jun 18 2008. 18 Lewis-Newby M, Wicclair M, Pope T, et al. An ofﬁcial American Thoracic Society policy statement: managing conscientious objections in intensive care medicine. Am J Respir Crit Care Med 2015;191:219–27. 19 Wicclair MR. Negative and positive claims of conscience. Camb Q Healthc Ethics 2009;18:14–22. 20 Cowley C. Provenance and peer review Not commissioned; externally peer reviewed. Provenance and peer review Not commissioned; externally peer reviewed. Open Access This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. See: http://creativecommons.org/licenses/ by/4.0/ Open Access This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited See: http://creativecommons org/licenses/ Open Access This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. See: http://creativecommons.org/licenses/ by/4.0/ Open Access This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. See: http://creativecommons.org/licenses/ by/4.0/ provided the original work is properly cited. See: http://creativecommons.org/licenses/ by/4.0/ Could conscientious treatment be accommodated? One option would be to allow individual clinicians to provide the rationed treatment as long as it imposed no greater cost on the public health system than currently funded alternatives. This may require patients to partly or completely fund their treat- ment. However, while the latter would accommodate objections, it would potentially undermine the point of the objection. (The objections in cases 3 and 4 are to the public health system or the insurer failing to provide treatment. A solution that means that the patient is paying for treatment herself does not seem to actually accommodate the objections at all.) It may also require physicians to provide their services pro bono. Alternatively, it may be possible to give clinicians discretion to provide non- standard treatment, as long as the incremental cost is within a reasonable limit. This may permit conscientious treatment only in a small subset of cases. on October 23, 2024 by guest. Protected by copyright. http://jme.bmj.com/ J Med Ethics: first published as 10.1136/medethics-2016-103795 on 12 October 2016. Downloaded from Paper Paper Contributors DW conceived and wrote this paper. trastuzumab emtansine (Kadcyla), which costs approximately £90 000 to extend life by 6 months.26 This is because the cost signiﬁcantly exceeds the usual cost-effectiveness threshold used in the UK to set a limit on affordable treatments (approximately £20–£30 000 per quality-adjusted life year saved). An individual doctor might disagree with the ofﬁcial assessment of Kadcyla, and choose to provide it for her patients with advanced breast cancer.x Yet, this would then potentially limit the ability of the health service to provide other less expensive (and potentially more effective) treatments. trastuzumab emtansine (Kadcyla), which costs approximately £90 000 to extend life by 6 months.26 This is because the cost signiﬁcantly exceeds the usual cost-effectiveness threshold used in the UK to set a limit on affordable treatments (approximately £20–£30 000 per quality-adjusted life year saved). An individual doctor might disagree with the ofﬁcial assessment of Kadcyla, and choose to provide it for her patients with advanced breast cancer.x Yet, this would then potentially limit the ability of the health service to provide other less expensive (and potentially more effective) treatments. Funding DW was supported for this work by a grant from the Wellcome trust WT106587/Z/14/Z. Funding DW was supported for this work by a grant from the Wellcome trust WT106587/Z/14/Z. Funding DW was supported for this work by a grant from the Wellcome trust WT106587/Z/14/Z. REFERENCES 1 Ubel PA, Arnold RM. The unbearable rightness of bedside rationing. Physician duties in a climate of cost containment. Arch Intern Med 1995;155:1837–42. 1 Ubel PA, Arnold RM. The unbearable rightness of bedside rationing. Physician duties in a climate of cost containment. Arch Intern Med 1995;155:1837–42. 2 Bognar G, Hirose I. The ethics of health care rationing: an introduction. Abingdon, Oxon: Routledge, 2014. 2 Bognar G, Hirose I. The ethics of health care rationing: an introduction. Abingdon, Oxon: Routledge, 2014. 3 Choudhry N, Slaughter P, Sykora K, et al. Distributional dilemmas in health policy: large beneﬁts for a few or smaller beneﬁts for many? J Health Serv Res Policy 1997;2:212–16. 3 Choudhry N, Slaughter P, Sykora K, et al. Distributional dilemmas in health policy: large beneﬁts for a few or smaller beneﬁts for many? J Health Serv Res Policy 1997;2:212–16. 4 Kapiriri L, Norheim OF. Criteria for priority-setting in health care in Uganda: exploration of stakeholders’ values. Bull World Health Organ 2004;82:172–9. 5 Bringedal B, Feiring E. On the relevance of personal responsibility in priority setting: a cross-sectional survey among Norwegian medical doctors. J Med Ethics 2011;37:357–61. 6 Gali V, Gupta N, Sivakumar S. Management of cardiac problems in trisomy 18—a major ethical dilemma; a case series review. Arch Dis Child 2011;96(Suppl 1):A72. 7 Janvier A, Okah F, Farlow B, et al. An infant with trisomy 18 and a ventricular septal defect. Pediatrics 2011;127:754–9. on October 23, 2024 by guest. Protec http://jme.bmj.com/ n 12 October 2016. Downloaded from p 8 Savulescu J. Conscientious objection in medicine. BMJ 2006;332:294–7. 8 Savulescu J. Conscientious objection in medic 9 Jotkowitz A, Glick S, Zivotofsky AZ. The case of Samuel Golubchuk and the right to live. Am J Bioeth 2010;10:50–3. 10 Morris N. Too fat to be a mother. The Guardian 2006 5/9/2006. 10 Morris N. Too fat to be a mother. The Guardian 2006 5/9/2006. Wilkinson D. J Med Ethics 2017;43:226–229. doi:10.1136/medethics-2016-103795 Against conscientious non-treatment Reasons that are provided against other forms of CO include that such objections violate core professional commitments, fail to protect vulnerable patients, create hardships for other clini- cians (where accommodated) or are discriminatory.18 Some of these might be cited against conscientious non-treatment. For example, we might believe that the doctor’s decision in case 1 conﬂicts with his duty to safeguard vulnerable patients, repre- sents a form of invidious discrimination, or violates professional commitments.18 Yet, it would be arguably acceptable (and com- patible with professional commitments) in case 1 or case 2 for the clinicians to withhold treatment if there were clear policies or guidelines supporting such an action. (Clearly, some who are opposed to rationing will dispute this. However, as noted earlier, I am focusing arguments in this paper on a setting in uest. Protected by copyright. Partly as a consequence of this, conscientious treatment appears to be unfair in a different way, since it imposes the costs of CO on the wider community and would have implications on access to treatment for others. For example, the UK National Health Service does not routinely fund the breast cancer drug 228 CONCLUSIONS A Defence of Conscientious Objection in Medicine: A Reply to Schuklenk and Savulescu. Bioethics 2016;30:358–64. 21 Wilkinson DJ, Truog RD. The luck of the draw: physician-related variability in end-of-life decision-making in intensive care. Intensive Care Med 2013;39:1128–32. Acknowledgements A draft of this paper was presented at a conference at the Brocher Foundation in June 2016 ‘The conscience of health professionals in the time of biotechnologies: present and future of conscientious objection in medicine’. I am grateful to Alberto Giubilini for organising the conference, and to participants at the meeting and two reviewers for very helpful suggestions. 22 Baerøe K. Priority setting in health care: on the relation between reasonable choices on the micro-level and the macro-level. Theor Med Bioeth 2008;29:87–102. 23 Daniels N. Just health: meeting health needs fairly. Cambridge: Cambridge University Press, 2008. 24 Daniels N, Sabin JE. Last chance therapies and managed care. Pluralism, fair procedures, and legitimacy. Hastings Cent Rep 1998;28:27–41. xIn the system as it stands, there would be no way for a physician to act on their conscientious belief and provide Kadcyla. If Conscientious Treatment were to be accepted, the idea is that individual physicians could override national or regional decisions not to fund a particular treatment by lodging a CO. p g y g p 25 Weinstock D. Conscientious refusal and health professionals: does religion make a difference? Bioethics 2014;28:8–15. 26 National Institute for Health and Care Excellence. Trastuzumab emtansine for treating HER2-positive, unresectable locally advanced or metastatic breast cancer after treatment with trastuzumab and a taxane. NICE, London, UK, 2015. 229 Wilkinson D. J Med Ethics 2017;43:226–229. doi:10.1136/medethics-2016-103795
https://openalex.org/W4361962745	https://figshare.com/articles/journal_contribution/Supplementary_Figure_from_First-in-Human_Dose-Escalation_Study_of_Cyclin-Dependent_Kinase_9_Inhibitor_VIP152_in_Patients_with_Advanced_Malignancies_Shows_Early_Signs_of_Clinical_Efficacy/22488158/1/files/39939764.pdf	English	null	Supplementary Figure from First-in-Human Dose-Escalation Study of Cyclin-Dependent Kinase 9 Inhibitor VIP152 in Patients with Advanced Malignancies Shows Early Signs of Clinical Efficacy	null	2,023	cc-by	359	Figure S1. Grade 3 and 4 adverse events occuring in cycles 0-2 versus cycles >2 Figure S1. Grade 3 and 4 adverse events occuring in cycles 0-2 versus cycles >2 Thirty-seven patients received 0 to 2 cycles of VIP152. The blue bars represent Grade 3 or 4 adverse events reported as starting any time from 0 to 2 cycles. Eighteen patients received >2 cycles (minimum 7 weeks, maximum 192 weeks) of VIP152. The red bars represent Grade 3 or 4 adverse events reported as starting any time after cycle 2. 0 10 20 30 40 50 60 70 80 90 100 Patients, % Acute kidney injury Blood bilirubin increased Cancer pain Cholangitis Decreased appetite Dehydration Device related infection Dyspnoea Fatigue Febrile neutropenia Hepatorenal syndrome Hyperglycaemia Hypertension Intestinal obstruction Mastitis Oesophageal cancer metastatic Orthostatic hypotension Patella fracture Portal vein thrombosis Pulmonary embolism Pyrexia Spinal operation Lymphocyte count decreased Neutrophil count decreased Non-cardiac chest pain Syncope Tumour pain Abdominal pain Blood alkaline phosphatase increased Hyponatraemia White blood cell count decreased Anaemia Neutropenia 0 10 20 30 40 50 60 70 80 90 100 Patients, % Acute kidney injury Blood bilirubin increased Cancer pain Cholangitis Decreased appetite Dehydration Device related infection Dyspnoea Fatigue Febrile neutropenia Hepatorenal syndrome Hyperglycaemia Hypertension Intestinal obstruction Mastitis Oesophageal cancer metastatic Orthostatic hypotension Patella fracture Portal vein thrombosis Pulmonary embolism Pyrexia Spinal operation Lymphocyte count decreased Neutrophil count decreased Non-cardiac chest pain Syncope Tumour pain Abdominal pain Blood alkaline phosphatase increased Hyponatraemia White blood cell count decreased Anaemia Neutropenia >2 (n=18) 0-2 (n=37) Cycles n=1 n=1 n=1 n=1 n=1 n=1 n=1 n=1 n=1 n=1 n=1 n=1 n=1 n=1 n=1 n=1 n=1 n=1 n=1 n=1 n=1 n=1 n=2 n=1 n=1 n=2 n=1 n=1 n=2 n=1 n=2 n=1 n=2 n=3 n=2 n=1 n=4 n=8 n=1 >2 (n=18) 0-2 (n=37) Cycles Thirty-seven patients received 0 to 2 cycles of VIP152. The blue bars represent Grade 3 or 4 adverse events reported as starting any time from 0 to 2 cycles. Eighteen patients received >2 cycles (minimum 7 weeks, maximum 192 weeks) of VIP152. The red bars represent Grade 3 or 4 adverse events reported as starting any time after cycle 2.
https://openalex.org/W2884048253	https://www.scielo.br/j/gmb/a/9mHkjyJTjB3KYWCQR5kMDZy/?lang=en&format=pdf	English	null	Decreased serum PON1 arylesterase activity in familial hypercholesterolemia patients with a mutated LDLR gene	Genetics and Molecular Biology	2,018	cc-by	6,250	Research Article Research Article Abstract Paraoxonase 1 (PON1) is a serum enzyme associated with high density lipoprotein (HDL) regulation through its paraoxonase and arylesterase activity. PON1 inhibits the oxidation of HDL and low density lipoprotein (LDL), and is involved in the pathogenesis of a variety of diseases including atherosclerosis. Conversely, mutations in the low den- sity lipoprotein receptor (LDLR) result in failure of receptor mediated endocytosis of LDL leading to its elevated plasma levels and onset of familial hypercholesterolemia (FH). In the current study we investigated the role of PON1 polymorphisms rs662; c.575A > G (p.Gln192Arg) and rs854560; c.163T > A (p.Leu55Met) in a large family having FH patients harboring a functional mutation in LDLR. Genotypes were revealed by RFLP, followed by confirmation through Sanger sequencing. PON1 activity was measure by spectrophotometry. Our results show significantly re- duced serum paraoxonase and arylesterase activities in FH patients compared with the healthy individuals of the family (p < 0.05). PON1 QQ192 genotype showed a significantly higher association with FH (p=0.0002). PON1 Q192 isoform was associated with reduced serum paraoxonase activity by in silico analysis and PON1 R192 exhibited higher serum paraoxonase and arylesterase activity than the other polymorphs. Our results highlight that the combi- nation of LDLR mutations and PON1 MMQQ genotypes may lead to severe cardiac events. Keywords: Paraoxonase-1, hypercholesteremia, arylesterase, LDLR mutation. Received: October 25, 2016; Accepted: January 16, 2018 Decreased serum PON1 arylesterase activity in familial hypercholesterolemia patients with a mutated LDLR gene Muhammad Idrees1, Abdul Rauf Siddiqi1, Muhammad Ajmal1, Muhammad Akram1, Rana Rehan Khalid1, Alamdar Hussain1, Raheel Qamar1 and Habib Bokhari1 Muhammad Idrees1, Abdul Rauf Siddiqi1, Muhammad Ajmal1, Muhammad Akram1, Rana Rehan Khalid1, Alamdar Hussain1, Raheel Qamar1 and Habib Bokhari1 1COMSAT Institute of Information Tecnology, Islamabad, Pakistan. 1COMSAT Institute of Information Tecnology, Islamabad, Pakistan. Send correspondence to Habib Bokhari. COMSAT Institute of Infor- mation Technology, Park Road, Chak Shadzad, Islamabad, Paki- stan. E-mail: habib@comsats.edu.pk. Introduction (HDL) in human serum and prevents oxidation of both low density lipoprotein (LDL) and HDL (Aviram et al., 1998; Durrington et al., 2001; Mackness et al., 1996). The inhibi- tion of LDL and HDL oxidation may protect against vari- ous pathologies including cardiovascular diseases (CVD); PON1, therefore, is also considered the gene of longevity (Lescai et al., 2009, Martinelli et al., 2013). A relationship between paraoxonase 1 (PON1) genotype status, anti- oxidant, and anti-atherogenic capacity of the enzyme has been suggested previously (Mackness et al., 1993, 2002; Rosenblat et al., 2006). In addition, PON1 arylesterase/pa- raoxonase activities have been shown to be inversely corre- lated to the risk of coronary heart diseases and hypercholes- terolemia (Humbert et al., 1993; Garin et al., 1997; Bryk et al., 2005). The human paraoxonase (PON) gene family located on the long arm of chromosome 17 consists of three mem- bers, each of which coding for three different calcium de- pendent esterases: PON1, PON2, and PON3 (La Du et al., 1993; Li et al., 2003; Mackness and Mackness, 2015). PON1 and PON3 are plasma HDL-associated enzymes with antioxidant activities, albeit with differences (Mack- ness et al., 1993). PON1 serum concentration is affected by inflammation and serum levels of oxidized-LDL (Ceron et al., 2014;Vakili et al., 2014; Mackness and Mackness, 2015). PON3 on the other hand is far less expressed and is not influenced by inflammation or oxidized lipids (Reddy et al., 2001). PON2 is an intracellular enzyme with ubiqui- tous expression and is thought to protect against oxidative stress (Ng et al., 2001). Functional mutations in LDLR gene cause the mono- genic form of familial hypercholesterolemia (FH) (Diakou et al., 2011; Ahmed et al., 2012). Recently, it was also shown that increased serum paraoxonase activity in LDLR (-/-) mice significantly inhibits progression of atherosclero- sis (Leckey et al., 2010). However, the role of PON1 activ- ity has not been studied previously in individuals with mutated LDLR. A large Pakistani family with LDLR associ- ated FH was investigated in this study to understand the role of PON1 in the protection against atherosclerosis In the current study we investigated PON1 in familial hypercholesteremia due to its role in diverse physiological and pathophysiological functions, including atherosclero- sis and inflammatory diseases (La Du, 1996; Li et al., 2003). PON1 is associated with high density lipoprotein Send correspondence to Habib Bokhari. Genetics and Molecular Biology, 41, 3, 570-577 (2018) Copyright © 2018, Sociedade Brasileira de Genética. Printed in Brazil DOI: http://dx.doi.org/10.1590/1678-4685-GMB-2016-0287 Introduction COMSAT Institute of Infor- mation Technology, Park Road, Chak Shadzad, Islamabad, Paki- stan. E-mail: habib@comsats.edu.pk. 571 Idrees et al. lowed a restriction site for HinfI (G/ANTC) to be intro- duced into the DNA amplification products in the presence of the polymorphisms arginine-PON1-192 or leucine- PON1-55. PON1 gene segments were amplified by PCR using 0.3 mM deoxyribonucleotide triphosphates (dNTPs), 1x PCR buffer (10 mM Tris–HCl pH 9.0, 50 mM KCl), 2.0 mM MgCl2, 0.5 mM of each primer (forward and reverse), 1.5 U of Taq Polymerase, and 50 ng of genomic DNA. The thermal cycling consisted of an initial denaturation at 95 °C for 4 min, followed by 35 cycles of amplification consisting of denaturing at 95 °C for 45 s, primer annealing at 55 °C for 1 min and chain extension at 72 °C for 1 min. A final ex- tension step was performed at 72 °C for 10 min. (Ajmal et al., 2010). In this study, we report on the role of PON1 coding sequences of single nucleotide poly- morphisms (SNPs) rs662 (c.575A > G; p.Gln192Arg) and rs854560 (c.163T > A (p.Leu55Met) in relation to resultant paraoxonase and arylesterase activity in hypercholesterol- emia patients with mutated LDLR. The structural and func- tional aspects of these SNPs have also been studied to explore how different allozymes affect and mediate the paraoxonase and arylesterase activities of the enzyme. (Ajmal et al., 2010). In this study, we report on the role of PON1 coding sequences of single nucleotide poly- morphisms (SNPs) rs662 (c.575A > G; p.Gln192Arg) and rs854560 (c.163T > A (p.Leu55Met) in relation to resultant paraoxonase and arylesterase activity in hypercholesterol- emia patients with mutated LDLR. The structural and func- tional aspects of these SNPs have also been studied to explore how different allozymes affect and mediate the paraoxonase and arylesterase activities of the enzyme. Subjects A large consanguineous Pakistani family (n=34) with LDLR mutation (c. 2416_2417InsG) presenting clinical FH was identified, including 10 patients suffering from FH and 24 healthy individuals (Ajmal et al., 2010). All subjects were screened for the presence/absence of CHD, diabetes, hypertension, malignant tumors, and acute or chronic in- fectious diseases. The study was approved by the Ethics Committee and Institutional Review Board of the Depart- ment of Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan. All patients and partici- pating healthy members of the family were informed about the study in their local language and written consent was obtained from them prior to inclusion in the study. Screening of the amplified products PCR products were purified using a DNA extraction kit (Fermentas Life Sciences, Burlington, Ontario, Canada) and subjected to restriction with Hinf1 enzyme to screen for the type of SNP in the target sequence. Digested fragments were resolved on 8% polyacrylamide gel to reveal the ge- notypes (Figure S1). The results were analyzed to compare the prevalence of PON1 L55M and PON1 Q192R SNPs. To confirm the DNA sequence, amplified products were also subjected to bidirectional sequencing by the Sanger se- quencing method to reveal the genotypes of all the individ- uals. Determination of PON1 SNPs Arylesterase activity was determined using an assay mixture containing 1.0 mM phenylacetate and 0.9 mM CaCl2 in 20 mM Tris-HCl buffer (pH 8.0). The rate of hy- drolysis was monitored at 270 nm. The results were calcu- lated using extinction coefficient 1310/Mcm. Genomic DNA was extracted from peripheral blood leukocytes using standard procedures (Helms et al., 1985). Two sets of primers were used for genotyping the poly- morphisms of codon 192 and codon 55 in the PON1 gene as described by Motti et al. (2001). The primer sequences for PON1 Q192R (rs662) and PON1 L55M (rs854560) were as follows: PON1 Q192R-forward primer TTG AAT GAT ATT GTT GCT GTG GGA CCT GAG and PON1 Q192R-reverse primer CGA CCA CGC TAA ACC CAA ATA CAT CTC CCA GaA; PON1 L55M -forward primer GAG TGATGT ATA GCC CCA GTT TC and PON1 L55M-reverse primer AGT CCATTA GGC AGTATC TCC g. The reverse primers contained mismatched nucleo- tides as indicated by lower case letters in italics. This al- Measurement of PON1 paraoxanase activity Blood (5 mL) was drawn after 12–14 h fasting and collected in separate tubes for DNA extraction by organic method (Helms et al., 1985) and serum separation for the determination of lipid profile and enzyme activities. For DNA extraction, blood was collected in acid citrate dex- trose (ACD) vacutainer (Becton–Dickinson, Franklin Lakes, NJ) and for serum separation, blood was collected in Z Serum Sep Clot Activator vacutainer tubes (Greiner bio- one, Munich, Germany). Serum was separated from clotted blood by centrifuging the vacutainers at 3000 rpm for 10 min, at 4 °C. Paraoxonase activity was determined by measuring the increase in absorbance at 412 nm (Thermo Scientific GENESYS 10 UV Scanning UV/Visible Spectropho- tometer) due to the formation of 4-nitrophenol (using paraoxon as substrate) (Zehra et al., 2009). The assay mix- ture contained 1.0 mM paraoxon and 1.0 mM CaCl2 in 50 mM glycine/NaOH buffer (pH 10.5). The amount of 4-nitrophenol liberated was calculated from the molar coef- ficient 18,290/Mcm. Results All the living individuals of the family (Figure 1) were sampled and their PON1 genotypes and phenotypes were determined. Demographic characteristics, lipid pro- files, and /insparaoxonase and arylesterase activities in hy- percholesterolemia patients and healthy individuals are summarized in Table 1. Other paramenters The lipid profile, including total cholesterol (TC), tri- glycerides (TG), LDL-cholesterol (LDL-C) and HDL cho- lesterol (HDL-C), of all the subjects was obtained using a Roche/Hitachi automated system with commercial kits for CHOL (Cholesterol CHOD-PAP), LDL-C plus 2nd gener- ation (LDL Cholesterol), HDL-C plus 3rd generation (HDL-Cholesterol) and TG (Triglyceride GPO-PAP) (all from Roche Diagnostics, Germany). 572 PON1 arylesterase activity Modeling the structure of PON allozymes Structures of the four different PON1 allozymes, L55, M55, Q192 and R192, were modeled at Modeller (Eswar et al., 2006) upon an already determined structure of human PON1 at 2.2 Å resolution with an R-factor 0.217 (Harel et al., 2004a,b). All four models were energy minimized and validated for various quality factors, which included Ramachandran plot for the backbone dihedral and , bad angles, bad bonds, steric clashes, and Z score of the model. Statistical analysis docking was done at Autodock, developed against Paraoxon and Phenyl Acetate and retrieved from Zinc data- base (Irwin and Shoichet, 2005; Morris et al., 2009). The docked conformations of the ligands were ranked on the ba- sis of binding affinity, analyzed, and assessed at Lig Plot+ and UCSF Chimera (Pettersen et al., 2004; Laskowski and Swindells, 2011). The allozymes L55 and M55 were also analyzed to explore why PON1 L55 has been reported to show high seral concentration than M55 polymorph (Bryk et al., 2005). Results for continuous variables are reported as mean SD. Differences among patients and healthy family mem- bers were assessed using Chi-square (2) and Fisher’s Ex- act test. P-values less than 0.05 were considered statistically significant. Continuous variables were ana- lyzed using ANOVA whereas categorical variables were compared by Chi-squared and Fisher’s Exact test. Assessing paraoxonase and arylesterase activities in silico There was no significant difference between patients and healthy individuals with respect to mean SD values of age, gender and body mass index (BMI). Routine labora- To assess the paraoxonase and arylesterase activities of PON1 allozymes Q192 and R192, in silico molecular Figure 1 - Pedigree of hypercholesterolemia family. Filled boxes and circles represent male and female patients respectively. Empty boxes and circles represent male and female carriers respectively. Modified from Ajmal et al. (2010). Figure 1 - Pedigree of hypercholesterolemia family. Filled boxes and circles represent male and female patients respectively. Empty boxes and circles represent male and female carriers respectively. Modified from Ajmal et al. (2010). 573 Idrees et al. Table 1 - Blood chemistry and clinical data of the affected and normal individuals of the studied family. Table 1 - Blood chemistry and clinical data of the affected and normal individuals of the studied family. Characteristics Patients (n = 10) Control (n = 24) P-value Age (Years) 22.5 16.8 26.5 14.6 NS Male: Female 5:5 12:12 1 BMI (kg/m2) 20 3.2 21 3.6 NS TC (mg/dL) 422.2 181.5 184.9 32.5 < 0.0001 TG (mg/dL) 167.6 67.1 135.7 80.1 0.28 LDL-C (mg/dL) 323.6 149.6 108.5 26.8 < 0.0001 HDL-C (mg/dL) 38 8.4 45.0 11.0 0.084 Paraoxonase activity (U/L) 116.5 40.3 172.5 61.6 0.001 Arylesterase activity (kU/L) 168.3 24.8 210.7 37.9 0.002 Xanthomas 1 (10%) 0 (0%) — CHD 1 (10%) 0 (0%) — Values are given as means SD (p < 0.05), continuous variables were analyzed using ANOVA whereas categorical variables were compared by Chi-square. NS: not statistically significant; BMI: body mass index; TC: serum total cholesterol; TG: serum total triglyceride; HDL-C: high-density lipo- protein cholesterol; LDL-C: low-density lipoprotein cholesterol; CHD: coronary heart disease risk. Values are given as means SD (p < 0.05), continuous variables were analyzed using ANOVA whereas categorical variables were compared by Chi-square. NS: not statistically significant; BMI: body mass index; TC: serum total cholesterol; TG: serum total triglyceride; HDL-C: high-density lipo- protein cholesterol; LDL-C: low-density lipoprotein cholesterol; CHD: coronary heart disease risk. previously (Motti et al., 2001). Sequencing results con- firmed the PCR-RFLP based identification of SNPs. The prevalence of various genotypes (PON1 SNPs and combi- nations) is given in Table 2. Assessing paraoxonase and arylesterase activities in silico PON1 M55 and Q192 allele prevalence was highly correlated with individuals with FH. Similarly, 80% of patients showed M55M55 genotype whereas 90% showed Q192Q192 genotype. PON1 L55 and R192 alleles were deficient in patients compared to healthy individuals of the family. Conversely, PON1 M55 and Q192 alleles were found significantly higher in patients. Similarly, the haplotype MM/QQ was prevalent in 80% of tory findings of lipid profile indicated significant differ- ences between the patients and healthy individuals for TC (p < 0.0001) and LDL-C (p < 0.0001) while differences be- tween the TG (p = 0.28) and HDL-C (p = 0.084) were not significant. Paraoxonase (p = 0.001) and arylesterase activ- ities (p = 0.002) were significantly lower in patients com- pared to healthy individuals of the family. p < 0.05 was considered statistically significant; data were analyzed using Fisher’s Exact Test. tistically significant; data were analyzed using Fisher’s Exact Test. Discussion This is the first study of PON1 activity and region polymorphisms coding in a family with FH with LDLR mu- tation, although in a couple of recent studies, the role of rs662 was assessed in relation to serum lipid levels and cor- onary artery disease (Liang, 2016; Chen et al., 2017). PON1 genotype and activity are not being monitored in routine clinical practice for the management of hypercho- lesterolemia. Understanding a possibly protective role of PON1 activity in patients susceptible to atherosclerosis can help in taking early preventive measures for improvement of life span of hypercholesterolemia patients. The binding pocket of PON1 is perfectly designed to spatially accommodate a lactone or arylester, with its distal end distributed by highly basic residues, N168 and N224, to interact with NO2 of paraoxon or OH of phenylacetate through hydrogen bonds. This arrangement helps position- ing the acyl bond in proximity to the catalytic dyad of histidine residues H115 and H134, which has been reported to deprotonate a water molecule to generate a hydroxyl ion, thereby hydroxylating the lactones (paraoxon) and arylesters (phenylacetate) (Khersonsky and Tawfik, 2006; Ben-David et al., 2012) . The contribution of PON1 in CVD is minor in healthy populations but it is known that genotypes with minor ef- fects in general population may have more pronounced ef- fects in patients, for example in FH cases (Leus et al., 2001; Wiegman et al., 2004). The beneficial effects of PON1 on the inhibition of atherosclerosis might be more pronounced in FH patients because they are more prone to develop ath- erosclerosis than the general population (van Himbergen et al., 2005). Our results suggest that position 192 lies near the in- ner lining of the PON1 binding pocket opening. This posi- tion is critical because of its sidechain lying closer to the Table 3 - Binding affinities of PON1 Q192 and R192 against paraoxon and phenyl acetate. PON1Allozyme Ligand Binding Energy (kcal/mol) R192 Paraoxon -6.10 Phenyl acetate -6.30 Q192 Paraoxon -5.60 Phenyl acetate -6.00 Table 3 - Binding affinities of PON1 Q192 and R192 against paraoxon and phenyl acetate. Low PON1 activity has been reported in previous studies as one of the leading factors causing atherosclerosis and myocardial infarction (Ayub et al., 1999; James et al., 2000; Mackness et al., 2002; van Himbergen et al., 2005; Bryk et al., 2005; Gur et al., 2006). Paraoxonase and arylesterase activities in silico Both paraoxon and phenyl acetate were docked against allozymes of the PON1, and showed a higher bind- ing affinity to PON1 R192 than Q192. The binding affinity of paraoxon to PON1 Q192 was observed to be -5.6 kcal/mol whereas for PON1 R192 the biding affinity has been significantly higher, i.e., 6.1 kcal/mol. In case of phenyl acetate (PA), the biding affinity for the allozyme R192 was observed to be 6.3 kcal/mol; however, it was 5.7 kcal/mol for Q192 (Table 3). PON1 polymporphisms genotyping data PCR amplification results and restriction fragment length polymorphism (RFLP) results of amplified products were in accordance with the specific band sizes reported Table 2 - Paraoxonase-1 allele, genotype, and haplotype distributions of L55M and Q192R polymorphisms in patients and healthy individuals of the hy- percholesterolemia family. Characteristics (Allele/Genotypes) Patients (n = 10) Controls (n = 24) p Alleles L55 2 (10%) 2 (4.2%) 0.336 M55 18 (90%) 46 (95.8%) 0.927 Q192 19 (95%) 33 (68.7%) 0.0164 R192 1 (5%) 15 (62%) 0.998 Genotypes L55L55 0 (0%) 0 (0%) 1 L55M55 2 (20%) 2 (8.3%) 0.334 M55M55 8 (80%) 22 (91.7%) 0.933 Q192Q192 9 (90%) 10 (41.7%) 0.0113 Q192R192 1 (10%) 13 (54.2%) 0.999 R192R192 0 (0%) 1 (4.2%) 1 Haplotypes M55M55/Q192Q192 8 (80%) 8 (33.3%) 0.017 L55M55/Q192Q192 1 (10%) 2 (8.3%) 0.661 L55M55/Q192R192 1 (10%) 0 (0%) 0.294 M55M55/Q192R192 0 (0%) 13 (54.2%) 1 M55M55/R192R192 0 (0%) 1 (4.2%) 1 574 PON1 arylesterase activity the FH patients whereas 10% of the FH patients exhibited LM/QQ and LM/QR haplotypes. the FH patients whereas 10% of the FH patients exhibited LM/QQ and LM/QR haplotypes. oxygen atoms of acyl group, which is a positively charged basic residue and has been selected by PON1 fold for this position over the course of time. A long sidechain of R192 with highly positively charged terminalguanidinium (HNC(NH2)2) mediates with acyl oxygen atoms then a small sized lysine sidechain with just one –NH3 +, R192 thus helps in positioning the acyl bond of the substrate to the cat- alytic dyad of H115 and H134, thus the higher arylesterase and paraoxonase activity of R192 allozyme (Figure 2). Discussion Thus, our re- sults combined with previously published data indicate the need of regular monitoring and upregulation of serum paraoxonase activity in patients with hypercholesterolemia for prevention of atherosclerosis. Our results highlight the importance of exploring PON1 as a therapeutic agent to ac- commodate the lower level of plasma PON1 in patients sus- ceptible to atherosclerosis. In the current study, the LDLR mutated patients had low level of PON1, which may indicate some gene-re- gulation action of LDLR and PON1, thus needing further investigations. With the current data, it is difficult to specu- late how LDLR and PON1 are involved in regulating each other. Patient V-6 was identified with xanthomas in addition to FH, but without any history of CVD. However, his levels of TC (917 mg/dL) and LDLC (728 mg/dL) were markedly high and HDL-C (22 mg/dL) was lower compared to other FH patients in the family. The presence of tendon xan- thomas is high risk factor of CVD among patients with FH, which along with decreased paraoxonase and arylesterase activities may lead to atherosclerosis (Jarvik et al., 2000; Mackness et al., 2003; Gur et al., 2006; Rosenblat et al., 2006; Soran et al., 2008; Sun et al., 2016; Verit et al., 2008). Both the hypercholesterolemia and PON1 deficiency are independent risk factors for the development of athero- sclerosis. In addition to controlling high levels of choles- terol, there is a need to regularly monitor PON1 status of hypercholesterolemia patients and normal family mem- bers. A better understanding of factors upregulating PON1 status in humans will have a significant public health im- pact by saving patients who are otherwise susceptible to atherosclerosis due to their deficient PON1 status. In the current study, controls from the general population were not screened, and this is a limitation of the study. However, based on the current findings, future studies can be de- signed to screen the population for PON1 and further inves- tigate its role in cardiovascular diseases. The low levels of serum paraoxonase and arylesterase activities of patients in this study may be due to their ge- netic makeup (PON1 coding region SNPs). The L55M polymorphism affects the enzyme concentration (plasma PON1 protein levels indicated by serum arylesterase activ- ity), whereas the Q192R polymorphism affects the catalytic efficiency (serum paraoxonase activity), but not the con- centration (Bryk et al., 2005). Discussion Numerous clinical stud- ies have shown an association of low PON1 activity with atherosclerosis and cardiovascular diseases (Jarvik et al., Figure 2 - Docked conformations of paraoxon in PON1 binding pocket. (A) Paraoxon bound in PON1 binding pocket. (B) Zoomed in binding pocket of PON1, paraoxon terminally making two hydrogen bonds with N168 and N224. Notice that R192 adopts a conformation more proximal to the binding pocket opening than that of Q192, steering the ligand to adopt the most suitable conformation for paraoxonase activity by H 115 and H134 dyad. (C) 2D il- lustration of paraoxon binding in PON1 binding pocket; paraoxon forms two hydrogen bonds with Asn 224 (3.00 Å) and Asn 168 (3.18 Å) shown in green while a number of hydrophobic interactions with the residues configure the binding pocket Figure 2 - Docked conformations of paraoxon in PON1 binding pocket. (A) Paraoxon bound in PON1 binding pocket. (B) Zoomed in binding pocket of PON1, paraoxon terminally making two hydrogen bonds with N168 and N224. Notice that R192 adopts a conformation more proximal to the binding pocket opening than that of Q192, steering the ligand to adopt the most suitable conformation for paraoxonase activity by H 115 and H134 dyad. (C) 2D il- lustration of paraoxon binding in PON1 binding pocket; paraoxon forms two hydrogen bonds with Asn 224 (3.00 Å) and Asn 168 (3.18 Å) shown in green while a number of hydrophobic interactions with the residues configure the binding pocket 575 Idrees et al. 2000; Mackness et al., 2003; Gur et al., 2006; Rosenblat et al., 2006; Soran et al., 2008; Sun et al., 2016; Verit et al., 2008). Thus, significantly decreased paraoxonase and arylesterase activities in patients compared to the healthy individuals in the present study, indicate an increased risk of atherosclerosis in patients. One of the patients (IV-4) in the current family had a history of CVD in addition to FH. He had premature coronary artery disease and had suffered from myocardial infarction at an early age, which may have been due to decreased paraoxonase and arylesterase activ- ity. The lipid profile of this 37-year-old male was not con- siderably elevated compared to other patients in this family. 2000; Rosenblat et al., 2011). Therefore, upregulation of PON1 levels by non-genetic factors (Rosenblat et al., 2011) can have a potential advantage in such cases for better and achievable protection against atherosclerosis. Discussion The PON1 M55 is associated with low plasma PON1 level (Blatter et al., 1993; Mack- ness et al., 1998a,b). The PON1 R192 allozyme hydrolyzes paraoxon more readily than Q192 (Costa et al., 2005). In our study, the high prevalence of M55 allele in the patients may have been responsible for the low levels of PON1 ac- tivity (Aviram et al., 2000). The frequency of the low- activity allele (Q192) and unstable form (M55 isoform, which is sensitive to proteolysis) was collectively higher in patients of this family indicated by high prevalence of MMQQ genotype (8, 80%), which may significantly re- duce the function of PON1 for protection against athero- sclerosis. The generally low frequency of the M55 is such that in some studies the MM genotype is not observed at all (Santos et al., 2005). M is not favored in human popula- tions, but it is kept in populations in heterozygous individu- als. The L55M mutation may considerably affect PON1’s stability and thereby account for the lower enzymatic activ- ity because M55 isoform is an unstable form (sensitive to proteolysis) (Harel et al., 2004a,b; Mackness et al., 1998a). Conclusion This study aimed at exploring the implications of PON1 polymorphism in the individuals affected by familial hypercholesterolemia (FH). The role of PON1 and its vari- ous polymorphs has not been studied previously in FH sub- jects. This work sought to investigate paraoxonase and arylesterase activity of various PON1 SNPs in individuals with mutated LDLR, thereby explore their role in the devel- opment of FH. The results suggest that most of the hyper- cholesterolemia patients with LDLR mutation have homo- zygous M55 and Q192 PON1 genotype, thus combination of MMQQ PON1 genotype and LDLR mutation might lead to a more severe disease outcome in the form of a fatal heart failure. Further studies are needed to explore the role of LDLR and PON1 pathways in the onset of hypercholester- olemia and atherosclerosis. Ahmed W, Ajmal M, Sadeque A, Whittall RA, Rafiq S, Putt W, Khawaja A, Imtiaz F, Ahmed N, Azam M, et al. (2012) Novel and recurrent LDLR gene mutations in Pakistani hy- percholesterolemia patients. Mol Biol Rep 39:7365-7372. 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Supplementary material The following online material is available for this article: Figure S1 - RFLP results of PON1 Q192R The following online material is available for this article: Figure S1 - RFLP results of PON1 Q192R Ng CJ, Wadleigh DJ, Gangopadhyay A, Hama S, Grijalva VR, Navab M, Fogelman AM and Reddy ST (2001) Parao- xonase-2 is a ubiquitously expressed protein with antioxi- dant properties and is capable of preventing cell-mediated Associate Editor: Mara H. Hutz License information: This is an open-access article distributed under the terms of the Creative Commons Attribution License (type CC-BY), which permits unrestricted use, distribution and reproduction in any medium, provided the original article is properly cited. References Verit FF, Erel O and Celik H (2008) Paraoxonase-1 activity in pa- tients with hyperemesis gravidarum. Redox Rep 13:134-138. 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Erratum In the article “Decreased serum PON1 arylesterase activity in familial hypercholesterolemia patients with a mutated LDLR gene”, with DOI number 10.1590/1678-4685-gmb-2016-0287, published in the jornal Genetics and Molecular Biol- ogy issue 41(3): 570-577, on page 570 the author name spelled as Abdul Rauf Siddiq should read Abdul Rauf Siddiqi. In the article “Decreased serum PON1 arylesterase activity in familial hypercholesterolemia patients with a mutated LDLR gene”, with DOI number 10.1590/1678-4685-gmb-2016-0287, published in the jornal Genetics and Molecular Biol- ogy issue 41(3): 570-577, on page 570 the author name spelled as Abdul Rauf Siddiq should read Abdul Rauf Siddiqi.
https://openalex.org/W4375940266	https://zenodo.org/records/7915537/files/2%20-%20Symposium%20-%20Bridging%20the%20Gap%20Highlights%20of%20the%201st%20Symposium%20of%20the%20Schools%20of%20Foreign%20Languages%20in%20the%20Aegean%20Region.pdf	English	null	Bridging the Gap: Highlights of the 1st Symposium of the Schools of Foreign Languages in the Aegean Region	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	6,914	Keywords Keywords School of Foreign Languages Accreditation Curriculum Development Aegean Region Bridging the Gap: Highlights of the 1st Symposium of the Schools of Foreign Languages in the Aegean Region Pınar GÜZEL GÜRBÜZa, Hüsem KORKMAZb, Ali CEYLANc, Yunus ÖZDEMİRd, Süheyla HEREKe, Seden ÖNSOYf, Asu PİNARg, Saffet DİNÇERh Pınar GÜZEL GÜRBÜZa, Hüsem KORKMAZb, Ali CEYLANc, Yunus ÖZDEMİRd, Süheyla HEREKe, Seden ÖNSOYf, Asu PİNARg, Saffet DİNÇERh Cite as: Güzel Gürbüz, P., Korkmaz, H., Ceylan, A. , Özdemir, Y., Herek, S., Önsoy, S., Pinar, A., & Dinçer, S. (2022). Bridging the Gap: Highlights of the 1st Symposium of the Schools of Foreign Languages in the Aegean Region. Manisa Celal Bayar University International Journal of English Language Studies. 1 (1); 3-16. a Corresponding: Pınar GÜZEL GÜRBÜZ, Prof. Dr., Manisa Celal Bayar University, Manisa, Türkiye, pinar.guzel@cbu.edu.tr, Orcid: 0000-0001- 5982-2816. b Hüsem KORKMAZ, Dr., Manisa Celal Bayar University, Manisa, Türkiye. Orcid: 0000-0002-5759-7392. c Ali CEYLAN, Dr., Manisa Celal Bayar University, Manisa, Türkiye, Orcid: 0000-0002-6509-7304. d Yunus ÖZDEMİR, Dr., Manisa Celal Bayar University, Manisa, Türkiye. e Süheyla HEREK, Instructor, Manisa Celal Bayar University, Manisa, Türkiye. f Seden , Instructor, Manisa Celal Bayar University, Manisa, Türkiye. g Asu PİNAR, Instructor, Manisa Celal Bayar University, Manisa, Türkiye. h Saffet DİNÇER, Instructor, Manisa Celal Bayar University, Manisa, Türkiye, Orcid: 0000-0002-5330-7925. a Corresponding: Pınar GÜZEL GÜRBÜZ, Prof. Dr., Manisa Celal Bayar University, Manisa, Türkiye, pinar.guzel@cbu.edu.tr, Orcid: 0000-0001- 5982-2816. Manisa Celal Bayar University International Journal of English Language Studies 2022; 1 (1); 3-16 h Saffet DİNÇER, Instructor, Manisa Celal Bayar University, Manisa, Türkiye, Orcid: 0000-0002-5330-7925. Introduction Scientific and academic events such as conferences, congresses, and symposiums are known to serve effectively to spread scientific knowledge by sharing the knowledge with the stakeholders and assessing it together in an academic sphere (Mercer, 1995). Moreover, such events are valuable opportunities for people from the same fields of study or interest to come together and be part of a common research and practice community (Wenger, 1998). In this way, they ensure that the cooperation and the knowledge-sharing process will continue after the event, as well. According to Hall and Longman (2008), scientific meetings encourage and spread new ideas and knowledge, and they have a central role in connecting researchers and practitioners from the same or similar professional and academic identities. For all these reasons, Manisa Celal Bayar University (MCBU) School of Foreign Languages (SFL) intended to bring the academicians of the schools of foreign languages working at 15 different universities in the Aegean Region of Türkiye together to bridge the gaps among the institutions in the same geographical region. It was, in particular, aimed to discuss the key issues in foreign language teaching, suggest solutions to the problems and experiences, and share good practices in the field of foreign language teaching. Bridging the Gap: Highlights of the 1st Symposium of the Schools of Foreign Languages in the Aegean Region Bridging the Gap: Highlights of the 1st Symposium of the Schools of Foreign Languages in the Aegean Region Bridging the Gap: Highlights of the 1st Symposium of the Schools of Foreign Languages in the Aegean Region 2022; 1 (1); MCBU IJELS\| Page 4 Abstract Sharing scientific knowledge with all the stakeholders of foreign language teaching is as important as producing it in a systematic way. Besides, sharing effective practices in English language teaching is also as essential as publishing the theoretical findings of the relevant research in the field. Therefore, scientific events such as symposiums in the present context play a crucial role in unearthing the strengths and weaknesses in educational practices in any participating institution. Among the rare attempts and the first one in the Aegean Region of Turkey, the present symposium brought together a dozen universities sharing a number of standard features but implementing different practices for the same or considerably similar learning outcomes. The concurrent sessions held during the symposium cast light on the good practices in various schools of foreign languages offering English language preparatory programs. Topics such as quality assurance and accreditation, assessment and evaluation, curriculum development, leadership and management, professional development, and administrative issues were among the topics visited during the concurrent sessions. In brief, it was concluded that each institution had instances of best English language teaching practices while all had much to learn from others and put into practice for further development. Cite as: Güzel Gürbüz, P., Korkmaz, H., Ceylan, A. , Özdemir, Y., Herek, S., Önsoy, S., Pinar, A., & Dinçer, S. (2022). Bridging the Gap: Highlights of the 1st Symposium of the Schools of Foreign Languages in the Aegean Region. Manisa Celal Bayar University International Journal of English Language Studies. 1 (1); 3-16. Method / Event The event took place in the form of a one-day symposium rather than a conference to ensure that all the participants representing their institutions could find opportunities to contribute to the discussions. A total of 15 schools of foreign languages were invited to the symposium with no participant quota restrictions. In the end, participants from 11 universities took part in the event which has been the first attempt to bring together the representatives of the schools of foreign languages in the region within an academic context. Dokuz Eylül University, Ege University, İzmir Bakırçay University, İzmir Democracy University, İzmir University of Economics, İzmir Katip Çelebi University, İzmir Institute of Technology, Muğla Sıtkı Koçman University, Pamukkale University, Yaşar University were represented in the symposium as well as the hosting institution Manisa Celal Bayar University. Within the frame of the event, the number of concurrent sessions was determined and held under the following headings: Assessment and Evaluation, Assessment and Evaluation, Curriculum Development, Leadership and Management, Administrative Issues & Student Affairs, Professional Development, Accreditation. The participants were allowed to choose the session they would join and were encouraged to actively participate in the sessions. At the end of the day, conclusions drawn from the sessions were compiled by the session reporters and presented by the moderators to all the participants in a debriefing session. Conclusions and Discussion Assessment and Evaluation Assessment and Evaluation 2022; 1 (1); MCBU IJELS\| Page 4 Güzel Gürbüz, Korkmaz, Ceylan, Özdemir, Herek, Önsoy, Pinar & Dinçer Assessment is one of the basic components of foreign language teaching. Accordingly, assessment and evaluation units are important in shaping language teaching in the schools of foreign languages or preparatory programmes at universities in Türkiye. Therefore, the assessment and evaluation session of the symposium mainly focused on the problems the assessment units faced and how to overcome or minimize these problems. The main conclusions drawn from the two different sessions were related to six key issues. Assessment is one of the basic components of foreign language teaching. Accordingly, assessment and evaluation units are important in shaping language teaching in the schools of foreign languages or preparatory programmes at universities in Türkiye. Therefore, the assessment and evaluation session of the symposium mainly focused on the problems the assessment units faced and how to overcome or minimize these problems. The main conclusions drawn from the two different sessions were related to six key issues. The first topic discussed was the excessive workload of the assessment unit members and the place and importance of the assessment units at the schools of foreign languages in Türkiye. Since The Council of Higher Education (YÖK) does not specify any items related to the structure and workload of the assessment and evaluation units and their members in its regulations, the members in the units face many difficulties in terms of their workload and responsibilities. The excessive workloads of academics in higher education in Türkiye have been addressed by many researchers and Cenkseven and Dost (2007) state that excessive workload is among the factors affecting faculty members negatively. Ercan Demirel and Cephe (2015) address the problem in the context of the language instructors working at three different universities in Türkiye and assert that excessive workload along with working in testing and similar units is among the reasons causing burnout for the language instructors. İpek and Kanatlar (2018) also research the causes affecting foreign language instructors’ motivation and conclude that excessive workload can be exhausting and demotivating. However, little or no research to date has focused on the workload of the assessment unit members in schools of foreign languages specifically. On the one hand, they have to work really hard in order to prepare tests and other assessment tools such as rubrics and in-class task evaluation forms. Assessment and Evaluation On the other hand, they try to keep up with their teaching responsibilities. All the participants in the assessment and evaluation sessions agreed that it is essential to re- evaluate and re-plan the workload of the unit members for effective assessment. In fact, this issue has had a long history in the context of higher education in Türkiye and has been addressed by many of the schools of foreign languages administrators in yearly meetings held across the country. Since their first meeting held at Sıtkı Koçman University in Muğla in 2008, the administrators of the schools of foreign languages have addressed the need for assessment units (Testing Offices) in the schools of foreign languages. In addition, starting from their sixth meeting held at Sabancı University in 2012 until their last meeting held at Bolu Abant Izzet Baysal University in 2021, they repeatedly included their suggestions about the assessment units in the final declarations of the meetings. They especially focused on the importance of reducing the workload of the assessment unit members and improving their working conditions in the declarations starting from 2012 and onward (YDYO-TR Yöneticiler Platformu, 2022). To the best of our knowledge, no progress has yet been made to solve the problem even though the issue continues to be one of the hot topics in the field. Increasing the language assessment literacy level of new assessment unit members and facilitating their professional development was another issue discussed in the symposium. Basic terms related to language assessment were presented by the session moderator and assessment concepts were discussed by the participants. Language assessment literacy refers to the language teachers’ knowledge about assessing a language (Malone, 2013). According to recent research in the field, language teachers in Türkiye do not have sufficient language assessment knowledge (Mede & Atay, 2017). Assessment and Evaluation Another research concludes that factors such as years of experience in language teaching, educational background whether or not instructors Page 5 \| 2022; 1 (1); MCBU IJELS Bridging the Gap: Highlights of the 1st Symposium of the Schools of Foreign Languages in the Aegean Region Bridging the Gap: Highlights of the 1st Symposium of the Schools of Foreign Languages in the Aegean Region are graduates from an English Language Teaching programme, being graduated from the BA programme including a testing course, and attending training sessions specifically focusing on testing and assessment do not have an effect on language assessment literacy level whereas working as an assessment unit member has an impact on language assessment literacy level of the teachers and there is a significant difference between the ones who are in the assessment units and the ones who are not. (Ölmezler-Öztürk & Aydın, 2019). In line with this study, Yastıbaş & Takkaç, (2018) place importance on self-improvement in language assessment literacy and state that self-improvement in assessment depends on peer-assessed exam preparation processes and gaining experience in test preparation. Therefore, in order to increase the language assessment literacy levels of new unit members it is important to engage them with the assessment processes after sharing with them the basic concepts regarding language assessment essentials. The next issue discussed in the symposium related to assessment was the exams such as YÖKDİL (Higher Education Foreign Language Test) and YDS (Foreign Language Proficiency Exam) which circumscribe assessment processes employed by the schools of foreign languages and preparatory programs at universities in Türkiye. According to the regulation published by The Council of Higher Education (YÖK) (2016), the students who can get passing scores on the nationwide language proficiency exams such as YÖKDİL and YDS are qualified to be exempt from language education in preparatory schools and can start their studies which are either partly or fully in English. However, this application seems to contradict the language assessment practices used by the preparatory school programmes. The language assessment procedures applied in these programmes include both formative and summative assessment applications in order to evaluate all foreign language skills of students throughout the education year. In this way, students gain confidence through these practices and use all foreign language skills effectively in their social lives and departmental studies. Assessment and Evaluation Concerning the exit level of the language programmes, the situation of the repeat students who fail a module and have to repeat it to get to the next level was also discussed. According to a study, students repeating the same module have serious motivation problems (Kuzu et al., 2022). Another study which specifically focuses on the burnout levels of repeat preparatory school students whose exit level is B1 points out that the burnout level of these students reaches the highest levels (Erakman & Mede, 2018). Besides, when the exit level is B2 or above, the motivation problem gets more complicated for the students failing the final module. Most of these students get discouraged and find it meaningless to study the last module twice because their departmental studies are partly in English which only requires thirty per cent of the courses in English. The participants in the session emphasised that the student motivation level in language teaching is very important as it directly affects the teachers’ motivation. As a result, the importance of a convenient exit level for a modular system was once more pointed out. Increasing the weight of formative assessment practices in evaluating writing assignments was the final topic discussed in the assessment session. Formative assessment is a means of assessing students’ work during their production processes and involves various strategies focusing on feedback and continuous student engagement (Heritage, 2010, p.19). In the context of higher education in Türkiye, Uzun and Ertok (2020) research the opinions of students about exam-based summative assessment approaches and task-based formative assessment approaches in English language teaching and conclude that majority of the students in the research favour formative assessment approaches over summative ones. The efficiency of using formative assessment tools for writing tasks has also been researched and using a writing portfolio system is proven to be an effective process. Caner (2010) points out the positive impact of a portfolio system in teaching writing skills along with other language skills but also states that it is regarded as a burden by students. This dimension of the portfolio assessment for writing was voiced by some participants in the assessment session. It was also stated that since it requires a lot of time and effort both on teachers’ and students’ parts, it is difficult to apply in a large scale. Assessment and Evaluation YÖKDİL and YDS exams, on the other hand, only focus on reading comprehension, translation studies, vocabulary and grammar knowledge of the test takers and lack any means of assessing listening skills along with the productive skills of writing and speaking. One study shows that the majority of the participants who took either the YÖKDİL or YDS exam stated that the exams did not really contribute to their foreign language learning processes and had a negative washback effect as they do not include all language skills (Polat, 2020). Therefore, all the participants in the session agreed that the re-evaluation of this application is required. The exit level for Progressive and Modular System students was also discussed in the symposium. Modular systems refer to modules including different tiered language skills and knowledge levels through which students reach a proficient language level that is gaining popularity in language teaching (Tercan, 2018). However, the exit level of the students learning English in a modular system is a controversial issue in language preparatory programmes in Türkiye. While some preparatory programmes apply B1+ as their exit level, others prefer the B2 level. According to the research conducted by the British Council (2015), most students starting preparatory programmes regardless of their programme types in Türkiye are at beginner levels in English and it is “impossible” for them to reach B2 level at the end of the language preparatory programs which last for eight months (p. 70). A more recent study focusing on the modular system employed at a state university in Türkiye also asserts that a modular system aligned with the Common European Framework of Reference for Languages (CEFR) at B1+ as an exit level serves high-quality language education although there are still some problems related to listening and speaking skills (Duru, 2021). In line with these findings, 2022; 1 (1); MCBU IJELS\| Page 6 Güzel Gürbüz, Korkmaz, Ceylan, Özdemir, Herek, Önsoy, Pinar & Dinçer another study suggests that for an effective language learning program B1 could be the exit level (Coşkun, 2013). Accordingly, the participants in the symposium stressed the importance of having a convenient exit level irrespective of the system employed by the schools of foreign languages. Also, it was generally agreed that B1+ would be a more manageable exit level for modular system programs applied by the schools of foreign languages in Türkiye. Assessment and Evaluation Given the large number of students in the preparatory schools in state universities in Türkiye, the application was regarded as impractical. The assessment session of the symposium was fruitful in that it provided the participants with the opportunity to exchange their ideas about different assessment practices employed in different schools of foreign languages in the Aegean Region. Furthermore, it demonstrated different assessment units in different schools have similar challenges related to assessment and evaluation processes. As a result, it provided insights for the participants and showed the importance of such events for further collaboration in the long run. Curriculum Development The curriculum session of the symposium aimed at supporting teachers to consider curriculum planning processes at their schools and sharing experiences to feel more confident Page 7 \| 2022; 1 (1); MCBU IJELS Bridging the Gap: Highlights of the 1st Symposium of the Schools of Foreign Languages in the Aegean Region Bridging the Gap: Highlights of the 1st Symposium of the Schools of Foreign Languages in the Aegean Region in their understanding of developing high-quality curricula and to improve the understanding of processes in constructing high-quality curricula. The session was held in two parts, each of which lasted 40 minutes. The participants in the sessions were mostly the instructors working in the curriculum units at their schools. Specifically, the session addressed four main themes: in their understanding of developing high-quality curricula and to improve the understanding of processes in constructing high-quality curricula. The session was held in two parts, each of which lasted 40 minutes. The participants in the sessions were mostly the instructors working in the curriculum units at their schools. Specifically, the session addressed four main themes: · The role of the curriculum in EFL settings, specifically at Preparatory Schools of Universities · The curriculum cycle · Curriculum planning issues in Modular Systems · The flexibility needed in Curriculum Planning in the changing world. The goal of a successful educational program and effective curriculum planning must meet the needs and demands of society, the expectations and aims of the educational institution, the beliefs and backgrounds of the teachers and the student profile. Therefore, the curriculum development process requires review, revision, and constant change (Johnson, 2001). In all participant schools, it was seen that the curriculum is in accordance with the descriptive and pedagogical principals of The Common European Framework of Reference for Languages (CEFR), covering all the areas above. The language proficiency levels in the curriculum at participant schools are therefore reflected as A1, A2 (Basic Users) and B1, B2 (Independent Users). In most schools, while the exit level is B1+, in some it is B2. The main purpose of all Preparatory Schools is to provide general English knowledge to students who are not proficient enough and to provide some basic skills for their departments’ academic language skills. Curriculum Development It was all agreed that while these two objectives can be performed for students at A2 and B1 levels at the beginning of the Preparatory programme, starting with a group that does not speak any English(A1) can cause problems. The other important issue mentioned was the use of formative assessment in the teaching process. Most participants stated that they have increased the weight of alternative measurement tools such as presentations and portfolios in the total evaluation rate. In all academic programmes, a curriculum design cycle includes needing analysis, setting objectives, material design, instructional activities, assessment, and evaluation parts. The dynamic nature of the cycle allows for curriculum modification or improvement via action plans and feedback. Throughout the year, the curriculum development units of the schools work on the components of the cycle to meet the needs and objectives of the school. During this part of the session, it was discussed how important it is to prepare weekly flow charts that direct the instructors on what to do and how to do it on each day of the curriculum plan. In this way, depending on the feedback from the instructors at the end of each week, the following flow can be rearranged and improved. At the end of this part, the participants of the session pointed out the fact that as the curriculum unit members of the schools must deal with all these issues in addition to their normal teaching duties, it increases their workload, which leads to exhaustion and motivational problems. The comparison between the participants from the state and private universities regarding their weekly lesson hours revealed that the number of lesson hours of curriculum members at private universities is much less than the ones at state universities. Most universities in Türkiye have a one-year compulsory English preparatory programme for students whose departments have English as the medium of instruction. Two systems in preparatory programs, the modular system and progressive system, characterize the 2022; 1 (1); MCBU IJELS\| Page 8 Güzel Gürbüz, Korkmaz, Ceylan, Özdemir, Herek, Önsoy, Pinar & Dinçer regulation and organization of courses, the assessment and evaluation procedures, classroom practices and material development and design (Eraslan, 2019). Curriculum Development The modular system can be defined as "a unit of work in a course of instruction that is virtually self-contained and a method of teaching that is based on the building up of skills and knowledge in discrete units" (Sejpal, 2013, p.169). While in a progressive system, English teaching is given throughout the year depending on learners’ English level according to the placement test done at the beginning of the education year, in a modular system English is taught in different modules at the same time. Students move forward or fall behind their current levels (Eraslan, 2019). Because the English levels of the preparatory class students at MCBU were not as good as expected, the modular system started to be implemented from the 2021-2022 academic year onwards. In the curriculum session, the modular system was discussed regarding the implementation of the curriculum, and it was agreed that the effectiveness of the system largely depends on the number of students enrolling in the preparatory schools as it requires more classrooms, instructors and materials. This conclusion supports the findings of Coşkun (2013), who in his research found out that the modular system was ineffective because the resources of the school could not provide repeat classes with the extra materials and academic assignments and the number of instructors was not enough. Regarding curricular issues, the programme for the students who repeat the same level in the Modular system was also discussed. While some schools run the repeat class curriculum with the same instructional materials, some change the textbooks in these classes. In addition, these students enrol in the same classes as the students who have moved up the same level for the first time in some schools, whereas in some preparatory programmes there are classes where all the repeat students follow a specific curriculum plan. The conclusions drawn from the discussion on these issues were that it is not so effective to use the same textbooks with these students as they have already used them, and also they get bored in the lessons since they do not encounter new tasks. Furthermore, placing repeat students in the same class as new students may embarrass repeaters or reduce the motivation of new students. Curriculum Development It was agreed that especially in State universities, expecting repeat students to buy new textbooks in the same module is not realistic, and it was suggested that the repeat class curriculum can be supplemented by the online materials of the books and more emphasis can be given to workbook tasks. Based on the social, economic, political and technological developments in the 21st century, expectations about the individual qualities needed are changing. In addition, these changes also affect education systems and the knowledge, skills and competencies that individuals must acquire (Cansoy, 2018). In this part of the session, it was discussed that it is necessary to make some necessary changes while planning the curriculum depending on the issues above. Firstly, the integration of technology in education is a real need for the students who grow up with the technology of the 21st century (Chapelle, 2003). Therefore, integrating the use of technological tools in the instructional activities to reach our curriculum objectives is a must, not a choice. Secondly, soft skills, also called generic skills, are emphasized in higher education today. These skills are personal and professional qualities that learners have in their professional lives in addition to their technical skills, and these skills such as leadership, communication, planning, adaptability, cultural awareness and relationship building can be emphasized more in our curriculum. Lastly, in today’s education, students are expected to be active learners in the learning process. Therefore, in addition to planning for their academic achievement, we need to help them improve skills such as communication and interaction with society. In this regard, collaborative learning is essential for developing students’ social Page 9 \| 2022; 1 (1); MCBU IJELS Bridging the Gap: Highlights of the 1st Symposium of the Schools of Foreign Languages in the Aegean Region Bridging the Gap: Highlights of the 1st Symposium of the Schools of Foreign Languages in the Aegean Region interaction skills (Ghavifekr, 2020). At the end of this part, all participants agreed on the fact that concepts such as digital integration, collaborative learning, and generic (soft) skills must be added to the curriculum planning process. interaction skills (Ghavifekr, 2020). At the end of this part, all participants agreed on the fact that concepts such as digital integration, collaborative learning, and generic (soft) skills must be added to the curriculum planning process. Curriculum Development The curriculum session was very productive and guided for the participants to exchange information, identify common problems and propose solutions. The common view of all participants was that such meetings and symposiums should be held more frequently and regularly because curriculum groups in the participating schools want to feel that they are not alone in the systems they apply and the decisions they make. Accreditation The term “accreditation” cannot actually be used interchangeably with “quality assurance” although they may easily be misinterpreted in educational settings. Reeves (2019) emphasizes the distinction by describing the state of being “accredited” as the ultimate resulting mark of an institution or accreditation program carrying out the audit process. In the accreditation session of the symposium, therefore, the focus was more on the accreditation rather than the quality assurance process in general. Internationalization of higher education all around the world has brought along the ever- growing interest in quality assurance and accreditation of educational practices by specialized bodies, and English language teaching programs were no exception, either (Harvey, 2006; Staub, 2019). Besides, there is evidence showing the need for a stronger focus on quality assurance in English language teaching in Türkiye (Staub, 2019) due to a number of deficiencies in foreign language learning (British Council, 2015). Lastly, the requirement of accountability as a result of the decrease in trust of state institutions has increased the popularity of quality assurance and accreditation endeavours (Kinser, 2014). Among the leading conclusions drawn from the discussions in the accreditation session, one is noteworthy since it also points to the difference between accreditation and quality assurance as two distinct but related concepts: “the focus should be on quality assurance; accreditation is the natural result”. The expression was further clarified by putting emphasis on the standardization of the practices and procedures, having an institutional policy of quality assurance, institutional transparency, and the learning outcome. Yet, above all, the quality itself should be put in the centre. Dr. Donald Staub, the Chair of the concurrent session, noted that quality in a program could be found in the learning, teaching, and management components. In other words, all the major processes in an English language program should reflect the quality. As the quality assurance labelling authorities, major accreditation schemes in Türkiye were also among the issues discussed. It was concluded that CEA, EAQUALS and DEDAK are the common schemes in the country and their standards are more or less the same although they are named differently. The examples presented by Dr. Staub were clear indicators of the similarity of the standards across different schemes. Another important point to consider is that accreditation standards are not prescriptive, and they do not force institutions to take any action, but the proof is required for any standard in all the accreditation schemes. Professional Development The Professional Development session of the symposium aimed at sharing ideas, experiences and practices of professional development units at preparatory schools of both state and private universities in the Aegean Region. The session was held in two parts, each of which lasted 40 minutes. The participants in the sessions were mostly the instructors working in the professional development units at their institutions. The first session addressed the practices of each institution whereas the second part addressed the problems and solutions during the practices. Leadership and Management The Leadership and management session of the symposium aimed to gather leaders including managers and unit heads to discuss the issues they had faced in managing their teams and the ways how to overcome those issues. The session lasted 50 minutes. There were 18 participants in the session and their roles varied from school directors to instructors. In the first part of the session, recent stressors related to their work were discussed. Two main themes emerged from the discussion and they were classified according to the duties of the instructors in their institutions. The first theme was named “Stressors for Staff with an Administrative Duty” and the second was named “Stressors for Staff without an Administrative Duty”. The issues under these headings are presented in Table 1. Table 1. Stressors for the Instructors Stressors for Staff with an Administrative Duty Stressors for Staff without an Administrative Duty Very busy schedule The high number of international students Lack of staff & contracted teachers Instructors refuse to get more lessons, extra hours and duties Workload Private problems of instructors (health or psychological) Some instructors come late & skip exam duties. Other instructors have to cover their lessons. Instructors refuse to teach evening classes because of money & long hours Lack of facilities Translation tasks are given by the upper administration Lack of classroom Workload Extra duties (like translation) Strict schedules Co-workers Coordinators Adaptation after Covid Adaptation to new materials Lack of facilities Short notice duties The difficulty of work and private life balance Table 1. Stressors for the Instructors In the later stages of the session, the psychological safety of the teachers was discussed. In this part, whether the psychological safety of the instructors was good or bad and how they could empower their psychological safety were the core of the session. In light of the discussions, delivering how to overcome those stressors and how to empower the psychological safety and well-being of the instructors may contribute to handling the issues mentioned in the session. 2022; 1 (1); MCBU IJELS\| Page 10 Güzel Gürbüz, Korkmaz, Ceylan, Özdemir, Herek, Önsoy, Pinar & Dinçer Accreditation In brief, the session provided the participants with insights into a number of major themes regarding quality assurance and accreditation in English language programs in Türkiye. Page 11 \| 2022; 1 (1); MCBU IJELS Bridging the Gap: Highlights of the 1st Symposium of the Schools of Foreign Languages in the Aegean Region 2022; 1 (1); MCBU IJELS\| Page 12 References British Council. (2015). The state of English in higher education in Türkiye: A baseline study November 2015. Türkiye Ekonomi Politikaları Araştırma Vakfı & British Council. Retrieved from https://www.britishcouncil.org.tr/sites/default/files/he_baseline_study_book_web_- _son.pdf. Caner, M. (2010). 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The Use of Plickers for Formative Assessment of Vocabulary Mastery. Ethical Lingua: Journal of Language Teaching and Literature, 7(2), 311-320. https://doi.org/10.30605/25409190.179 MohdAsraf, R & Supian, N. (2017). Metacognition and Mobile – Assisted Vocabulary Learning. Arab World English Journal, 8(2). DOI: https://dx.doi.org/10.24093/awej/vol8no2.2. Sasmiko, A. R., Noni, N., ve Salija, K. (2019). The Use of Plickers to Enhance Students’ English Reading Comprehension at SMP Muhammadiyah 6 Makassar. In. Stockwell, G. (2008). Investigating Learner Preparedness for and Usage Patterns of Mobile Learning. ReCALL, 20(3), 253–270. Stockwell, G., & Hubbard, P. (2013). Some emerging principles for mobile-assisted language learning. Monterey, CA: The International Research Foundation for English Language Education. Wang, B. T., Teng, C. W., & Chen, H. T. (2015). Using iPad to Facilitate English Vocabulary Learning. 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https://openalex.org/W4291476188	http://bcpublication.org/index.php/BM/article/download/1479/1490	English	null	Research on Intelligent Parking Operation and Management Planning Based on Revenue Model Function	BCP business & management	2,022	cc-by	3,788	1. Introduction Under reasonable and necessary assumptions, considering only one parking space, a revenue model function is built to calculate the possible billing gains and losses that may result from mobile video capture vehicle billing. The solution is to build a mathematical function model and run it using python code to derive the relevant data[1]. We address certain areas of Shenyang, ranging from Shenyang Station-Taiyuan Street area (west to Shengli Street, east to Nanjing Street, north to North 5th Road (including the south side of North 5th Road), and south to South 8th Road). Based on considering the characteristics of the area, we eventually only need to analyze the specific time required for a scan car to go around and the number of scan cars for the two requested quantities, and establish a mathematical model to plan the mobile video capture vehicle's route[2]. Still still consider The region uses a two-dimensional scatter distribution diagram to establish a mathematical model to rationalize the toll collection method under different circumstances, and to re- plan the route of the mobile video capture vehicle in the section where manual toll collection has been determined[3]. On the basis of reasonable assumptions and consideration of necessary labor and volume maintenance fees and other costs (the service life of the vehicle is calculated by 10 years), an optimized comprehensive billing scheme is designed. The continuous improvement of people's living standard and the quality of life have been changed dramatically, our travel mode has also changed dramatically, and we can clearly feel the pressure of resources as well as travel pressure[4]. Let's take an area in Shenyang as an example for analysis: with the continuous improvement of the living standard of Shenyang residents, the civilian car ownership in Shenyang reached 2.635 million by the end of 2020, among which, 2.334 million private cars were owned, and the per capita car ownership ranked the tenth in China. The growth of car ownership has brought great convenience to the transportation of citizens, but at the same time, it has also brought considerable pressure to road traffic and parking. Since 2018, Shenyang City has started to develop smart parking in order to alleviate the problems of "difficult parking" and "indiscriminate parking". The "smart parking" uses Internet technology to realize the functions of finding parking spaces, booking parking spaces, route navigation, shared parking, automatic timing, and sensorless parking. Research on Intelligent Parking Operation and Management Planning Based on Revenue Model Function Yidi Wang College of Mathematics, Liaoning Normal University, Dalian, Liaoning, 116029 China Yidi Wang Abstract: With the development of the world, people's living standard has also improved significantly. At present, self-driving has become one of the popular ways to travel, but this improvement in living standard has also brought some problems such as "difficult parking" and "indiscriminate parking" to the society. In order to solve the problem, our country has launched the decision to develop smart parking. In the case of Shenyang, starting from 2018, due to the substantial growth of civilian car ownership in Shenyang, in order to alleviate the problems of "difficult parking" and "indiscriminate parking", the relevant areas in Shenyang began to develop smart parking. The current smart parking service mainly adopts two billing methods: mobile video collection vehicle billing and manual billing, and this paper analyzes the route planning and possible benefits of the smart parking service by establishing an appropriate mathematical model to obtain the final optimized design scheme. Keywords: Smart parking; Mobile video capture vehicle billing; Manual billing; Python eywords: Smart parking; Mobile video capture vehicle billing; Manual billing; Python BCP Business & Management Volume 23 (2022) BCP Business & Management Volume 23 (2022) GEBM 2022 2.1.1 Model Building First of all, we make reasonable assumptions about the question: assume that the scanning interval of the scanning car is 30min, and since the question has divided the areas and car models we are about to process and analyze, we set the class I, class II and class III areas as A1, A2 and A3 respectively, and the small and medium-sized cars and large cars as x1 and x2, and the number of times the scanning car scans the same car is n. We assume that the scanning We assume that the scanning car can input the number of scans n and the parking lot parking class A and the corresponding car type directly into the Python code of our calculation, we execute different conditions of the if statement according to the different input information, and finally, according to the result of the sum of the accumulation, we can calculate a quantity of car in a parking lot charges. We input the function into Matlab to plot the solution. At the same time, for new energy vehicles we handle it differently, that is, we only need to divide by 2 on the basis of the final charge result, that is, the charge result of new energy vehicles. If the owner of the parking space chooses "monthly" parking, 600 RMB/month for the first class area, 400 RMB/month for the second class area, and 180 RMB/month for the third class area, the charge is fixed[8]. 1. Introduction At present, smart parking mainly uses the inventory of existing resources to provide parking services, parking billing methods are mainly mobile video collection car billing and manual billing two. For different road sections, we set different billing services, design the parking scheme as reasonably as possible, adopt the optimized billing 961 BCP Business & Management Volume 23 (2022) GEBM 2022 method, and improve the effectiveness and reliability of the smart parking management service is what we need to consider[5-7]. method, and improve the effectiveness and reliability of the smart parking management service is what we need to consider[5-7]. 2.2.1 Model Building Since the smart parking scheme firstly has different rates for different areas, the Shenyang Station- Taiyuan Street area (west to Shengli Street, east to Nanjing Street, north to North 5th Street (including the south side of North 5th Street) and south to South 8th Street) given in the title needs to be divided. On the basis of getting the area map, the specific discussion of each type of area is carried out. As different areas of parking spaces, vehicle parking hours will have different characteristics, more Since the smart parking scheme firstly has different rates for different areas, the Shenyang Station- Taiyuan Street area (west to Shengli Street, east to Nanjing Street, north to North 5th Street (including the south side of North 5th Street) and south to South 8th Street) given in the title needs to be divided. Since the smart parking scheme firstly has different rates for different areas, the Shenyang Station- Taiyuan Street area (west to Shengli Street, east to Nanjing Street, north to North 5th Street (including the south side of North 5th Street) and south to South 8th Street) given in the title needs to be divided. On the basis of getting the area map, the specific discussion of each type of area is carried out. As different areas of parking spaces, vehicle parking hours will have different characteristics, more typical are: near residential and work units, vehicles may be parked for a long time; near commercial areas, the vehicle parking hours in the middle; and near fast food restaurants and farmers markets[9], etc., may be parked for a short time. So the whole big area needs to be divided according to the Since the smart parking scheme firstly has different rates for different areas, the Shenyang Station- Taiyuan Street area (west to Shengli Street, east to Nanjing Street, north to North 5th Street (including the south side of North 5th Street) and south to South 8th Street) given in the title needs to be divided. On the basis of getting the area map, the specific discussion of each type of area is carried out. 2.1.2 Model solving For different areas, different vehicles and the number of times they are scanned, we analyze the gain function separately and compile it on Matlab software to obtain the corresponding gain function relationship, which is very intuitive and clear for solving the problem. Figure 1 shows the mathematical model of the gain function. Figure 1 Mathematical model of the revenue function 2.2 Mathematical model for planning the route of a mobile video capture vehicle 2 2 1 Model Building Figure 1 Mathematical model of the revenue function 2.2 Mathematical model for planning the route of a mobile video capture vehicle 2.2 Mathematical model for planning the route of a mobile video capture vehicle 2.2.1 Model Building 2.2.1 Model Building Planning of Class II area This type of area belongs to the on-street parking lot in the second class area of the peace zone, and the route of the parking space is also marked on the found second class area, and the route of the existing parking space is shown in Figure 3. Figure 2 Class I area parking space distribution map g 2. Planning of Class II area g 2. Planning of Class II area This type of area belongs to the on-street parking lot in the second class area of the peace zone, and the route of the parking space is also marked on the found second class area, and the route of the existing parking space is shown in Figure 3. This type of area belongs to the on-street parking lot in the second class area of the peace zone, and the route of the parking space is also marked on the found second class area, and the route of the existing parking space is shown in Figure 3. Figure 3 Class II area parking space distribution map l Figure 3 Class II area parking space distribution map 3. Planning of Class III areas Figure 3 Class II area parking space distribution map Figure 3 Class II area parking space distribution map III g 3. Planning of Class III areas This type of area belongs to the three types of on-street parking in the peace area, and the route of the parking space is marked on the found type of area to get the route of the existing parking space as Figure 4. Figure 4 Three types of regional parking space distribution map Figure 4 Three types of regional parking space distribution map 2.2.1 Model Building As different areas of parking spaces, vehicle parking hours will have different characteristics, more typical are: near residential and work units, vehicles may be parked for a long time; near commercial areas, the vehicle parking hours in the middle; and near fast food restaurants and farmers markets[9], etc., may be parked for a short time. So the whole big area needs to be divided according to the On the basis of getting the area map, the specific discussion of each type of area is carried out. As different areas of parking spaces, vehicle parking hours will have different characteristics, more typical are: near residential and work units, vehicles may be parked for a long time; near commercial areas, the vehicle parking hours in the middle; and near fast food restaurants and farmers markets[9], etc., may be parked for a short time. So the whole big area needs to be divided according to the On the basis of getting the area map, the specific discussion of each type of area is carried out. As different areas of parking spaces, vehicle parking hours will have different characteristics, more typical are: near residential and work units, vehicles may be parked for a long time; near commercial areas, the vehicle parking hours in the middle; and near fast food restaurants and farmers markets[9], etc., may be parked for a short time. So the whole big area needs to be divided according to the 962 BCP Business & Management Volume 23 (2022) GEBM 2022 characteristics, and discuss the characteristics you meet in each area separately, and on this basis the route planning of the video collection vehicle is carried out to give the required number of vehicles. characteristics, and discuss the characteristics you meet in each area separately, and on this basis the route planning of the video collection vehicle is carried out to give the required number of vehicles. 1 Planning of a class one area 1. Planning of a class one area The class area belongs to a class area in the peace area on-street parking, in the class area has been found on the parking space route labeling, to get the existing parking space route as Figure 2. Figure 2 Class I area parking space distribution map 2 Planning of Class II area Figure 2 Class I area parking space distribution map 2. 2.2.2 Model solving We assume that there are parking spaces along each street and the length of the parking spaces is assumed to be 6 m. After the division, we calculated the length of each path separately and formed a closed curve (for the scanning route of the scanning vehicle) for the relevant paths. We performed 963 BCP Business & Management Volume 23 (2022) GEBM 2022 the path planning separately for the scanning vehicles in different zones. The purpose of these steps is to find the solution that minimizes the number of scanning vehicles needed and maximizes the number of scans. the path planning separately for the scanning vehicles in different zones. The purpose of these steps is to find the solution that minimizes the number of scanning vehicles needed and maximizes the number of scans. Based on the total distance of the route we want to travel measured according to the figure, because the speed of the collection vehicle is certain, so we may assume that the speed of the collection vehicle is v m/s, so that the time of each section of the journey we can calculate, respectively, t1, t2, t3, ..., and then according to the type of different lots, the length of the parking time, set the relevant detection vehicle travel The interval time T1, T2, T3, and then compare the detection car in the road travel time and detection interval, if 0 < t1 < T1, send a detection car on; if T1 < t2 < 2T1, then send two detection cars in the corresponding interval to share the interval, and then the corresponding analogy g p g Figure 5 Class Ⅰ regional mobile video truck collection route Figure 6 Class II regional mobile video truck collection route Figure 7 Class III regional mobile video truck collection route Ultimately, according to the design of the route, taking into account the collection vehicle a class I area is recommended to require five collection vehicles, a class II area require collection vehicles, and a class III area requires three collection vehicles. 2.2.2 Model solving Figure 5 Class Ⅰ regional mobile video truck collection route Figure 5 Class Ⅰ regional mobile video truck collection route Figure 5 Class Ⅰ regional mobile video truck collection route Figure 5 Class Ⅰ regional mobile video truck collection route Figure 6 Class II regional mobile video truck collection route Figure 6 Class II regional mobile video truck collection route Figure 7 Class III regional mobile video truck collection route Ultimately, according to the design of the route, taking into account the collection vehicle travel, a class I area is recommended to require five collection vehicles, a class II area requires three collection vehicles, and a class III area requires three collection vehicles. 964 BCP Business & Management Volume 23 (2022) GEBM 2022 2.3 Mathematical modeling using two-dimensional scatter plots in the region We collect the relevant data about how much the number of traffic flow of parking spaces in three types of districts in Shenyang on the network, and by analyzing the data, we can design the relevant two-dimensional scatter plot through Matlab. Through the analysis of the two-dimensional scatter plot, we can know that the number of point traffic flow is positively related to the number of collected cars[10]. The scatter plot of the relationship between the change of traffic flow and the number of scanned cars is shown in Figure 8. Figure 8 Scatter plot of traffic flow variation versus the number of scanned vehicles Let's assume that Figure 8 Scatter plot of traffic flow variation versus the number of scanned vehicles Let's assume that Let us assume that the value of the change in the number of parkings at nine time points on a given day in a Class III area is set to the matrix A. 𝑎 𝑎 𝑎 Let us assume that the value of the change in the number of parkings at nine time points on a given day in a Class III area is set to the matrix A. 𝑎 𝑎 𝑎 𝐴ൌ൭ 𝑎ଵଵ 𝑎ଵଶ 𝑎ଵଷ 𝑎ଶଵ 𝑎ଶଶ 𝑎ଶଷ 𝑎ଷଵ 𝑎ଷଶ 𝑎ଷଷ ൱ (1) (1) ଷଵ ଷଶ ଷଷ Assuming that we have enough acquisition vehicles to scan, we set the 9 times interval matrix T of these working scanning vehicles. 𝑡 Assuming that we have enough acquisition vehicles to scan, we set the 9 times interval matrix T of these working scanning vehicles. 𝑡 𝑇ൌ൭ 𝑡ଵ 𝑡ଶ 𝑡ଷ ൱ (2) ൭ 𝑎ଵଵ 𝑎ଵଶ 𝑎ଵଷ 𝑎ଶଵ 𝑎ଶଶ 𝑎ଶଷ 𝑎ଷଵ 𝑎ଷଶ 𝑎ଷଷ ൱*൭ 𝑡ଵ 𝑡ଶ 𝑡ଷ ൱=𝐶 (3) (2) (3) where C matrix is the change in the number of vehicles during this day. where C matrix is the change in the number of vehicles during this day. If C<0, then we need to add more collection vehicles in that time period, if C>0, we don't need to increase the number of collection vehicles. 2.3.2 Model solving 2.3.2 Model solving On the basis of the known actual distance of the road section concerned, we looked up the information and concluded that it takes about 2-3 min for a normal person to walk 200 m. Therefore, for the actual length of the road section <600 m, manual billing is used. The road sections marked as 1, 2, 3, 14, 15,, 76, 79 use manual billing. The rest are billed using the video mobile car, so the route of the mobile video collection vehicle planned in question two should also be changed, and the re- planned route is shown in the following figure. The red line segment in Figure 9-11 represents the video capture vehicle billing method, and the blue line segment represents the manual billing method. 965 GEBM 2022 GEBM 2022 BCP Business & Management Volume 23 (2022) BCP Business & Management ( ) Figure 9 Roadmap for the redesign of a class I area Figure 10 Roadmap for the redesign of a class II area Figure 11 Roadmap for the redesign of a class III area Consideration of cost Figure 9 Roadmap for the redesign of a class I area Figure 9 Roadmap for the redesign of a class I area Figure 10 Roadmap for the redesign of a class II area Figure 10 Roadmap for the redesign of a class II area Figure 11 Roadmap for the redesign of a class III area Consideration of cost. Labor costs: mainly including the salaries and welfare costs of the acquisition personnel, the purchase and depreciation costs of the electronic equipment required by the acquisition personnel, and the office space for the acquisition personnel, etc. Vehicle costs: fixed loss of fuel, maintenance costs, and depreciation costs of mobile video acquisition vehicles, wages and welfare costs of personnel driving acquisition vehicles, etc. 4. Conclusion The current smart parking service mainly adopts two billing methods, mobile video capture vehicle billing and manual billing. In this paper, the route planning and possible gains of the smart parking service are analyzed by establishing an appropriate mathematical model, and the final optimized design scheme is obtained. First, for a parking space, the possible gains as well as losses from its detection are analyzed. Firstly, the gains are analyzed. Since the actual problem is divided into different time periods, different types of areas, and different car models, we use flowcharts for logical analysis, and then use python to code the different cases to calculate the possible gains, and also discuss the losses arising from the uncertainty of the detection time in the process. Secondly, the actual road length is mapped according to the characteristics of typical areas, and the number of parking spaces and the moving speed of mobile video acquisition vehicles are assumed, whereby the acquisition time and interval time of the acquisition vehicles are obtained, and then a reasonable 966 BCP Business & Management Volume 23 (2022) GEBM 2022 number of vehicles and acquisition routes are determined. Finally, re-planning of certain road sections, using manual billing method on the road sections with short distances and few parking spaces, and using mobile video collection vehicles on the road sections with long distances and denser parking spaces, and then re-routing the three areas and considering certain costs, designing a comprehensive billing scheme and giving the related gains and losses. number of vehicles and acquisition routes are determined. Finally, re-planning of certain road sections, using manual billing method on the road sections with short distances and few parking spaces, and using mobile video collection vehicles on the road sections with long distances and denser parking spaces, and then re-routing the three areas and considering certain costs, designing a comprehensive billing scheme and giving the related gains and losses. References [1] Yan Huijia, Research and design of laser distance measurement based smart parking management system, Laser Journal [J], Vol. 8, No. 40, 2019 [2] Lijun Tang, Modeling research of intelligent parking system, School of Mathematics and Computer Science, Ningxia University [3] Ma Snap Yun, Research on the interaction design of smart parking system in the context of smart city, Hebei University of Technology [4] Bai Qizheng, Mathematical Modeling Case Study [M], Ocean Press, 2000 [4] Bai Qizheng, Mathematical Modeling Case Study [M], Ocean Press, 2000 [5] Daoyuan Zhu, Selected Cases in Mathematical Modeling [M], Science Publishing House, 2003 [5] Daoyuan Zhu, Selected Cases in Mathematical Modeling [M], Science Publishing House, [6] Cai Lockzhang, Mathematical Modeling: Principles and Methods [M], Ocean Press, 2000 ] Li Junyu. City-level smart parking platform based on big data [J]. Internet of things, 2020, 10 [7] Li Junyu. City-level smart parking platform based on big data [J]. Internet of things, 2020, 10(1):83-87 [8] Fu Hui. Smart city construction starts from smart parking [J]. Strait Science and Industry, 2014, (7):86- 90 [8] Fu Hui. Smart city construction starts from smart parking [J]. Strait Science and Industry, 2014, (7):86- 90 [9] He Yang. A preliminary investigation of intelligent parking management application [J]. Reform and Innovation, 2021, 35(1) [10] Liu Na. Urban intelligent parking[J]. Urban governance and planning 2018, (12) [10] Liu Na. Urban intelligent parking[J]. Urban governance and planning 2018, (12) 967
https://openalex.org/W2605035492	https://europepmc.org/articles/pmc5461536?pdf=render	English	null	Protease-Inhibitor Interaction Predictions: Lessons on the Complexity of Protein–Protein Interactions	Molecular & cellular proteomics	2,017	cc-by	11,639	Protease-Inhibitor Interaction Predictions: Lessons on the Complexity of Protein–Protein Interactions□ S Fortelny‡§¶‡‡, Georgina S. Butler¶, Christopher M. Overall‡¶§§, P lidi §‡‡¶¶ Nikolaus Fortelny‡§¶‡‡, Georgina S. Butler¶, Christopher M. Overall‡¶§§, and Paul Pavlidis§‡‡¶¶ Protein interactions shape proteome function and thus biology. Identification of protein interactions is a major goal in molecular biology, but biochemical methods, al- though improving, remain limited in coverage and accu- racy. Whereas computational predictions can guide bio- chemical experiments, low validation rates of predictions remain a major limitation. Here, we investigated compu- tational methods in the prediction of a specific type of interaction, the inhibitory interactions between proteases and their inhibitors. Proteases generate thousands of pro- teoforms that dynamically shape the functional state of proteomes. Despite the important regulatory role of pro- teases, knowledge of their inhibitors remains largely in- complete with the vast majority of proteases lacking an annotated inhibitor. To link inhibitors to their target pro- teases on a large scale, we applied computational meth- ods to predict inhibitory interactions between proteases and their inhibitors based on complementary data, includ- ing coexpression, phylogenetic similarity, structural infor- mation, co-annotation, and colocalization, and also sur- veyed general protein interaction networks for potential inhibitory interactions. In testing nine predicted interac- tions biochemically, we validated the inhibition of kal- likrein 5 by serpin B12. Despite the use of a wide array of complementary data, we found a high false positive rate of computational predictions in biochemical follow-up. Based on a protease-specific definition of true negatives derived from the biochemical classification of proteases and inhibitors, we analyzed prediction accuracy of indi- vidual features, thereby we identified feature-specific lim- itations, which also affected general protein interaction prediction methods. Interestingly, proteases were often not coexpressed with most of their functional inhibitors, contrary to what is commonly assumed and extrapolated predominantly from cell culture experiments. Predictions of inhibitory interactions were indeed more challenging than predictions of nonproteolytic and noninhibitory inter- actions. In summary, we describe a novel and well-de- fined but difficult protein interaction prediction task and thereby highlight limitations of computational interaction prediction methods. Molecular & Cellular Proteomics 16: 10.1074/mcp.M116.065706, 1038–1051, 2017. Identification of protein interactions is an important goal in molecular biology yet one that remains difficult. Approaches such as yeast-2-hybrid, coimmunoprecipitation and newer experimental methods (1, 2) are highly productive and scal- able. However, limited accuracy from false positives and cov- erage that is context dependent remain problematic (3, 4). From the ‡Department of Biochemistry and Molecular Biology; §Michael Smith Laboratories; ¶Centre for Blood Research; Depart- ment of Oral Biological and Medical Sciences, Faculty of Dentistry; Department of Psychiatry, University of British Columbia, Vancou- ver, British Columbia, Canada Author’s Choice—Final version free via Creative Commons CC-BY license. Received November 21, 2016, and in revised form, March 24, 2017 Published April 6, 2017, MCP Papers in Press, DOI 10.1074/mcp.M116.065706 Author contributions: N.F., G.S.B., C.M.O., and P.P. designed the research; N.F. performed the research; N.F. contributed new reagents or analytic tools; N.F. analyzed data; and N.F., C.M.O., and P.P. wrote the paper. Research lsos Research lsos Research lsos Research Author’s Choice © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org 1 The abbreviations used are: TP, true positives; TN, true negatives; PPI, protein-protein interaction; MMP, matrix metalloproteinase; AUC, area under the curve; ROC, receiver operating characteristic; RPKM, reads per kilobase of transcript per million mapped reads; GO, Gene Ontology; EXP, Inferred from Experiment; IDA, Inferred from Direct Assay; IPI, Inferred from Physical Interaction; IGI, Inferred from Genetic Interaction; IMP, Inferred from Mutant Phenotype; IEP, In- ferred from Expression Pattern; TAS, Traceable Author Statement. Protease-Inhibitor Interaction Predictions: Lessons on the Complexity of Protein–Protein Interactions□ S Computational methods have been developed to predict protein–protein interactions, commonly linking together pro- teins on the basis of shared features such as patterns of conservation, expression, or annotations (5–8)—a version of guilt by association. A second class of approaches uses protein structural features to identify potential physical inter- action interfaces (9). These approaches can be combined. However, their practical utility remains unclear. In the meth- ods cited above, accuracy was estimated by cross-validation or by validating a small number of hand-picked test cases (5, 6). Estimates of the true efficacy of prediction methods in structured evaluations, such as those that exist for function prediction (critical assessment of protein function annotation algorithms (10)), structure prediction (critical assessment of protein structure prediction (11)), or for structural docking (critical assessment of prediction of interactions (12)), are lacking for protein interaction prediction methods. If compu- tational predictions of interactions were sufficiently accurate, biochemical assays could be targeted more efficiently by focusing on predicted pairs (9), but to date, computational predictions do not appear to have played a major role in interaction discovery or prioritization (13). We hypothesized that studying a specific subset of protein interactions and Molecular & Cellular Proteomics 16.6 1038 Bioinformatics Prediction of Protease–Inhibitor Interactions combining computational prediction and biochemical valida- tion will grant deeper insights into the pitfalls and state of the art for general protein interaction predictions. number of proteins. Protease inhibitors are therefore often secreted in the plasma or distal tissues to block proteases delivered by diffusion, secretion, or leakage from tissues to the circulation. Considering the key role of proteases in cell signaling pathways, identifying additional, physiologically rel- evant protease–inhibitor pairs would greatly benefit our un- derstanding of protease biology. We focused on the prediction of interactions between pro- tease inhibitors and proteases—a problem that has not re- ceived specific attention to our knowledge—despite being characterized by covalent or low-KD noncovalent interactions (low nM or pM) and hence, in principle, being more tractable for identification than high-KD noncovalent, general protein– protein interactions. Previous cell culture and transcript anal- yses have suggested that known protease–inhibitor pairs are often coexpressed and coregulated (14, 15). It is therefore hypothesized that protease–inhibitor coexpression plays a major role in the regulation of the detrimental activities of a protease. Inverse protease–inhibitor coexpression is thought to amplify protease activity but has only been observed for relatively few protease–inhibitor pairs (16, 17). Protease-Inhibitor Interaction Predictions: Lessons on the Complexity of Protein–Protein Interactions□ S Overall, it is currently a common assumption that protease–inhibitor co- expression is evidence for an inhibitory interaction, but this concept has not been tested comprehensively. Important questions in interaction prediction methods are which input data to use for predictions and how to evaluate performance (in contrast, the prediction algorithm used plays relatively little role (22)). To evaluate performance of a predic- tor, efficacy in separating predefined true positives (TP)1 and true negative (TN) examples is measured. For example, in interaction prediction, if most true interacting proteins are coexpressed and noninteractors are not coexpressed, then coexpression is a good predictor of interaction. The better the separation of the two groups, the better the predictive per- formance. In general, TPs are readily found in biological da- tabases, but the definition of TNs is a challenge, especially for weak interactions having low mM KDs, and more practically since a lack of interaction is rarely established and docu- mented. Common approaches therefore use unlikely interac- tions as TNs, for example, random interactions (based on the assumption that true interactions are a small subset of all possible interactions) or interactions between proteins local- ized to different cellular compartments according to annota- tion (4). An advantage of the protease–inhibitor prediction task is the ability to define TP and TN inhibitions more accurately. Protease inhibitors are characterized by tight interactions with their cognate proteases, thus providing a clear separation between true and false interactors. Further, proteases and their inhibitors are organized into families based on their pri- mary sequence and into clans based on the structure of their active site and reactive site, respectively (21). Families and clans of inhibitors can mostly be assigned specifically to one or two target protease classes. Thus, it is possible to define TN pairs, where the inhibitor cannot inhibit the protease based on known chemical and structural constraints. As examples, a serpin will not inhibit a metalloprotease, and a tissue inhibitor of metalloproteinases will neither inhibit a serine protease nor aspartate, threonine, or cysteine proteases. However, matrix metalloproteinases (MMPs) cleave and inactivate many ser- pins and so transiently are also interactors before peptide bond scission, albeit with a moderate KD ( Km) (18, 23). A further advantage of selecting this group of proteins in the analysis of prediction methods is the accuracy of biochemical concept has not been tested comprehensively. Molecular & Cellular Proteomics 16.6 Protease-Inhibitor Interaction Predictions: Lessons on the Complexity of Protein–Protein Interactions□ S Mappings were binarized into 0 (absent) and 1 (pres- ent) for the binary networks before calculating the fraction of agree- ment (where the genes are absent or present in both organisms), Pearson correlation (cor package in R (29)) or mutual information (entropy package in R) for all pairs of genes. The Bits network was constructed by multiplying InParanoid scores with the bit score for each cluster and the calculating Pearson correlation (cor package in R). Here, we defined TP inhibitions (n 294) as those inhibi- tions annotated in MEROPS (21). We defined TN inhibitions (n 6,990) as enzymatically implausible inhibitor/protease pairs that are known not to be inhibitory. Using this gold standard, we evaluated the predictive power of common interaction prediction methodology to predict protease– inhibitor pairs in the protease web. Predictions were based on protein–protein interaction data, coannotation, coexpression, phylogenetic similarity, and colocalization as input data. In- terestingly, we report that coexpression is surprisingly low for many functional protease–inhibitor pairs, contrary to what is commonly assumed. In particular, we employed 40 interac- tion predictors based on coexpression values derived from different input data and correlation metrics, all of which we found suffered from weak predictive power. Nonetheless, we predicted 270 protease–inhibitor pairs, examined 9 of these predicted inhibitions biochemically, and validated the novel inhibition of kallikrein 5 (KLK5) by serpin B12 (SERPINB12), previously an orphan inhibitor. Machine Learning—Machine learning algorithms were run using the caret package. 60% of pairs were used for training and 40% for testing. Parameters picked by cross-validation were mtry of 2 for random forest and C of 0.1 and sigma of 0.2 for the radial support vector machine. Biochemical Validation Experiments—Coagulation factor 11 (F11), coagulation factor 12 (F12), plasma kallikrein (KLKB1), and the chro- mogenic substrates for F11 (2366 Catalogue# S821090), F12 (Cata- logue# S820340), and for KLKB1 (S2302) were from DiaPharma. Chromogenic substrates were measured at an emission wavelength of 405 nm as recommended by the suppliers. KLK5 (Catalogue# 1108-S.E.-010) and its quenched fluorescent substrate (Catalogue# ES011) and kallikrein 5 (KLK7, Catalogue# 2624-S.E.-010) and its quenched fluorescent substrate (Catalogue# ES002) were from R&D Systems. Cleavage of quenched fluorescent substrates was meas- ured using excitation/emission wavelengths of 380/460 nm for KLK5 and 320/405 nm for KLK7 as recommended by the suppliers. SERPINB12 was kindly provided by Dr. G. A. Silverman (Children’s Hospital of Pittsburgh); serpin A4 was kindly provided by Dr. J. Protease-Inhibitor Interaction Predictions: Lessons on the Complexity of Protein–Protein Interactions□ S Proteases are a critical component of the posttranslational regulatory machinery in cells and therefore promising drug targets. However, drug targeting of proteases has been ham- pered by complex protease biology that is often poorly un- derstood. One aspect of this complexity is the organization of proteases in dense interaction networks of protease cleavage and interaction (18). Proteases regulate the activity of other proteases by direct cleavage or by cleaving their endogenous inhibitors, which in turn influences additional distal cleavage events. Thus, proteases can potentially indirectly influence the cleavage of substrates other than their direct substrates. We recently established a graph model of protease web interac- tions based on existing biochemical data that can be used to predict proteolytic pathways (19). However, the network is far from its full potential because cleavage and inhibition interac- tion data underlying the model are incomplete. This is mainly due to the lack of studies of proteases and inhibitors but also to the lack of uploading of existing data to community data- bases. Computational prediction could provide a means to accelerate the addition of interactions to this network. How- ever, large-scale computational prediction efforts in protease interaction biology have been limited to the use of molecular features of proteases and their substrates to predict protease cleavage (20) and have largely ignored protease inhibition. Therefore, the whole realm of protease inhibition is underex- plored, with 354 (80%) of 444 human proteases lacking annotated inhibitors and 13 (14%) of 94 inhibitors without any annotated targets (orphan inhibitors) in the MEROPS protease database (21). Proteases are regulated by multiple mechanisms other than inhibition such as autodegradation, reversible activation, substrate-induced activation, and other allosteric activators. However, protease inhibitors are often present in adjacent compartments to block and clear excess proteases that could rapidly and irreversibly cleave a large Molecular & Cellular Proteomics 16.6 1039 Bioinformatics Prediction of Protease–Inhibitor Interactions package in R. Full datasets or subsets were used as inputs as explained in the results section and in supplemental Table S5. testing of the predictions by measuring inhibition of the cat- alytic activity of the protease. Phylogenetic Profiling—Phylogenetic profile data were constructed by downloading mappings from human proteins to other species from InParanoid (32). EXPERIMENTAL PROCEDURES Proteases and Protease Inhibitor Data—Protease and protease inhibitor data and coexpression matrices used throughout the anal- yses are available for download at http://hdl.handle.net/11272/ 10472. Protease and inhibitor class, family, cleavage, and inhibitor information were extracted from the MEROPS database (http://mer- ops.sanger.ac.uk/) (21) version 9.9 on September 30, 2013. MEROPS identifiers were used to classify proteases and inhibitors into classes and families as described on the MEROPS website. Protein–Protein Interaction Networks—Protein-protein interaction (PPI) data from Human Integrated Protein-Protein Interaction Refer- ence (24) version 1.5 were downloaded on June 12, 2013. PPI data from high-throughput experiments were downloaded from BioGRID (25) on October 11, 2013. PPI data from (26) were downloaded on October 11, 2013. Experiments with up to 100 identified PPIs were considered low throughput, those with 100–1,000 PPIs were labeled medium throughput, and those with more than 1,000 PPIs were deemed high throughput. Protease-Inhibitor Interaction Predictions: Lessons on the Complexity of Protein–Protein Interactions□ S Chao (Medical University of South Carolina); murine serpin B8 was from Sino Biological, Inc. (Catalogue# 50215-M08B); and serpin B7 was from Creative BioMart (Catalogue# SERPINB7–2596H). Protease ac- tivity was measured after incubation for 1 h at 37 °C with and without serpins in a POLARstar OPTIMA plate reader (BMG Labtech). Sub- strate cleavage and protease inhibition assays were also analyzed by 10% SDS-PAGE and silver staining of proteins after incubation at a 1:1 ratio protease:inhibitor (w/w) for 1 h at 37 °C. RESULTS Our results are organized around a presentation of our investigation of the predictive power of each of several data types we considered. We then describe a combined predic- tion approach that attempts to improve predictive power, biochemical validation of selected predictions, and finally a more in-depth investigation of coexpression patterns of pro- teases and inhibitors. Protein Localization—Protein localization information was down- loaded from three sources: LocDB (27) (data downloaded November 19, 2013), the Human Protein Atlas (28) (downloaded November 12, 2013.), and Gene Ontology (GO) annotation using the hgu95av2.db package in R (29) (downloaded August 8, 2013). For each dataset, annotations were mapped to GO terms and annotation trees for each protein were generated using the GOstats package in R (29). For LocDB, primary and secondary localization information was com- bined for each protein. Main and other localization data from the Human Protein Atlas were used if the reliability was annotated as High, Medium, or Supportive. GO annotations were retained if the evidence code was one of EXP, IDA, IPI, IGI, IMP, IEP, or TAS. PPIs—Our goal in considering general PPI data was to identify any relevant interactions that are not included in MER- OPS. We analyzed PPI networks of proteases and their inhib- itors from the databases HIPPIE (24) (supplemental Table S1) and BioGRID (25) (supplemental Table S2), and a literature- curated PPI network (26) (supplemental Table S3). Comparing 559 annotated cleavage and 325 inhibition interactions be- tween proteases (including inactive proteases) and inhibitors from MEROPS to PPI data from HIPPIE (24), we found a PPI between protease and substrate for 187 known cleavages (33%) and between inhibitor and protease for 88 known inhi- bitions (27%). Figs. 1 and S1A show interactions annotated in Coexpression Networks—Genome Tissue Expression Atlas (GTEx) data (30) were downloaded on January 31, 2013. Gene Expression Omnibus Series 7307 expression data were downloaded from the database Gemma (31) on June 26, 2013. Other microarray-based expression datasets used in meta-coexpression analysis were down- loaded from Gemma on January 18, 2013 and are listed in supple- mental Table S6. Gene correlation was calculated using the cor function in R (29). Partial correlation was calculated using the ppcor Molecular & Cellular Proteomics 16.6 1040 Bioinformatics Prediction of Protease–Inhibitor Interactions FIG. 1. Protein–protein interaction (PPI) networks of proteases and inhibitors. PPI network of proteases and inhibitors based on the HIPPIE database with a HIPPIE score cutoff of 0.7. RESULTS Nodes are colored according to their MEROPS class (proteases: green—threonine; blue—metallo; yellow—serine; orange—cysteine; purple—aspartic; inhibitors: red). Red edges are enzymatic interactions. Thickness of edges corresponds to the HIPPIE score of the interaction. Bioinformatics Prediction of Protease–Inhibitor Interactions FIG. 1. Protein–protein interaction (PPI) networks of proteases and inhibitors. PPI network of proteases and inhibitors based on the HIPPIE database with a HIPPIE score cutoff of 0.7. Nodes are colored according to their MEROPS class (proteases: green—threonine; blue—metallo; yellow—serine; orange—cysteine; purple—aspartic; inhibitors: red). Red edges are enzymatic interactions. Thickness of edges corresponds to the HIPPIE score of the interaction. FIG. 1. Protein–protein interaction (PPI) networks of proteases and inhibitors. PPI network of proteases and inhibitors based on the HIPPIE database with a HIPPIE score cutoff of 0.7. Nodes are colored according to their MEROPS class (proteases: green—threonine; blue—metallo; yellow—serine; orange—cysteine; purple—aspartic; inhibitors: red). Red edges are enzymatic interactions. Thickness of edges corresponds to the HIPPIE score of the interaction. HIPPIE between well-defined groups of proteases and inhib- itors such as the proteasome, cathepsins, serum serine pro- teases, MMPs, and deubiquitin hydrolases. Confirming our earlier findings, (18) the connectivity in this network is heavily influenced by protease inhibitors (supplemental Fig. S1B). in MEROPS and examined all the original publications that served as references for these interactions in HIPPIE (supple- mental Table S4). In 28 cases (29%), an inhibition of protease activity was observed in the original paper; in 20 (21%), an inhibition was inferred from complex formation in the source publication; and 14 (15%) interactions were based on a cleav- age event of the inhibitor. Taken together, 62 (65%) of the 96 interactions were known protease web interactions that were simply not annotated in MEROPS, confirming the incomplete status of protease and protease inhibitor annotations reported We investigated the possibility that novel protease inhibi- tors of proteases might be hidden in the high-throughput PPI screens, which identify thousands of interactions but often without functional follow up. We collected 96 protease– inhibitor PPIs not already annotated as inhibitory interactions Molecular & Cellular Proteomics 16.6 1041 Bioinformatics Prediction of Protease–Inhibitor Interactions previously (18). Of the remaining 34 interactions, 18 (19% of total) reflected a PPI not related to inhibition or cleavage, 3 (3%) were unclear interactions, and 13 (14%) PPIs were phys- ical interactions between proteases and inhibitors with no mention of inhibitory activity and therefore potentially inter- esting novel inhibitions (Supplementary Results). Thus, PPI data not only reflect known protease interactions but also are potentially useful to predict novel inhibitory interactions. To identify additional inhibitory interactions, we further ana- lyzed a BioGRID-derived (25) (supplemental Table S2, Fig. Predictive power thus needs to be assessed sepa- rately for each matrix. We investigated the possibility of exploiting gene expres- sion patterns to predict protease–inhibitor interactions. It has been observed in cell culture that expression of a protease inhibitor positively correlates with its target protease, which is suggested to counterbalance the cleavage potential of the protease (14, 15) or negatively correlates to facilitate proteol- ysis (16, 17). Such correlated expression (coexpression) is promising as a prediction tool because RNA expression is generally measured for all genes simultaneously, and it is thus less biased than e.g. protein interaction data (22). We calcu- lated correlation values for all protease–inhibitor pairs (a co- expression matrix) and compared these to our gold standard of TP and TN inhibitory interactions derived from MEROPS. We compared the ability of coexpression matrices to pre- dict protease inhibition (inhibitory protease–inhibitor pairs). We measured the area under the curve (AUC) of the receiver- operator characteristic for separating predefined TP pairs (an- notated) from TN protease–inhibitor pairs (enzymatically im- plausible) using a given coexpression matrix. Fig. 5A shows that almost all matrices had some predictive value (better than random picking with AUC 0.5). TP pairs thus had higher coexpression than TN pairs on average. However, consider- ing the common perception that protease inhibitors are co- expressed with their target protease, this signal was surpris- ingly low (AUCs 0.7). One explanation for this discrepancy might be that RNA levels do not correspond to protein levels, and thus proteins can be coexpressed whereas their mRNAs are not. We tested this possibility by creating a coexpression network based on proteomics quantification data in the Hu- man Proteome Map (36), but this performed worse than the RNA networks (AUC of 0.6, data not shown). This poor per- formance might be due to noise in protein quantification or the small sample size of Human Proteome Map compared with GTEx. It thus remains unclear if protein data could be more informative than mRNA data. We aimed to capture the variety of possible coexpression patterns observed (Fig. 2). Therefore, we generated 40 coex- pression matrices based on different data and correlation methods to (summarized in supplemental Table S5). As dem- onstrated in supplemental Fig. S4, Pearson correlation (which is strongly influenced by samples with small values or zeros), captures tissue-specific expression patterns whereas Spear- man correlation requires correlation across all tissues. S1C) and a literature-curated PPI network (26) (supplemen- tal Table S3, Fig. S1D). However, these networks were subject to study biases as described previously (33) and did not yield interesting predictions (Supplementary Results). These results motivated us to consider additional data types for inhibitory interaction prediction. proteins (GTEX_All_Max). We then generated coexpression matrices (Pearson, Spearman, and maximum of both) using partial correlation, which might help resolve complex correla- tion patterns between multiple variables (genes). To capture tissue-specific correlation, we generated coexpression matri- ces in subsets of GTEx limited to only one tissue and one matrix representing the average of the tissue-specific matri- ces (each both for Pearson and Spearman correlation). For comparison, we also measured Pearson and Spearman cor- relation coefficients in a large dataset based on mRNA mi- croarrays (GEO-ID: GSE7307, 677 samples from over 100 tissues, supplemental Fig. S5B). Finally, with the aim of de- riving a more robust coexpression result, we performed a meta-analysis (34, 35) of gene expression over multiple mi- croarray datasets (listed in supplemental Table S6 and shown in Fig. S5C) across all tissues and in a tissue-specific manner in two ways: (i) datasets were merged into one large dataset (Merged) and (ii) Pearson correlation coefficients were ob- tained from each dataset and then averaged for each gene pair (Averaged (22)). Coexpression Patterns—We explored tissue expression profiles of proteases and inhibitors to seek useful patterns of coexpression, primarily in the GTEx (30) due to its high cov- erage (RNA-Seq data for 26 different tissues distributed over 1,660 samples). Expression patterns (Fig. 2) distinguish tis- sue-specific genes and housekeeping genes expressed in most or all tissues. For example, serpins either have a broad expression pattern across tissues (e.g. serpins E1, F1, and G1) or are specific to one or two tissues (e.g. serpins A1, C1, and D1), matching with known targets such as coagulation proteins and kallikreins (Fig. 2). Further examples are shown in supplemental Figs. S2 and S3. The resulting coexpression matrices differed strongly in content depending on the methods and data used (Fig. 3, Supplementary Results). Coexpression values between some matrices were highly correlated overall (matrices GTEX_All_ Pcc and GTEX_All_Scc with r 0.67) as shown in Fig. 4, indicating similarity between methods. Yet, if predicting inter- acting pairs by applying a coexpression cutoff (blue lines in Fig. 4), these predictions resulted in a small overlap (top right corner). We therefore generated coexpression matrices using both meas- ures on the entire GTEx dataset (supplemental Fig. S5A). In addition, to simultaneously capture both patterns of coex- pression, we generated a matrix using the maximum of Pear- son and Spearman correlation coefficients for each pairs of Similar results were found when measuring accuracy of interaction prediction within the top 10% of coexpressed Molecular & Cellular Proteomics 16.6 1042 Bioinformatics Prediction of Protease–Inhibitor Interactions FIG. 2. Expression patterns. Tissue RNA expression levels of groups of proteases and their inhibitors showing tissue-specific and broad expression patterns as well as correlation of expression patterns between serpins and serine proteases. Log10 transformed reads per kilobase of transcript per million mapped reads (log10(RPKM)) as obtained from GTEx (30) shown on the left. Zero values were set to 0.01 before log10 transformation. Normalized RPKMs for each gene are shown on the right and plotted as standard deviation from the mean (SD). Values were averaged across samples of each tissue. FIG. 2. Expression patterns. Tissue RNA expression levels of groups of proteases and their inhibitors showing tissue-specific and broad expression patterns as well as correlation of expression patterns between serpins and serine proteases. Log10 transformed reads per kilobase of transcript per million mapped reads (log10(RPKM)) as obtained from GTEx (30) shown on the left. Zero values were set to 0.01 before log10 transformation. Normalized RPKMs for each gene are shown on the right and plotted as standard deviation from the mean (SD). Values were averaged across samples of each tissue. FIG. 2. Expression patterns. Tissue RNA expression levels of groups of proteases and their inhibitors showing tissue-specific and broad expression patterns as well as correlation of expression patterns between serpins and serine proteases. Log10 transformed reads per kilobase of transcript per million mapped reads (log10(RPKM)) as obtained from GTEx (30) shown on the left. Zero values were set to 0.01 before log10 transformation. Normalized RPKMs for each gene are shown on the right and plotted as standard deviation from the mean (SD). Values were averaged across samples of each tissue. Molecular & Cellular Proteomics 16.6 1043 Bioinformatics Prediction of Protease–Inhibitor Interactions FIG. 3. Coexpression and phylogenetic similarity matrices. Heatmap of correlations values (agreement) show similarity and dissimilarity of matrices described in Supplementary results and Table S5. For two matrices, the Pearson correlation coefficient across all coexpression values in both matrices is shown. FIG. 3. Coexpression and phylogenetic similarity matrices. Heatmap of correlations values (agreement) show similarity and dissimilarity of matrices described in Supplementary results and Table S5. For two matrices, the Pearson correlation coefficient across all coexpression values in both matrices is shown. ize to other genes. The high AUC and accuracy of GTEX_ All_Max demonstrated the usefulness this network, which combined tissue specific and across-tissue coexpression, making it the better candidate for prediction. pairs of each matrix (Fig. 5B). Here, Array_Averaged_Liver and GTEX_All_Max methodologies had the highest signal. The strong correlation of Array_Averaged_Liver with protease web data was expected because many interactions are known between proteases and inhibitors that are expressed in liver as part of the well-understood complement and coagulation systems. However, this network is therefore biased to a sub- set of the protease web and performance would not general- pairs of each matrix (Fig. 5B). Here, Array_Averaged_Liver and GTEX_All_Max methodologies had the highest signal. The strong correlation of Array_Averaged_Liver with protease web data was expected because many interactions are known between proteases and inhibitors that are expressed in liver as part of the well-understood complement and coagulation systems. However, this network is therefore biased to a sub- set of the protease web and performance would not general- Phylogenetic Profiles—Phylogenetic similarity of two genes is a measure for their co-occurrence across a range of taxa and is reported to reflect functional relations (37). We created similarity matrices of phylogenetic profiles of proteases and Molecular & Cellular Proteomics 16.6 1044 Bioinformatics Prediction of Protease–Inhibitor Interactions FIG. 4. Effect of threshold on predicted pairs in two corre- lated matrices. Binned scatterplot of GTEX_All_Pcc (Pearson) and GTEX_All_Scc (Spearman) coexpression values for all pairs of pro- teins. Counts are number of pairs in each bin. Blue lines indicate a threshold of the mean plus two standard deviations. The overlap in predicted pairs above threshold (top right corner) is small despite the high correlation of these matrices. comention as predictive features but dismissed both on the- oretical and practical grounds. As we observed above for PPI data, utilizing this approach it is hard to distinguish between results that are de novo predictions, where novel interactions or functions are predicted, and mere retrieval of information already present in the literature but not yet annotated to databases (13). A related serious difficulty with this approach is that annotation is strongly biased by patterns of publication and gene annotation (40). Furthermore, if there were GO an- notations linking a protease–inhibitor pair or in the literature, the interaction would likely already have been characterized biochemically. Estimation of prediction performance based on coannotation would thus appear overoptimistic. Predicted pairs would represent examples of information retrieval and not de novo predicted pairs. Because of the bias in annota- tion, poorly characterized proteins would likely never be pre- dicted to be associated. Furthermore, many proteases and inhibitors are functionally related in cascades or biological processes, without having physical interactions, while here we were only interested in direct interactions, especially be- tween previously unconnected cascades. Overall, comention and coannotation lack the coverage and detail required to make predictions about particular novel candidate pairs, and so we disregarded this feature. FIG. 4. Effect of threshold on predicted pairs in two corre- lated matrices. Binned scatterplot of GTEX_All_Pcc (Pearson) and GTEX_All_Scc (Spearman) coexpression values for all pairs of pro- teins. Counts are number of pairs in each bin. Blue lines indicate a threshold of the mean plus two standard deviations. The overlap in predicted pairs above threshold (top right corner) is small despite the high correlation of these matrices. inhibitors from the InParanoid (32) database using correlation of sequence similarity scores as well as correlation and mu- tual independence measures of binary presence/absence val- ues of proteases across 162 species represented in the da- tabase (supplemental Table S5). The resulting matrices were very similar to each other (correlation 0.7–0.98), but dissimilar from the coexpression matrices (Fig. 3), indicating that this data source could be complementary to coexpression. Pre- diction performance of phylogenetic similarity matrices was generally comparable to coexpression matrices but weaker than GTEX_All_Max in the top pairs (Fig. 5B). inhibitors from the InParanoid (32) database using correlation of sequence similarity scores as well as correlation and mu- tual independence measures of binary presence/absence val- ues of proteases across 162 species represented in the da- tabase (supplemental Table S5). The resulting matrices were very similar to each other (correlation 0.7–0.98), but dissimilar from the coexpression matrices (Fig. 3), indicating that this data source could be complementary to coexpression. Pre- diction performance of phylogenetic similarity matrices was generally comparable to coexpression matrices but weaker than GTEX_All_Max in the top pairs (Fig. 5B). Predicting Novel Inhibitions—In the face of poor prediction performance of the individual input data types assayed above, we hypothesized that a combination of matrices would im- prove prediction. However, we did not observe improvement when combining the different prediction matrices in machine learning classifiers (Supplementary Results, Fig. S6). There- fore, we focused on the best performing individual matrix for prediction, GTEX_Max, selecting a coexpression threshold of 0.6. This threshold enriching TP 5.5-fold in comparison to TN as shown by TP to TN ratio (TP:TN, Table II). We attempted to combine coexpression with colocalization. Colocalization of protease–inhibitor pairs further enriched TPs threefold and antilocalization enriched results 2.4-fold. Colocalization—We next evaluated subcellular colocaliza- tion, which might enrich for interacting proteins. We obtained localization annotations LocDB (27), the Human Protein Atlas (28), and GO (38). We defined four groups of localization: (i) CO, colocalized for proteins sharing localization annotation; (ii) AT, antilocalized where one protein was extracellular and the other was in the cytosol or where one protein was in an organelle and the other in the cytosol (so that they meet upon cellular stimuli); (iii) NC, not colocalized where neither of the above was the case; and (iv) NA, not annotated, where sub- cellular localization was not annotated for one or both of the proteins. The use of colocalization enriched TP interactions considerably (Table I). However, colocalization also reduced the number of remaining pairs (the search space) significantly, mostly because of lack of annotation (NA). Using colocaliza- tion for novel predictions might increase specificity but sub- stantially reduce sensitivity. Nonetheless, colocalization information was missing for many proteins, thus limiting and biasing predictions. We therefore also included pairs where no localization information was available for one of the proteins. Finally, we removed all enzymatically implausible pairs, retaining only those where (i) it is known that the inhibitor blocks a protease from the same family as the predicted target protease or (ii) it is known that inhibition of the protease occurs by an inhibitor from the same family as the predicted cognate inhibitor. These two filters reduced the search space from 1,239 coexpressed pairs to 270 pairs. We anticipated that the incorporation of enzymatic constraint would greatly increase the precision of predictions. TABLE I TABLE I tion of factor XI (F11), factor XII (F12), and KLKB1 by kallistatin (SERPINA4) (three pairs) as well as inhibition of KLK5 and kallikrein 7 (KLK7) by serpins B7, B8, and B12 (six pairs). It is important to note that we evaluated predictions without biases resulting from selecting the most likely pairs. Instead, we selected pairs of biologically interest and availability of reagents. GTEx showed high expression of kallistatin in liver together with the coagulation proteases factor XI, factor XII, and plasma kallikrein. Whereas this indicated an interesting new role for kallistatin, the predictions were indeed risky since all of the four proteins are well studied and newly discovered inhibition is therefore unlikely. Moreover, liver is known to express and secrete many serum proteases and inhibitors, and so there was additional likelihood that these predicted pairs would not be true. On the other hand, ser- pins B7, B8, and B12 are little studied inhibitors and thus biologically interesting interaction partners of KLK5 and KLK7. TABLE I TP, TN, and remaining inhibitor–protease pairs after applying colocal- ization filters TN TP TP:TN Remaining pairs All 6,990 294 4.21% 32,368 Colocalized 430 65 15.12% 1,747 Antilocalized 466 30 6.44% 1,757 Information missing 5,338 159 2.98% 25,135 Remaining 756 40 5.29% 3,729 TABLE II TP, TN, and remaining inhibitor–protease pairs after applying colocal- ization and coexpression filters TP TN TP:TN Remaining pairs Total 294 6,990 1:24 32,368 Coexpressed (R 0.6) 46 205 1:4.5 1,237 Coexpressed and colocalized 10 18 1:1.8 112 Coexpressed and antilocalized 6 14 1:2.3 73 TP, TN, and remaining inhibitor–protease pairs after applying colocal- ization filters TABLE II TABLE II TP, TN, and remaining inhibitor–protease pairs after applying colocal- ization and coexpression filters TABLE II TP, TN, and remaining inhibitor–protease pairs after applying colocal- ization and coexpression filters TP TN TP:TN Remaining pairs Total 294 6,990 1:24 32,368 Coexpressed (R 0.6) 46 205 1:4.5 1,237 Coexpressed and colocalized 10 18 1:1.8 112 Coexpressed and antilocalized 6 14 1:2.3 73 We tested all pairs by fluorescent substrate cleavage as- says in vitro. We found no new targets for kallistatin among the three serum proteases but did observe inhibition of KLK5 by SERPINB12 (Fig. 6A), which we confirmed by analysis of covalent complex formation on SDS-polyacrylamide gels (Fig. 6B). Given that these proteins are coexpressed and therefore Inhibitor–protease pairs meeting these criteria are shown in supplemental Fig. A loss of sensitivity is possible if all target protease families of an inhibitor or all inhibitor family members of a target protease are not annotated as such, but we considered this unlikely since relevant inhibitor families are known for most proteases. Coannotation and Comentioning in the Literature—Interact- ing genes often participate in similar cellular functions, and so it is possible to predict gene interactions based on their annotation patterns (39). We considered coannotation and Molecular & Cellular Proteomics 16.6 1045 Bioinformatics Prediction of Protease–Inhibitor Interactions FIG. 5. Performance of coexpression and phylogenetic similarity matrices in predicting protease inhibition. TPs are inhibitions (n 218); TNs are specific inhibitor/protease pairs, where inhibition is enzymatically implausible. TNs were subsampled to reflect the number of TPs (n 218, 200 times) to avoid effects resulting from an unbalanced gold standard. AUC values obtained from each sample are represented as boxplots. (A) AUC of the receiver/operator curve. (B) Accuracy of prediction (percentage of correct classifications), when predicting interactions as the top 10% of pairs of each matrix. FIG. 5. Performance of coexpression and phylogenetic similarity matrices in predicting protease inhibition. TPs are inhibitions (n 218); TNs are specific inhibitor/protease pairs, where inhibition is enzymatically implausible. TNs were subsampled to reflect the number of TPs (n 218, 200 times) to avoid effects resulting from an unbalanced gold standard. AUC values obtained from each sample are represented as boxplots. (A) AUC of the receiver/operator curve. (B) Accuracy of prediction (percentage of correct classifications), when predicting interactions as the top 10% of pairs of each matrix. FIG. 5. Performance of coexpression and phylogenetic similarity matrices in predicting protease inhibition. TPs are inhibitions (n 218); TNs are specific inhibitor/protease pairs, where inhibition is enzymatically implausible. TNs were subsampled to reflect the number of TPs (n 218, 200 times) to avoid effects resulting from an unbalanced gold standard. AUC values obtained from each sample are represented as boxplots. (A) AUC of the receiver/operator curve. (B) Accuracy of prediction (percentage of correct classifications), when predicting interactions as the top 10% of pairs of each matrix. TABLE I Thus, the lower molecular weight forms of the serpin–KLK5 covalent complex likely represent cleavage of N or C peptides of the serpin in addition to the inhibitory cleavage in the reactive center loop. Further autocleavage of KLK5 results in lower molecular weight forms of a still active protease, which may also form lower molecular weight inhibitory complexes. FIG. 6. Inhibition of KLK5 by SERPINB12 (A) Cleavage of the quenched fluorescent substrate ES011 by KLK5 was followed over time after preincubation with different mole ratios of SERPINB12 (as indicated). A decrease in KLK5 activity (A.U.—arbitrary units) with increasing SERPINB12 confirms that SERPINB12 inhibits KLK5 as predicted. (B) Silver-stained 10% SDS-PAGE gel of KLK5, SERPINB12, and the inhibitory KLK5:SERPINB12 complex, indicating that the serpin is covalently bound to the protease. Serpins form metastable kinetically trapped folding states that are crucial for the inhibitory mechanism. Thus, serpins can occur in multiple folded states, some of which expose the reactive center loop as bait for protease cleavage. The presence of other folded states also indicates that N or C-terminal loops and strands may be cleaved by KLK5 depending on the conformations of the serpin present, which may shield or expose other potential cleavage sites. Thus, the lower molecular weight forms of the serpin–KLK5 covalent complex likely represent cleavage of N or C peptides of the serpin in addition to the inhibitory cleavage in the reactive center loop. Further autocleavage of KLK5 results in lower molecular weight forms of a still active protease, which may also form lower molecular weight inhibitory complexes. pression analysis (Fig. 7A). Yet, these interactions are bio- chemically meaningful, as serpins are exported from the liver to the serum and thus transported through the body, where they encounter and inhibit proteases with uncorrelated ex- pression patterns. Thus, as we showed before, coexpression in exocrine tissues is limited in predicting such protein interactions. found in the same tissue, we conclude that this interaction is also likely to be physiologically relevant. The interaction could also represent an interesting drug target since KLK5 is a major regulator in a number of diseases (41). Limited Coexpression of Proteases and Their Inhibitors— We were intrigued by the low coexpression of protease– inhibitor pairs (Fig. 5) and the limited success in validating predicted coexpression pairs. TABLE I To analyze differences in pre- dictions of our prediction matrices, we compared the true inhibitions that are retrieved when collecting only the most coexpressed protease–inhibitor pairs (top 10%) from each matrix. This analysis confirmed that GTEX_All_Max captured most pairs predicted by other coexpression matrices and was thus the better choice for prediction (supplemental Fig. S8, Supplementary Results). We also tested whether a more com- prehensive dataset would improve predictions by basing our predictions using updated versions of GTEx (version 4 instead of 3 with 2,921 instead of 1,660 samples) and MEROPS (version 11.0) but did not see any improvement (supplemental Figs. S9 and S10). To further understand the low correlation between coexpression and the protease inhibition, we inves- tigated coexpression of exemplary, well-studied inhibitor– protease pairs. First, we examined serpins that regulate coagulation (e.g. antithrombin III (SERPINC1) and alpha- 2-antiplasmin (SERPINF2)). Indeed, these serpins were co- expressed with coagulation proteases in liver, which, as noted above, is the case for many serum proteins destined for secretion to the circulation. However, these serpins are anno- tated to inhibit many additional proteases. For example, SERPINF2 inhibits KLK 4, 5, 7, 13, and 14 that are not highly expressed in liver, have uncorrelated expression patterns from SERPINF2 (Fig. 2) and are thus not retrieved by coex- These observations were not limited to serpins but applied to all groups of inhibitors: For example, TIMP 2 (Fig. 7B) was not coexpressed with any of its annotated target proteases with a few exceptions (MMP2 or MMP14), which were drowned out by the number of other annotated targets. In the extreme case, alpha-2-macroglobulin (Fig. 7C), a highly mul- tifunctional protease inhibitor that inhibits multiple classes of proteases, is also only coexpressed with a small portion of proteases. However, alpha-2-macroglobulin is present in blood plasma and so can reach most tissues (especially in inflammation were blood vessel permeability is increased) (23) and inhibit extracellular proteases or intracellular proteases that are secreted (e.g. by neutrophils (e.g. serine proteases and MMPs)) or released from damaged cells. These examples show that expression patterns of inhibitors are often corre- lated with the expression pattern of some (possibly the most relevant) targets but uncorrelated with additional biologically relevant targets. Protease–Inhibitor Predictions Comparison to General Pro- tein Interaction Predictions—We compared the performance of our coexpression matrices in predicting protease–inhibitor interactions to the performance achieved when predicting general protein interactions in the HIPPIE network (Fig. TABLE I S7 and listed in supplemental Table S7. To evaluate the predicted protease–inhibitor pairs, we se- lected a number of pairs for biochemical experiments: inhibi- Molecular & Cellular Proteomics 16.6 1046 Bioinformatics Prediction of Protease–Inhibitor Interactions FIG. 6. Inhibition of KLK5 by SERPINB12 (A) Cleavage of the quenched fluorescent substrate ES011 by KLK5 was followed over time after preincubation with different mole ratios of SERPINB12 (as indicated). A decrease in KLK5 activity (A.U.—arbitrary units) with increasing SERPINB12 confirms that SERPINB12 inhibits KLK5 as predicted. (B) Silver-stained 10% SDS-PAGE gel of KLK5, SERPINB12, and the inhibitory KLK5:SERPINB12 complex, indicating that the serpin is covalently bound to the protease. Serpins form metastable kinetically trapped folding states that are crucial for the inhibitory mechanism. Thus, serpins can occur in multiple folded states, some of which expose the reactive center loop as bait for protease cleavage. The presence of other folded states also indicates that N or C-terminal loops and strands may be cleaved by KLK5 depending on the conformations of the serpin present, which may shield or expose other potential cleavage sites. Thus, the lower molecular weight forms of the serpin–KLK5 covalent complex likely represent cleavage of N or C peptides of the serpin in addition to the inhibitory cleavage in the reactive center loop. Further autocleavage of KLK5 results in lower molecular weight forms of a still active protease, which may also form lower molecular weight inhibitory complexes. FIG. 6. Inhibition of KLK5 by SERPINB12 (A) Cleavage of the quenched fluorescent substrate ES011 by KLK5 was followed over time after preincubation with different mole ratios of SERPINB12 (as indicated). A decrease in KLK5 activity (A.U.—arbitrary units) with increasing SERPINB12 confirms that SERPINB12 inhibits KLK5 as predicted. (B) Silver-stained 10% SDS-PAGE gel of KLK5, SERPINB12, and the inhibitory KLK5:SERPINB12 complex, indicating that the serpin is covalently bound to the protease. Serpins form metastable kinetically trapped folding states that are crucial for the inhibitory mechanism. Thus, serpins can occur in multiple folded states, some of which expose the reactive center loop as bait for protease cleavage. The presence of other folded states also indicates that N or C-terminal loops and strands may be cleaved by KLK5 depending on the conformations of the serpin present, which may shield or expose other potential cleavage sites. TABLE I 1), using interactions annotated in HIPPIE as TPs and random interactions as TNs. Prediction of protein interactions showed Molecular & Cellular Proteomics 16.6 1047 Bioinformatics Prediction of Protease–Inhibitor Interactions FIG. 8. Prediction of PPI and predictions of protease inhibition. Area under the receiver-operator curve for predictions of protease inhibitions (Protease Web), protein–protein interactions from HIPPIE (PPI, Fig. 1), and interactions of proteasome components in HIPPIE (Proteasome, Fig. 1). Predictions are based on the coexpression matrices GTEX_All_Pcc (Pearson), GTEX_All_Scc (Spearman), and GTEX_All_Max (Max). FIG. 8. Prediction of PPI and predictions of protease inhibition Area under the receiver operator curve for predictions of protease FIG. 8. Prediction of PPI and predictions of protease inhibition. Area under the receiver-operator curve for predictions of protease inhibitions (Protease Web), protein–protein interactions from HIPPIE (PPI, Fig. 1), and interactions of proteasome components in HIPPIE (Proteasome, Fig. 1). Predictions are based on the coexpression matrices GTEX_All_Pcc (Pearson), GTEX_All_Scc (Spearman), and GTEX_All_Max (Max). FIG. 8. Prediction of PPI and predictions of protease inhibition. FIG. 8. Prediction of PPI and predictions of protease inhibition. Area under the receiver-operator curve for predictions of protease inhibitions (Protease Web), protein–protein interactions from HIPPIE (PPI, Fig. 1), and interactions of proteasome components in HIPPIE (Proteasome, Fig. 1). Predictions are based on the coexpression matrices GTEX_All_Pcc (Pearson), GTEX_All_Scc (Spearman), and GTEX_All_Max (Max). FIG. 7. Recovery of known inhibitor–protease pairs in matrices. Recovered pairs among the top 10% of ranked pairs in each matrix marked in black. Columns are coexpression and phylogenetic simi- larity matrices from supplemental Table S5. Rows are genes of pro- teases that are annotated to be inhibited by (A) SERPINF2 (alpha-2- antiplasmin), (B) TIMP2 (tissue inhibitor of metalloproteinases), and (C) A2M (alpha 2 macroglobulin). To assess whether predictions of generic protein interac- tions in state-of-the-art methodology are useful in predicting protease inhibitions, we obtained predicted interaction part- ners of protease inhibitors from PrePPI (42). Without our en- zymatic constraints, these predictions included many predic- tions that were irrelevant for the identification of protease inhibitors. For example, predicted partners of kallistatin (SER- PINA4) in PrePPI included many proteins that were not pro- teases or were themselves protease inhibitors (e.g. SER- PIND1 and SERPINC1). Focusing on interactions between inhibitors and proteases (predicted probability of 0.9 or higher), we identified 241 predictions. 41 interactions over- lapped with our 270 predictions despite the use of different methodology and underlying data. TABLE I It is not possible to deter- mine the accuracy of PrePPI predictions since PrePPI uses structural constraints and GO annotations, which partially correspond to the annotations we used to build the gold standard. However, despite these filters, 7 of the 51 interac- tions of serpins included metalloproteases, and none of the eight predicted interactions of cystatins included cysteine proteases, indicating that these predictions lack specificity and are not easily applicable to the prediction of inhibitory protease interactions. We observed that coexpression was very limited in utility in predicting protease–inhibitor pairs, contrary to previous find- ings suggesting proteases and their inhibitors have correlated expression (14 and 15). Our results suggest this is not a general principle, at least at the RNA level. We did not observe specific coexpression of many protease inhibitors with their target proteases despite our extensive efforts in calculating coexpression across human tissues and within tissues, based on proteomics and using machine learning to combine net- works. As demonstrated by our analysis (Fig. 7), we deci- phered a biological reason for the delinking of protease– inhibitor coexpression in the mobility of proteins in an open FIG. 7. Recovery of known inhibitor–protease pairs in matrices. Recovered pairs among the top 10% of ranked pairs in each matrix marked in black. Columns are coexpression and phylogenetic simi- larity matrices from supplemental Table S5. Rows are genes of pro- teases that are annotated to be inhibited by (A) SERPINF2 (alpha-2- antiplasmin), (B) TIMP2 (tissue inhibitor of metalloproteinases), and (C) A2M (alpha 2 macroglobulin). higher accuracy than predictions of protease inhibitions (Fig. 8). This difference was even stronger when focusing on pro- teasomal protein complex, demonstrating that prediction of protease inhibitions is indeed a difficult task compared with the prediction of general protein interactions. Molecular & Cellular Proteomics 16.6 1048 Bioinformatics Prediction of Protease–Inhibitor Interactions in realistic settings can explain the limited use of prediction tools in guiding biochemical experiments. biological system with interactions between tissues and cells. While coexpression might thus be better observed at the protein level, we also did not observe this, possibly due to the limited quality and extent of protein quantification. We further hypothesize that in complex networks, where an inhibitor inhibits many proteases and vice versa, the expression pat- tern of one gene represents a combination of the patterns of its interactors and is thus not clearly correlated with any one of them individually. However, our attempt to address this by using partial correlations did not provide support for this notion. We also revealed strengths and weaknesses of individual input features in our focused analysis. One important input feature was the prior knowledge of structural constraints, namely the knowledge of protease and inhibitor classes. We hypothesized that this feature would greatly simplify the in- hibitor prediction task by narrowing and focusing the search space compared with the general protein interaction predic- tion problem. Without enzymatic constraints, common protein– protein interaction predictions included many irrelevant pre- dictions, as we demonstrated in the case of PrePPI. In our analysis, protease-specific structural features significantly re- duced the search space to relevant interactions but also did not discern specificity between closely related protein family members. We conclude that such structural information as well as the related sequence-based input used in phyloge- netic similarity decreases the search space to plausible pairs but is not specific enough to identify individual interactions. In addition to many true inhibitor–protease pairs not coex- pressing, we also found that coexpressed pairs are often not inhibitory. We hypothesize that this is due to pathways of genes, where a gene can be coexpressed with other genes in the same pathway that are not direct interactors. In our data, one example is the coexpression of SERPINB12 with KLK5 and KLK7. SERPINB12 inhibits KLK5 but not KLK7. KLK5 is also an activator of KLK7 (41). Whereas SERPINB12 and KLK7 are involved in the same pathway and coexpressed, they do not interact directly. Nonetheless, inhibition of KLK5 by SERPINB12 prevents activation of KLK7 and thus indi- rectly inhibits KLK7. Thus, network effects (18) might explain some false positives in protein interaction prediction studies. Overall, we conclude that computational interaction predic- tion remains challenging and with the current state of data, and methods seem unable to accomplish the task with suffi- cient specificity to reliably replace biochemical experiments. Improvements may come from more comprehensive expres- sion studies and proteomics quantification as well as a better definition and larger numbers of TP and TN examples used for training to identify pertinent patterns. This will improve as more data are uploaded to community databases such as MEROPS and TopFIND for proteases and inhibitors. We cau- tion against overoptimistic estimates of performance based on aggregating diverse data in a black box algorithm and overtrusting cross-validations based on flawed gold stand- ards (43, 45). Equally problematic are biases toward well- studied proteins (40) by relying on functional or localization data that can lead to valid but less interesting interactions. By carefully selecting and combining features in a transparent method that is more analogous to a biologist’s reasoning, we can clarify limitations of features and thus should build con- fidence among the users of predictions. p p p Due to the complexity of biological networks, identification of biochemically relevant protein interactions remains an in- tricate problem. Prediction of inhibitors of proteases provided in-depth insights into the related difficulties since it allows a cleaner definition of TP and TN examples than general protein interaction prediction. We emphasize that biochemical evalu- ations are useful to give realistic estimates of expected per- formance because computational evaluation such as cross- validation performance is a very poor predictor of how a guilt-by-association method will do in reality (43). Our per- formance results are in line with many previous evaluations of guilt-by-association methods (43), and we conclude that much higher validation rates are unrealistic in unbiased bio- chemical experiments. Indeed, no prediction method has been adopted by biochemists for routine prioritization (13), despite reports of improved prediction performance (9, 42). The best performing biochemically validated protein interac- tion prediction method (PrePPI) (9, 42) was reported to be successful in 15 of 19 experiments (suggesting 79% preci- sion). However, in that case, predictions were guided by GO annotation, possibly reflecting information retrieval rather than the harder de novo predictions, and were hand selected for validation based on plausibility, which can significantly bias performance evaluation. Therefore, the reported performance for PrePPI is likely overoptimistic. Indeed, in a recent mass spectrometry screen of interaction partners of adenosine monophosphate-activated protein kinase-1 and -1 (44), only 63 of the 381 biochemically identified interaction partners overlapped with the 1,235 interactors predicted by PrePPI, giving only 5% precision. We posit that low validation rates Acknowledgments—We thank Dr. Ed Pryzdial and Scott Meixner who kindly provided F11, F12, and KLKB1 as well as their chromo- genic substrates. We further thank Dr. Silverman (Children’s Hospital of Pittsburgh), who provided SERPINB12, and Dr. Chao (Medical University of South Carolina), who provided serpin A4. 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https://openalex.org/W2319507205	https://www.scielo.br/j/rbef/a/NBQcLKwDcmzphrmRVdQ6nQG/?lang=pt&format=pdf	Portuguese	null	Atividades experimentais com enfoque no processo de modelagem científica: Uma alternativa para a ressignificação das aulas de laboratório em cursos de graduação em física	Revista Brasileira de Ensino de Física	2,016	cc-by	12,097	Recebido em 7 de agosto de 2015. Aceito em 17 de outubro de 2015 Ainda hoje s˜ao frequentes em aulas de laborat´orio de cursos de gradua¸c˜ao em f´ısica atividades que levam os estudantes a agir mecanicamente na execu¸c˜ao de roteiros excessivamente dirigidos, sem reﬂetir suﬁcientemente sobre os fundamentos te´oricos que embasam seus experimentos. Muitas dessas atividades s˜ao amparadas em concep¸c˜oes epistemol´ogicas inade- quadas, favorecendo a constru¸c˜ao, por parte dos estudantes, de concep¸c˜oes ingˆenuas sobre a natureza da ciˆencia. Frente a esse quadro, faz-se necess´ario propostas para aulas de laborat´orio que coloquem os estudantes como protagonistas em suas investiga¸c˜oes e que evidenciem aspectos importantes do fazer experimental. Focados nessa necessidade, apresentamos neste artigo quatro atividades, envolvendo oscila¸c˜oes mecˆanicas, ﬂuidos e termodinˆamica, delineadas com o objetivo de ressigniﬁcar as atividades experimentais por meio do que denominamos de epis´odios de modelagem. Nessas atividades, os estudantes s˜ao postos frente a situa¸c˜oes que: i) evidenciam aspectos importantes do processo de modelagem cient´ıﬁca, dando oportunidade para que eles construam concep¸c˜oes epistemol´ogicas n˜ao ingˆenuas, e ii) demandam uma postura ativa, possibilitando o desen- volvimento de competˆencias relacionadas com a cria¸c˜ao de quest˜oes de pesquisa, o delineamento de experimentos, a execu¸c˜ao cr´ıtica de procedimentos experimentais e a an´alise de dados. Espera-se que essas atividades possam inspirar professores e pesquisadores que tenham o intuito de delinear e realizar atividades experimentais que favore¸cam o engajamento e a reﬂex˜ao dos estudantes durante as suas investiga¸c˜oes, utilizando conhecimentos cient´ıﬁcos para enfrentar problemas sobre eventos reais. Palavras-chave: atividades experimentais, modelagem cient´ıﬁca, laborat´orio de f´ısica. Today, laboratory classes are still frequent in physics undergraduate courses: activities that lead students to act me- chanically in the execution of excessively guided scripts, without reﬂecting enough on the theoretical fundaments that give base to their experiments. Many of these activities are supported by inadequate epistemological conceptions, fostering naive views about the nature of science. In face of this situation, it is necessary to propose laboratory classes that make students the protagonists of their own inquiries and highlight important aspects of the practice of experimentation. With focus on this necessity, we present in this article four activities that involve mechanical oscillations, ﬂuids and thermodynamics. The activities have been designed with the purpose of reframe experimental activities through what we call Modeling Episodes. ∗Endere¸co de correspondˆencia: leonardo@heidemann.com.br. Copyright by Sociedade Brasileira de F´ısica. Printed in Brazil. Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504 (2016) www.scielo.br/rbef DOI: http://dx.doi.org/10.1590/S1806-11173812080 Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504 (2016) www.scielo.br/rbef DOI: http://dx.doi.org/10.1590/S1806-11173812080 Desenvolvimento em Ensino de F´ısica cbnd Licenc¸a Creative Commons Atividades experimentais com enfoque no processo de modelagem cient´ıﬁca: Uma alternativa para a ressigniﬁca¸c˜ao das aulas de laborat´orio em cursos de gradua¸c˜ao em f´ısica g ¸ Experimental activities with focus on the process of scientiﬁc modeling: An alternative for the laboratory classes in physics undergraduate courses Instituto de F´ısica, Universidade Federal do Rio Grande do Sul, Porto Algre, RS, Brasil Recebido em 7 de agosto de 2015. Aceito em 17 de outubro de 2015 1. Introdu¸c˜ao O editor da revista Physics Today, Charles Day, em recente publica¸c˜ao no blog The Dayside [1], exp˜oe uma reﬂex˜ao sobre o motivo pelo qual n˜ao se tor- nou um f´ısico experimental. Relata que, quando estudante de gradua¸c˜ao em f´ısica, no tradicional Imperial College London, cursou disciplinas de la- borat´orio obrigat´orias t˜ao entediantes que ele nem mesmo lembra dos experimentos realizados. Fica n´ıtido em seu relato que, em sua forma¸c˜ao como f´ısico, Day participou de aulas de laborat´orio que n˜ao lhe trazem boas lembran¸cas, podendo ter con- tribu´ıdo decisivamente para a sua escolha de n˜ao trabalhar como f´ısico experimental. Neste artigo, nossa proposta ´e enfrentar o pro- blema da dissocia¸c˜ao teoria-pr´atica em disciplinas experimentais de cursos de gradua¸c˜ao em f´ısica. Mais especiﬁcamente, descrevemos exemplos de ativida- des que oportunizam avan¸cos nas aulas de labo- rat´orio no sentido de superar cr´ıticas repetidamente presentes na literatura [3-6], se diferenciando das abordagens tradicionais: a) pelo enfoque dos proble- mas propostos, delineados com o intuito de propor- cionar situa¸c˜oes que promovam o enriquecimento das concep¸c˜oes epistemol´ogicas dos estudantes, e b) pelas tarefas incumbidas aos estudantes que, al´em de n˜ao serem detalhadas em um roteiro, envolvem desde a cria¸c˜ao de quest˜oes de pesquisa pertinen- tes at´e a constru¸c˜ao de conclus˜oes baseadas em evidˆencias experimentais. N˜ao h´a d´uvida de que existem estudantes que se adaptam `a metodologia de ensino empregada em ati- vidades experimentais tradicionais, desfrutando de boas experiˆencias em suas aulas de laborat´orio. No entanto, o relato de Day pode ser entendido como mais uma evidˆencia de que a forma como muitas ati- vidades experimentais s˜ao desenvolvidas em cursos de gradua¸c˜ao em f´ısica, muitas vezes com enfoque no preenchimento de tabelas e na constru¸c˜ao de gr´aﬁcos previstos em roteiros fortemente dirigidos, costuma n˜ao ser bem aceita pelos estudantes [2-3]. Devido `a forma como s˜ao estruturadas, essas ati- vidades podem ser realizadas mecanicamente, pois n˜ao estimulam os estudantes a reﬂetir suﬁciente- mente sobre as constru¸c˜oes te´oricas que fundamen- tam os experimentos realizados [3-6]. Em fun¸c˜ao disso, s˜ao frequentemente desenvolvidas dissociadas dos conte´udos abordados nas aulas te´oricas de f´ısica, favorecendo um ensino dicotomizado, em que teoria e pr´atica s˜ao tratados de forma desagregada. Essa dissocia¸c˜ao, j´a denunciada por Feynmann [7] como um dos principais problemas do ensino de f´ısica no Brasil, contribui para que os estudantes tenham signiﬁcativas diﬁculdades para relacionar teorias e realidade. 1. Introdu¸c˜ao Para isso, expomos a estrutura metodol´ogica e os objetivos das atividades, que denominamos de epis´odios de modelagem, e, em seguida, as espe- ciﬁcidades de quatro atividades sobre diferentes conte´udos de f´ısica (oscila¸c˜oes mecˆanicas, ﬂuidos e termodinˆamica) intituladas como: i) Pˆendulos, ii) Sistema de Amortecimento Automotivo, iii) Ar- quimedes e a coroa do rei, e iv) Resfriamento de Sistemas. Recebido em 7 de agosto de 2015. Aceito em 17 de outubro de 2015 In these activities, students face situations that: i) highlight important aspects of the process of scientiﬁc modeling, giving them the opportunity to construct epistemological concepts that are wise and not naive, and ii) demand an active role of the students, allowing the development of competences related to the creation of research questions, the designing of experi- ments, the critical execution of experimental procedures and data analysis. We hope these activities may inspire teachers and researchers to design and execute experimental activities that are favor engagement and reﬂection by the students during their inquiries, allowing them to use their scientiﬁc knowledge to tackle real problems originated in real events. Keywords: experimental activities, scientiﬁc modeling, physics laboratory. ds: experimental activities, scientiﬁc modeling, physics laboratory. 1504-2 Atividades experimentais com enfoque no processo de modelagem cient´ıﬁca fornece uma contribui¸c˜ao importante para o ensino de ciˆencias, preconizando investiga¸c˜oes que levem os estudantes a serem protagonistas em suas apren- dizagens por meio da procura por respostas para problemas sobre a natureza. No entanto, o enfoque das SEI n˜ao ´e especiﬁcamente para o Ensino Su- perior e grande parte das atividades desenvolvidas com base nesse referencial tem como alvo o Ensino Fundamental e M´edio. Nesses casos, n˜ao h´a explici- tamente uma maior preocupa¸c˜ao com a formaliza¸c˜ao dos conhecimentos abordados, o que ´e fundamental no contexto de disciplinas experimentais de cursos de gradua¸c˜ao. Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 DOI: http://dx.doi.org/10.1590/S1806-11173812080 2. Epis´odios de modelagem: Estrutura metodol´ogica e objetivos O cen´ario destacado aqui evidencia a necessidade de inser¸c˜ao no ensino de f´ısica de atividades expe- rimentais que demandem n˜ao apenas a execu¸c˜ao de experimentos previamente constru´ıdos, mas que tamb´em envolvam, em algum grau, a proposi¸c˜ao de quest˜oes de pesquisa, o delineamento de experimen- tos e a an´alise de dados com base em modelos ci- ent´ıﬁcos, a coleta de dados experimentais e, por ﬁm, a constru¸c˜ao de conclus˜oes a partir de evidˆencias. Nesse sentido, a proposta de Sequˆencias de Ensino Investigativas (SEI), de Pessoa de Carvalho [8-9], Os quatro epis´odios de modelagem propostos neste artigo foram delineados com base na Modelagem Did´atico-Cient´ıﬁca (MDC) [10]. Esse referencial te´orico-epistemol´ogico parte da tese de que a mo- delagem cient´ıﬁca pode ser entendida como um campo conceitual subjacente aos campos conceitu- ais espec´ıﬁcos da f´ısica para propor conhecimentos de referˆencia que os estudantes precisam mobilizar DOI: http://dx.doi.org/10.1590/S1806-11173812080 Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 1504-3 1504-3 Heidemann et al. quatro quest˜oes relacionadas com a leitura sugerida. Essa entrega pode ser realizada eletronicamente, por meio de, por exemplo, uma plataforma de ensino `a distˆancia como o Moodle1 ou de um formul´ario online constru´ıdo com o Google Drive2 [16]. Espera- se ent˜ao que o professor analise as respostas dos estudantes e que, antes dos epis´odios de modelagem, fa¸ca uma pequena discuss˜ao sobre as diﬁculdades dos alunos nas quest˜oes da tarefa de leitura, que pode ser entendida como uma introdu¸c˜ao ao epis´odio de modelagem. quando enfrentam situa¸c˜oes que demandam a cons- tru¸c˜ao, o uso e a valida¸c˜ao de modelos cient´ıﬁcos. Desse modo, propor situa¸c˜oes que possibilitem que os estudantes desenvolvam esses conhecimentos ´e um dos objetivos principais dos epis´odios de modelagem. Adotando a MDC, nos aliamos aos pesquisado- res que defendem que os modelos cient´ıﬁcos s˜ao os mediadores entre as teorias e a realidade [11-13]. En- tendemos que, para resolver problemas sobre eventos reais, que, diferentemente das quest˜oes frequente- mente abordadas nas aulas de f´ısica, s˜ao problemas n˜ao idealizados, os estudantes precisam ter clareza sobre as simpliﬁca¸c˜oes da realidade assumidas nos modelos cient´ıﬁcos que constroem e/ou exploram. Decorre da´ı nossa decis˜ao de, nos epis´odios de mo- delagem, buscar envolver os estudantes de f´ısica com aspectos centrais do processo de modelagem ci- ent´ıﬁca, dando oportunidade para que desenvolvam competˆencias que envolvem desde a constru¸c˜ao de representa¸c˜oes simpliﬁcadas de eventos reais, at´e a contrasta¸c˜ao dessas representa¸c˜oes com eventos reais por meio de investiga¸c˜oes experimentais. 2. Epis´odios de modelagem: Estrutura metodol´ogica e objetivos Evidente- mente, n˜ao ´e nossa expectativa que os estudantes desenvolvam essas competˆencias em algumas poucas atividades de laborat´orio. O caminho a ser percor- rido at´e que se tornem aptos a realizar de forma independente as a¸c˜oes necess´arias para uma inves- tiga¸c˜ao experimental ´e bastante longo, demandando um tempo muito maior do que o usualmente desti- nado para uma disciplina de laborat´orio. Por isso, entendemos que os objetivos de aprendizagem das atividades exempliﬁcadas neste artigo precisam per- mear muitas das disciplinas ao longo de todo o curso de f´ısica. Inspirados nos ciclos de modelagem de Hestenes [17-18], os epis´odios de modelagem s˜ao estruturados em trˆes etapas, quais sejam: i) discuss˜ao inicial, ii) investiga¸c˜ao e iii) discuss˜ao ﬁnal. Usaremos aqui o epis´odio de modelagem “Pˆendulos” para exempliﬁcar as caracter´ısticas de cada uma delas, destacando os pap´eis do professor e dos alunos nas atividades. p p p Na discuss˜ao inicial o problema a ser enfrentado ´e compartilhado com os estudantes. A apresenta¸c˜ao desse problema, realizada pelo professor a partir dos conhecimentos mobilizados na tarefa de leitura, envolve dois aspectos: i) a problematiza¸c˜ao das in- vestiga¸c˜oes a serem realizadas pelos estudantes e ii) a simpliﬁca¸c˜ao dos eventos que motivam essas in- vestiga¸c˜oes. Por exemplo, na atividade “Pˆendulos”, o professor desenvolve uma problematiza¸c˜ao ana- lisando processos que podem ser melhor compre- endidos por meio de estudos sobre pˆendulos. S˜ao exemplos desses processos: i) a medida da latitude em um ponto da Terra utilizando um pˆendulo de Foucault, ii) a procura por petr´oleo com o uso de grav´ımetros pendulares e iii) a mensura¸c˜ao de in- tervalos de tempo com rel´ogios de pˆendulo. Ap´os a problematiza¸c˜ao, ´e previsto que o professor, em constante debate com os alunos, argumente que in- vestigar o movimento de oscila¸c˜ao de um pˆendulo constitu´ıdo por uma bola suspensa em um ﬁo pos- sibilita melhor entendimento dos processos descri- tos. Em outras palavras, espera-se que o professor conduza um processo de simpliﬁca¸c˜ao dos eventos problematizados em eventos menos complexos, que podem ser mais facilmente manipulados em labo- rat´orio, possibilitando uma maior compreens˜ao dos eventos problematizados. O enfrentamento e at´e mesmo a compreens˜ao das situa¸c˜oes propostas nos epis´odios de modela- gem demanda dos estudantes o uso de uma s´erie de conhecimentos te´oricos e pr´aticos. A ﬁm de mobili- zar alguns dos conhecimentos b´asicos fundamentais para as atividades que desenvolver˜ao, ´e previsto que, anteriormente `a aula, realizem uma tarefa de leitura. 1Dispon´ıvel em https://moodle.org. Acesso em 20/7/2015. 2Dispon´ıvel em https://www.google.com/intl/pt-BR/ drive/. Acesso em 20/7/2015. Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 DOI: http://dx.doi.org/10.1590/S1806-11173812080 2. Epis´odios de modelagem: Estrutura metodol´ogica e objetivos Aos moldes do que ´e proposto na metodo- logia intitulada Ensino sob Medida [14], que tem alcan¸cado bons resultados em termos do est´ımulo `a leitura antes das aulas por parte dos estudantes [15], ´e solicitado aos alunos na semana anterior aos epis´odios de modelagem a leitura de um texto curto, com aproximadamente cinco p´aginas. Eles enviam ao professor at´e o dia anterior ao in´ıcio dos epis´odios de modelagem suas repostas a um conjunto de trˆes ou A problematiza¸c˜ao e a simpliﬁca¸c˜ao realizadas pelo professor norteiam o compartilhamento do pro- DOI: http://dx.doi.org/10.1590/S1806-11173812080 Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 1504-4 Atividades experimentais com enfoque no processo de modelagem cient´ıﬁca blema a ser enfrentado no epis´odio de modelagem, que sempre tem enfoque em aspectos do processo de modelagem cient´ıﬁca. Na atividade “Pˆendulos”, ´e su- gerido o uso do modelo de pˆendulo simples [19] para representar o movimento de uma bola que oscila suspensa por um ﬁo. Prop˜oe-se ent˜ao que os estu- dantes avaliem o dom´ınio de validade desse modelo por meio de investiga¸c˜oes experimentais realizadas com o intuito de identiﬁcar as condi¸c˜oes em que predi¸c˜oes de per´ıodos constru´ıdas por meio do mo- delo de pˆendulo simples diferem de no m´aximo 5% dos per´ıodos de pˆendulos reais. O objetivo deste epis´odio de modelagem ´e mostrado na Tabela 1, jun- tamente com os objetivos das outras trˆes atividades descritas neste artigo. me¸cam o seu per´ıodo diversas vezes utilizando am- plitudes diferentes em cada uma das medidas. Cabe ressaltar que os estudantes tˆem liberdade para rea- lizar investiga¸c˜oes diferentes daquelas sugeridas nos guias de atividade, desde que elas contribuam para uma melhor compreens˜ao do problema proposto na discuss˜ao inicial. Uma s´erie de materiais e instrumentos s˜ao dis- ponibilizados aos estudantes para que delineiem e conduzam suas investiga¸c˜ao. Por exemplo, no caso do epis´odio de modelagem “Pˆendulos”, sugere-se que sejam disponibilizados ﬁos com distintas elas- ticidades e densidades, bolas com diferentes densi- dades e diˆametros, hastes, mufas, sargentos, trenas, r´eguas, paqu´ımetros, cronˆometros, photogates. N˜ao h´a obrigatoriedade de uso de todos esses materiais (sugest˜oes de materiais a serem disponibilizados nos v´arios epis´odios s˜ao apresentados na Ref. [20]). Cabe ressaltar que, como as investiga¸c˜oes dos estudantes podem ser imprevis´ıveis, ´e dif´ıcil antecipar todos os materiais que ser˜ao necess´arios, sendo recomen- dado apresentar op¸c˜oes diversiﬁcadas. Mesmo assim, ´e poss´ıvel que seja preciso providenciar materiais durante as investiga¸c˜oes. 2. Epis´odios de modelagem: Estrutura metodol´ogica e objetivos Na segunda etapa dos epis´odios de modelagem, de- nominada investiga¸c˜ao, os estudantes, organizados em pequenos grupos, realizam investiga¸c˜oes experi- mentais a ﬁm de resolver o problema proposto na discuss˜ao inicial. O trabalho ´e norteado por um guia de atividade em que s˜ao apresentados: i) a problema- tiza¸c˜ao do epis´odio de modelagem, ii) conhecimentos cient´ıﬁcos b´asicos ´uteis para as investiga¸c˜oes e iii) op¸c˜oes de investiga¸c˜ao. Por exemplo, no guia de atividade do epis´odio de modelagem “Pˆendulos”, que pode ser consultado na Ref. [20], s˜ao expostos, al´em da problematiza¸c˜ao da atividade e de aspectos gerais do modelo de pˆendulo simples, trˆes op¸c˜oes de investiga¸c˜ao. Em uma delas, por exemplo, ´e suge- rido que os estudantes avaliem o quanto a amplitude de oscila¸c˜ao de um pˆendulo real inﬂuencia no seu per´ıodo. Prop˜oe-se que construam um pˆendulo e g Pequenos quadros brancos (90 cm x 60 cm) s˜ao uti- lizados pelos estudantes para compartilharem suas ideias durante as investiga¸c˜oes. O papel do professor nesta etapa ´e de auxiliar os estudantes no deline- amento, na coleta e na an´alise de dados das suas investiga¸c˜oes, auxiliando-os em suas diﬁculdades e alertando-os sobre os equ´ıvocos que possam cometer. Tamb´em ´e papel do professor avaliar o planejamento das investiga¸c˜oes dos estudantes, autorizando-os a coletar dados somente ap´os a sua aprova¸c˜ao. Para Tabela 1: Objetivos gerais dos epis´odios de modelagem. Os objetivos espec´ıﬁcos das atividades podem ser consultados na Ref. [20]. Epis´odio de modelagem Objetivo Geral: proporcionar situa¸c˜oes-problemas para que os estudantes compreendam... Pˆendulos ...o car´ater representacional dos modelos te´oricos da f´ısica, e que o grau de precis˜ao e o dom´ınio de validade desses modelos dependem das simpliﬁca¸c˜oes da realidade consideradas. Sistema de amorteci- mento automotivo ...que os modelos te´oricos da f´ısica s˜ao construc¸˜oes com o prop´osito de representar eventos reais, ou supostos como tais, e que as simpliﬁcac¸˜oes da realidade consideradas nesses modelos inﬂuenciam fortemente o delineamento de experimentos, pois procuramos utilizar arranjos experimentais onde os aspectos desconsiderados no modelo te´orico de referˆencia inﬂuenciam minimamente. Arquimedes e a coroa do rei ...que o ato de medir pressup˜oe o uso de modelos te´oricos auxiliares e que a escolha desses modelos inﬂuencia diretamente na precis˜ao das medidas. Resfriamento de sis- temas ...que quando o grau de precis˜ao de um modelo te´orico ´e insuﬁciente os modelos podem ser modiﬁcados por meio de altera¸c˜oes nas simpliﬁca¸c˜oes da realidade consideradas e/ou na teoria geral que o ampara. 2. Epis´odios de modelagem: Estrutura metodol´ogica e objetivos Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 DOI: http://dx.doi.org/10.1590/S1806-11173812080 Tabela 1: Objetivos gerais dos epis´odios de modelagem. Os objetivos espec´ıﬁcos das atividades podem ser consultados na R f [20] Arquimedes e a coroa do rei ...que o ato de medir pressup˜oe o uso de modelos te´oricos auxiliares e que a escolha desses modelos inﬂuencia diretamente na precis˜ao das medidas. ...que o ato de medir pressup˜oe o uso de modelos te´oricos auxiliares e que a escolha desses modelos inﬂuencia diretamente na precis˜ao das medidas. Cabe a ele: i) criar um ambiente de respeito e colabora¸c˜ao entre os estudantes, incentivando-os a explicitarem suas ideias e racioc´ınios para que se estabele¸cam as bases para o compartilhamento de signiﬁcados entre os participantes do processo de ensino e aprendiza- gem, ii) introduzir termos t´ecnicos na medida em que sejam necess´arios para aprimorar a qualidade do discurso dos estudantes, iii) introduzir novas fer- ramentas de representa¸c˜ao na medida em que os estudantes estiverem preparados para fazer bom uso delas, e iv) promover debates expl´ıcitos sobre as- pectos relacionados com o processo de modelagem cient´ıﬁca, dando oportunidade para que os estudan- tes evoluam em suas concep¸c˜oes sobre a natureza da ciˆencia. J´a os estudantes precisam: i) compartilhar seus resultados com o grande grupo, dirimindo as d´uvidas dos colegas e do professor, ii) dirigir pergun- tas aos seus colegas sobre as investiga¸c˜oes realizadas. Deseja-se com isso que os estudantes desenvolvam competˆencias para se expressarem na forma oral, construindo argumentos fundamentados em conhe- cimentos cient´ıﬁcos e em evidˆencias experimentais. A ´ d b t b i ti ˜ d di Assim como em qualquer atividade de ensino, a avalia¸c˜ao ´e um aspecto fundamental nos epis´odios de modelagem. Adotamos o princ´ıpio de que a ava- lia¸c˜ao da aprendizagem deve ser coerente com os ob- jetivos e com os procedimentos de ensino, conforme preconizam Valadares e Gra¸ca [21]. Para tanto, cons- tru´ımos, baseados nos objetivos de aprendizagem es- tabelecidos para cada uma das etapas dos epis´odios de modelagem, um protocolo de avalia¸c˜ao para ser usado pelos professores na corre¸c˜ao dos relat´orios experimentais produzidos pelos estudantes. Como pode ser constatado no Tabela 2, esse protocolo ´e constitu´ıdo por trˆes colunas. Nas duas primeiras s˜ao apresentados, respectivamente, os aspectos dos relat´orios aos quais os crit´erios de avalia¸c˜ao se refe- rem e os pr´oprios crit´erios de avalia¸c˜ao. A terceira coluna ´e reservada para que o professor avalie os re- lat´orios em trˆes n´ıveis usando os seguintes s´ımbolos: ", 1/2, e %, conforme considera que o crit´erio foi bem, medianamente ou mal atendido no relat´orio avaliado, respectivamente. No ﬁnal do protocolo, ´e reservado ainda um espa¸co para que o professor fa¸ca coment´arios ressaltando os aspectos positivos e as deﬁciˆencias mais marcantes dos relat´orios. ´ ´E previsto que os estudantes tenham conheci- mento do protocolo de avalia¸c˜ao antes dos epis´odios de modelagem. ...que o ato de medir pressup˜oe o uso de modelos te´oricos auxiliares e que a escolha desses modelos inﬂuencia diretamente na precis˜ao das medidas. Resfriamento de sis- temas ...que quando o grau de precis˜ao de um modelo te´orico ´e insuﬁciente os modelos podem ser modiﬁcados por meio de altera¸c˜oes nas simpliﬁca¸c˜oes da realidade consideradas e/ou na teoria geral que o ampara. DOI: http://dx.doi.org/10.1590/S1806-11173812080 Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 1504-5 1504-5 Heidemann et al. esse planejamento, os estudantes precisam, em al- gum n´ıvel: de dados produzidos em simula¸c˜oes computacionais em que s˜ao consideradas idealiza¸c˜oes e aproxima¸c˜oes diferentes das assumidas no modelo investigado. Es- ses resultados podem ser consultados com detalhes na Ref. [18]. • Criar quest˜oes de pesquisa; • Construir ou escolher um modelo te´orico de referˆencia para a investiga¸c˜ao; ´E importante ressaltar aqui que, ainda que te- nhamos nos inspirado nos ciclos de modelagem de Hestenes, os epis´odios de modelagem possuem ca- racter´ısticas que os distinguem. Por exemplo, ainda que os ciclos de modelagem envolvam problemas mais abertos que o usual, possibilitando que os es- tudantes assumam uma postura mais ativa, eles se aproximam das atividades tradicionais quando, diferentemente dos epis´odios de modelagem, n˜ao colocam os estudantes frente a situa¸c˜oes que de- mandam reﬂex˜oes expl´ıcitas sobre as idealiza¸c˜oes e aproxima¸c˜oes consideradas nos modelos cient´ıﬁcos utilizados. Al´em disso, apesar de apresentarem um referencial te´orico-epistemol´ogico para suas ativida- des, Hestenes e seus colaboradores n˜ao explicitam com clareza como suas atividades possibilitam que os estudantes desenvolvam conhecimentos cient´ıﬁcos e habilidades de modelagem para enfrentar situa¸c˜oes que demandam a constru¸c˜ao, o uso e/ou a valida¸c˜ao de representa¸c˜oes simpliﬁcadas de eventos reais. • Delinear um arranjo experimental e procedi- mentos de coleta de dados; • Planejar a an´alise dos dados que ser˜ao coleta- dos. Ap´os a aprova¸c˜ao dos seus planejamentos, os estu- dantes necessitam, ainda na etapa de investiga¸c˜ao: • Construir os arranjos experimentais delineados; • Coletar dados experimentais; • Construir os arranjos experimentais delineados; • Coletar dados experimentais; • Coletar dados experimentais; • Analisar os dados coletados com base no modelo te´orico de referˆencia; ; • Avaliar as diferen¸cas entre predi¸c˜oes e evidˆencias; • Construir conclus˜oes com base nas evidˆencias da investiga¸c˜ao. A ´ultima etapa dos epis´odios de modelagem ´e a discuss˜ao ﬁnal. Nela, os estudantes exp˜oem seus resultados ao grande grupo, utilizando os peque- nos quadros brancos que permitem a visualiza¸c˜ao simultˆanea dos resultados de v´arios grupos. O papel do professor nesse momento ´e de mediador. Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 DOI: http://dx.doi.org/10.1590/S1806-11173812080 ...que o ato de medir pressup˜oe o uso de modelos te´oricos auxiliares e que a escolha desses modelos inﬂuencia diretamente na precis˜ao das medidas. A op¸c˜ao por realizar tal procedi- mento ´e baseada em resultados de pesquisa que evidenciam que protocolos de avalia¸c˜ao possibilitam que os estudantes desenvolvam conhecimentos me- tacognitivos (para uma revis˜ao da literatura, veja Ref. [22]). Mais precisamente, o conhecimento dos Ap´os os debates sobre as investiga¸c˜oes dos diver- sos grupos, o professor exp˜oe uma solu¸c˜ao para o problema enfrentado na atividade, relacionando-a com os resultados obtidos pelos alunos. Na atividade “Pˆendulos”, por exemplo, o professor apresenta resul- tados de investiga¸c˜oes em que o dom´ınio de validade do modelo de pˆendulo simples ´e avaliado por meio 1504-6 Atividades experimentais com enfoque no processo de modelagem cient´ıﬁca Atividades experimentais com enfoque no processo de modelagem cient´ıﬁca Tabela 2: Protocolo de avaliac¸˜ao para os relat´orios experimentais dos epis´odios de modelagem. Aspecto Crit´erio de avalia¸c˜ao do relat´orio Avalia¸c˜ao do professor Objetivo da atividade Relaciona o objetivo da atividade com um modelo te´orico. Faz referˆencia somente a grandezas, objetos, rela¸c˜oes te´oricas ou eventos f´ısicos previamente deﬁnidos no relat´orio. Referencial te´orico Explicita as aplica¸c˜oes de leis e/ou princ´ıpios de uma teoria geral na situa¸c˜ao f´ısica investigada, construindo um modelo te´orico adequado para o experimento realizado. N˜ao apresenta erros conceituais. Ressalta as implica¸c˜oes das simpliﬁca¸c˜oes da realidade consideradas durante a aplica¸c˜ao das leis e/ou princ´ıpios de uma teoria geral `a situa¸c˜ao f´ısica investigada. Explicita os objetos reais do experimento realizado que s˜ao considerados no mo- delo te´orico adotado, n˜ao confundindo objetos com as grandezas utilizadas para representar suas propriedades. Procedimento experimental Explicita as grandezas que foram medidas. Explicita os instrumentos de medida utilizados. Explicita o arranjo experimental utilizado. Explicita o evento f´ısico investigado. Explicita procedimentos tomados para se controlar vari´aveis, ou seja, procedimentos realizados para que os fatores desprezados pelo modelo te´orico adotado inﬂuenciem minimamente os dados experimentais. Apresenta¸c˜ao e an´alise dos dados experimentais Explicita como o modelo te´orico adotado dirigiu a an´alise dos dados experimentais. Utiliza ferramentas de representa¸c˜ao (gr´aﬁcos, tabelas, ﬁguras pict´oricas, etc.) para representar os dados coletados experimentalmente de forma adequada (e.g., explicita as grandezas representadas nos eixos dos gr´aﬁcos, escolhe escalas adequadas para tais eixos, etc.). Interpreta as representa¸c˜oes apresentadas corretamente. Explicita corretamente as incertezas de medida relacionadas com as imprecis˜oes dos instrumentos de medida utilizados. Calcula corretamente as incertezas propagadas das imprecis˜oes intr´ınsecas dos instrumentos de medida utilizados. Interpreta as incertezas de medida dos dados coletados experimentalmente, utili- zando o n´umero adequado de algarismos signiﬁcativos para represent´a-los. Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 DOI: http://dx.doi.org/10.1590/S1806-11173812080 ...que o ato de medir pressup˜oe o uso de modelos te´oricos auxiliares e que a escolha desses modelos inﬂuencia diretamente na precis˜ao das medidas. De modo diferente, a investiga¸c˜ao proposta no epis´odio de modelagem “Pˆendulos” tem como prop´osito avaliar a adequa¸c˜ao de um modelo te´orico para representar eventos reais. Mais especi- ﬁcamente, ´e sugerido que os estudantes avaliem o dom´ınio de validade do modelo de pˆendulo simples [19]. Com isso, procura-se salientar aos alunos que os modelos cient´ıﬁcos s˜ao representa¸c˜oes simpliﬁcadas, e n˜ao c´opias especulares da realidade, como uma parcela signiﬁcativa deles acredita [26-27]. onalmente despendido com um mesmo experimento em disciplinas experimentais. Esse ´e o pre¸co a se pagar ao dar-se certa autonomia aos estudantes e exigir-lhes desde o delineamento do experimento at´e a exposi¸c˜ao oral de seus resultados. Esse tempo de- pende substancialmente do n´ıvel de conhecimento dos alunos e da maturidade para o trabalho colabora- tivo com certa independˆencia. A t´ıtulo de ilustra¸c˜ao, mencionamos que, em uma turma com sete estudan- tes que cursavam a segunda disciplina experimental do curso de f´ısica [23], foram necess´arias aproxi- madamente quatro horas para o desenvolvimento do epis´odio de modelagem mais curto (Arquimedes e a coroa do rei) e seis horas e meia para o mais longo (Resfriamento de sistemas), sendo que cerca de 30 minutos eram usados para os debates sobre as tarefas de leitura e as discuss˜oes iniciais e 40 minutos para as discuss˜oes ﬁnais. O modelo de pˆendulo simples [19] pressup˜oe uma s´erie de idealiza¸c˜oes. O corpo suspenso ´e considerado pontual; o ﬁo de sustenta¸c˜ao, sem massa e inel´astico; for¸cas resistivas s˜ao desprezadas. ´E usual, ainda, a aproxima¸c˜ao de que o pˆendulo simples oscila com pequenas amplitudes, o que possibilita predizer o seu per´ıodo T pela equa¸c˜ao p Nesta se¸c˜ao, apresentamos as caracter´ısticas co- muns aos quatro epis´odios de modelagem propostos neste artigo, usando a atividade “Pˆendulos” para exempliﬁcar aspectos centrais dos epis´odios de mode- lagem. Nas pr´oximas se¸c˜oes, trataremos de particu- laridades referentes aos quatro epis´odios exempliﬁca- dos. N˜ao ´e nosso objetivo esmiu¸car os procedimentos que o professor e os alunos realizam durante cada uma das etapas dos epis´odios de modelagem. Esses detalhes podem ser consultados na Ref. [20], onde s˜ao disponibilizados tamb´em materiais para a rea- liza¸c˜ao das atividades, incluindo tarefas de leitura, guias de atividades, apresenta¸c˜oes de slides para as discuss˜oes iniciais e ﬁnais, e sugest˜oes de objetos e instrumentos que podem ser disponibilizados aos estudantes. ...que o ato de medir pressup˜oe o uso de modelos te´oricos auxiliares e que a escolha desses modelos inﬂuencia diretamente na precis˜ao das medidas. Ressalta as principais fontes de incerteza relacionadas com a imprecis˜ao dos instru- mentos de medida utilizados. Ressalta as consequˆencias das simpliﬁca¸c˜oes consideradas no modelo te´orico adotado que n˜ao foram completamente respeitadas no experimento. Conclus˜oes Avalia o modelo te´orico adotado no experimento. Apresenta somente conclus˜oes que contam com amparo de evidˆencias experimentais. Analisa as poss´ıveis contribui¸c˜oes dos resultados experimentais para a resolu¸c˜ao do problema que norteou a investiga¸c˜ao realizada. Reda¸c˜ao N˜ao apresenta erros ortogr´aﬁcos ou gramaticais. N˜ao apresenta frases cuja falta de clareza comprometem a sua compreens˜ao. Est´a estruturado em se¸c˜oes divididas apropriadamente. Coment´arios: , ) Interpreta as representa¸c˜oes apresentadas corretamente. Explicita corretamente as incertezas de medida relacionadas com as imprecis˜oes dos instrumentos de medida utilizados. Calcula corretamente as incertezas propagadas das imprecis˜oes intr´ınsecas dos instrumentos de medida utilizados. itens sobre os quais ser˜ao avaliados possibilita que os estudantes controlem suas a¸c˜oes a ﬁm de alcan¸car um objetivo pr´e-estabelecido. Desse modo, usando o protocolo de avalia¸c˜ao, facilitamos a regula¸c˜ao dos processos de aprendizagem, o que tamb´em ´e um princ´ıpio da avalia¸c˜ao da aprendizagem proposto por Valadares e Gra¸ca. para a forma¸c˜ao de bons cientistas. As discuss˜oes ﬁnais das atividades d˜ao oportunidade para que o professor avalie a capacidade dos seus estudantes de apresentar com clareza o delineamento e a execu¸c˜ao de suas investiga¸c˜oes assim como de expor suas conclus˜oes com base em evidˆencias. Nessa etapa das atividades o professor pode avaliar tamb´em se os seus estudantes desenvolveram concep¸c˜oes n˜ao ingˆenuas sobre a natureza da ciˆencia, promovendo debates sobre o assunto na medida do necess´ario. Al´em do uso do protocolo do Apˆendice A, para avaliar as competˆencias dos estudantes relacionadas com a modelagem cient´ıﬁca, espera-se tamb´em que, nos epis´odios de modelagem, os alunos sejam avalia- dos tamb´em em termos de suas competˆencias para explicitar ideias na forma oral, que s˜ao essenciais Destaca-se que a realiza¸c˜ao de epis´odios de mo- delagem como os propostos neste artigo demanda maior tempo, dentro e fora de aula, do que o tradici- DOI: http://dx.doi.org/10.1590/S1806-11173812080 Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 1504-7 1504-7 Heidemann et al. leis cient´ıﬁcas a partir de dados coletados experi- mentalmente. Existem casos ainda em que os estu- dantes s˜ao incentivados a realizar um experimento com o objetivo de “provar” empiricamente uma te- oria cient´ıﬁca, o que tamb´em ´e um grave equ´ıvoco epistemol´ogico. ...que o ato de medir pressup˜oe o uso de modelos te´oricos auxiliares e que a escolha desses modelos inﬂuencia diretamente na precis˜ao das medidas. Neste artigo, nos limitamos a debater os aspectos mais gerais dos epis´odios de modelagem, destacando como cada um deles tem potencial para: possibilitar que os estudantes superem concep¸c˜oes ingˆenuas sobre o fazer cient´ıﬁco; coloc´a-los frente a situa¸c˜oes que podem dar sentido tanto aos concei- tos cient´ıﬁcos de diferentes campos da f´ısica quanto aos conceitos envolvidos no processo de modelagem cient´ıﬁca; e tamb´em favorecer o desenvolvimento de competˆencias relacionadas `a constru¸c˜ao, uso e valida¸c˜ao de modelos cient´ıﬁcos por parte dos estu- dantes. T = 2π. s l g, (1) (1) onde l ´e o comprimento do ﬁo de sustenta¸c˜ao do pˆendulo e g ´e a acelera¸c˜ao gravitacional local. Com frequˆencia, as idealiza¸c˜oes e aproxima¸c˜oes consideradas no modelo de pˆendulo simples n˜ao s˜ao devidamente destacadas no ensino de f´ısica. Desse modo, cria-se um contexto que favorece que os es- tudantes concebam o referido modelo como uma constru¸c˜ao te´orica que tem como referˆencia imedi- ata a realidade quando, de fato, ele se refere a uma descri¸c˜ao simpliﬁcada de eventos reais. Procurando destacar o car´ater representacional dos modelos, a atividade “Pˆendulos” tem por objetivo avaliar o dom´ınio de validade do modelo de pˆendulos simples por meio da contrasta¸c˜ao de predi¸c˜oes constru´ıdas com base na Eq. (1) com dados coletados experimen- talmente. O enunciado do problema compartilhado com os estudantes na discuss˜ao inicial do epis´odio de modelagem ´e exposto abaixo. At´e que ponto podemos dizer que a ampli- tude de um pˆendulo ´e pequena? Quando podemos dizer que as dimens˜oes do corpo suspenso s˜ao desprez´ıveis? At´e que ponto a for¸ca de arrasto com o ar inﬂuencia no per´ıodo de um pˆendulo? Quando podemos dizer que a massa do ﬁo de sustenta¸c˜ao ´e desprez´ıvel? Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 3. Pˆendulos Nesta atividade, distanciando-se das atividades ex- perimentais que favorecem o desenvolvimento de con- cep¸c˜oes empiristas-indutivistas ingˆenuas por parte dos estudantes, prop˜oe-se uma avalia¸c˜ao da ade- qua¸c˜ao do modelo de pˆendulo simples para represen- tar eventos reais, possibilitando que os estudantes compreendam que os modelos cient´ıﬁcos s˜ao repre- senta¸c˜oes simpliﬁcadas da realidade, e que por isso possuem um grau de precis˜ao e um dom´ınio de validade. Al´em disso, os estudantes podem compre- ender que as diferen¸cas entre predi¸c˜oes constru´ıdas com base em modelos cient´ıﬁcos e dados coletados experimentalmente decorrem n˜ao apenas da impre- cis˜ao dos instrumentos de medida utilizados, mas tamb´em das simpliﬁca¸c˜oes da realidade considera- das no modelo te´orico de referˆencia do experimento. As rela¸c˜oes entre os per´ıodos de pˆendulos reais e suas amplitudes, dimens˜oes dos corpos suspensos e massas dos seus ﬁos de sustenta¸c˜ao s˜ao exemplos de conhecimentos da f´ısica que os estudantes tˆem a oportunidade de compreender com maior profundi- dade com a atividade “Pˆendulos”. Ademais, eles s˜ao postos frente a situa¸c˜oes que podem dar sentido a conceitos como, por exemplo, de per´ıodo, frequˆencia e amplitude, assim como de dom´ınio de validade e grau de precis˜ao. Os estudantes podem ainda avaliar a inﬂuˆencia das dimens˜oes do corpo suspenso sobre o per´ıodo de pˆendulos reais. Para isso, eles podem medir o per´ıodo de um pˆendulo diversas vezes modiﬁcando o comprimento do ﬁo de sustenta¸c˜ao em cada conjunto de medidas. Desse modo, pode-se comparar as raz˜oes entre o diˆametro do corpo que oscila dividido pelos comprimentos do ﬁo de sustenta¸c˜ao, que fornecem estimativas de qu˜ao razo´avel ´e considerar o corpo suspenso pontual em cada um dos casos investigados, com os per´ıodos medidos do pˆendulo. p p Uma terceira possibilidade de experimento pode ser realizada com o intuito de avaliar a inﬂuˆencia da for¸ca resistiva do ar sobre o per´ıodo de pˆendulos reais. Para isso, pode-se medir o per´ıodo de um pˆendulo diversas vezes utilizando corpos suspensos com distintas dimens˜oes, variando as suas se¸c˜oes retas. ´E importante debater em todas as atividades, mas principalmente nesse experimento, sobre pro- cedimentos de controle de vari´aveis. Por exemplo, se para se alterar a se¸c˜ao reta do corpo suspenso for alterada tamb´em a sua distribui¸c˜ao de massa, al´em da se¸c˜ao reta do corpo suspenso ser´a modiﬁ- cado tamb´em o momento de in´ercia do pˆendulo em rela¸c˜ao ao seu ponto de sustenta¸c˜ao. 3. Pˆendulos O entendimento de que as leis cient´ıﬁcas s˜ao cons- tru´ıdas por meio de generaliza¸c˜oes realizadas a par- tir de um conjunto de observa¸c˜oes, que ´e um dos pilares da concep¸c˜ao empirista-indutivista, ´e pro- fundamente questionado por ﬁl´osofos da ciˆencia h´a bastante tempo [24-25]. No entanto, frequentemente aulas de f´ısica geral experimental se sustentam nessa concep¸c˜ao, sendo desenvolvidas atividades estrutura- das com o intuito de levar os estudantes a redescobrir Nesta tarefa exploraremos o contexto de validade do modelo de pˆendulo simples. Para isso, queremos que vocˆes explorem os limites nos quais a diferen¸ca entre o DOI: http://dx.doi.org/10.1590/S1806-11173812080 Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 1504-8 Atividades experimentais com enfoque no processo de modelagem cient´ıﬁca Atividades experimentais com enfoque no processo de modelagem cient´ıﬁca Os softwares Tracker3 e Modellus4 podem ser utili- zados nos experimentos para, respectivamente, cole- tar dados experimentais por meio de videoan´alises e para emular o comportamento do modelo de pˆendulo simples por meio de simula¸c˜oes computacionais. Mais detalhes sobre o uso desses softwares na ativi- dade pˆendulos podem ser consultados na Ref. [18]. per´ıodo predito por meio do modelo de pˆendulo simples e o per´ıodo de pˆendulos reais ´e menor do que 5%. N˜ao se espera que os estudantes respondam todas as quest˜oes propostas no enunciado da atividade. Tampouco espera-se que eles encontrem uma res- posta precisa para as perguntas apresentadas. O fundamental ´e que, durante a fase de investiga¸c˜ao, os estudantes realizem ao menos um experimento, que pode ser um dos trˆes sugeridos no guia da ativi- dade, com o intuito de coletar dados para serem con- trastados com predi¸c˜oes constru´ıdas com a Eq. (1). Por exemplo, procurando avaliar a inﬂuˆencia da amplitude de um pˆendulo real em seu per´ıodo, os estudantes podem construir um pˆendulo e medir o seu per´ıodo de oscila¸c˜ao diversas vezes utilizando amplitudes diferentes. Os per´ıodos mensurados po- der˜ao ent˜ao ser contrastados com o per´ıodo predito por meio da Eq. (1), possibilitando uma avalia¸c˜ao da amplitude para a qual a diferen¸ca entre o per´ıodo medido e o predito ´e maior do que 5%. 3Dispon´ıvel em http://modellus.com. Acesso em 20/7/2015. 4Dispon´ıvel em https://www.cabrillo.edu/˜dbrown/ tracker/. Acesso em 20/7/2015. 3. Pˆendulos Desse modo, n˜ao ser´a poss´ıvel avaliar se as varia¸c˜oes no per´ıodo do pˆendulo real ser˜ao decorrentes de mudan¸cas na se¸c˜ao reta do corpo suspenso ou no momento de in´ercia do pˆendulo investigado. Para controlar o mo- mento de in´ercia do corpo suspenso, pode-se alterar sua se¸c˜ao reta revestindo-o com bolas de isopor le- ves, por exemplo. Desse modo, utilizando bolas de isopor muito mais leves do que o corpo suspenso, a altera¸c˜ao da se¸c˜ao reta do pˆendulo acarretar´a em varia¸c˜oes desprez´ıveis no seu momento de in´ercia. Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 4. Sistema de amortecimento automotivo O papel dos conhecimentos pr´evios no desenvol- vimento da ciˆencia ´e amplamente destacado por muitos ﬁl´osofos [24, 28]. A concep¸c˜ao defendida por empiristas ingˆenuos de que o pesquisador pode (ou deve) abster-se de suas ideias pr´evias para conduzir uma investiga¸c˜ao experimental, baseando-se exclu- sivamente em observa¸c˜oes, n˜ao condiz com o que efetivamente ´e feito pelos cientistas. Experimentos s˜ao sempre realizados com base em conhecimentos j´a existentes [29]. Analisando especiﬁcamente as aulas de laborat´orio, s˜ao os conhecimentos pr´evios dos estudantes que os tornam capazes ou n˜ao de identi- ﬁcar os aspectos chave dos experimentos propostos [30]. Destacando este ponto, Arruda e Labur´u [31] argumentam que, “se levarmos nossos alunos para o laborat´orio e dissermos ‘observem!’, eles certa- mente ir˜ao perguntar ‘observar o quˆe?”’. Mais preci- Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 DOI: http://dx.doi.org/10.1590/S1806-11173812080 1504-9 Heidemann et al. samente, sem conhecimentos sobre vers˜oes did´aticas de modelos cient´ıﬁcos, os estudantes n˜ao s˜ao capa- zes de delinear, executar ou analisar experimentos cient´ıﬁcos. Modelagem e experimenta¸c˜ao, portanto, s˜ao processos profundamente imbricados. Apesar disso, pesquisadores tˆem identiﬁcado diﬁculdades dos estudantes para reconhecer rela¸c˜oes entre a ex- perimenta¸c˜ao e o processo de modelagem cient´ıﬁca [32-34]. Procurando destacar essas rela¸c˜oes aos es- tudantes, a atividade “Sistema de Amortecimento Automotivo” tem como um de seus objetivos eviden- ciar que os experimentos cient´ıﬁcos s˜ao, em maior ou menor grau, executados em meios artiﬁciais deli- neados com base em modelos cient´ıﬁcos e, com isso, destacar o papel dos conhecimentos pr´evios em in- vestiga¸c˜oes experimentais. Especiﬁcamente, tem-se o intuito de possibilitar que os estudantes ampliem seus conhecimentos sobre o sistema de amorteci- mento de autom´oveis por meio de experimentos de- lineados com base em um modelo te´orico constru´ıdo para representar o sistema massa-mola amortecido. por uma mola que se move imerso em um ﬂuido? Como esses fatores inﬂuenciam na oscila¸c˜ao desse corpo? Do que dependem esses fatores? Para representar o comportamento do arranjo ex- perimental sugerido, prop˜oe-se o uso de um modelo te´orico constru´ıdo com base no objeto-modelo de sistema massa-mola amortecido e na mecˆanica new- toniana [35], que denominamos aqui apenas como modelo de sistema massa-mola amortecido. Sugere- se ent˜ao aos estudantes que eles, durante a fase de investiga¸c˜ao, baseiem-se nesse modelo para de- linear experimentos com o objetivo de explorar o comportamento de um corpo suspenso por uma mola que oscila imerso em um ﬂuido. 4. Sistema de amortecimento automotivo Trˆes op¸c˜oes de experimentos s˜ao apresentadas no guia de ati- vidade. Por exemplo, como o modelo de sistema massa-mola amortecido pressup˜oe que o amorteci- mento do movimento de um corpo que oscila em um ﬂuido depende das propriedades desse ﬂuido, ´e proposto que os estudantes realizem medidas das constantes de amortecimento dos movimentos de um mesmo corpo quando ele oscila preso a uma mesma mola imerso em diferentes ﬂuidos, com diferentes densidades e/ou viscosidades. ´ Frequentemente, cientistas constroem aparatos experimentais simples com o objetivo de investi- gar sistemas reais mais complexos. Assim ocorre quando, por exemplo, um cientista produz em labo- rat´orio centelhas com um gerador el´etrico em um ambiente controlado com o objetivo de investigar o comportamento de raios na atmosfera terrestre. Evidentemente, a complexidade dos aparatos pode variar muito dependendo das quest˜oes que se al- meja responder e de aspectos relacionados com a infraestrutura dispon´ıvel. ´E previsto que o professor proponha que os estu- dantes ﬁlmem os eventos investigados e, por meio de videoan´alises realizadas com o software Trac- ker, coletem dados da posi¸c˜ao do corpo que oscila em fun¸c˜ao do tempo. Al´em disso, ele pode suge- rir tamb´em que os participantes analisem os dados coletados com uma planilha eletrˆonica, inferindo a curva exponencial que melhor modula a sen´oide que descreve o movimento do sistema massa-mola amor- tecido, possibilitando a mensura¸c˜ao da constante de amortecimento desse sistema. Esse procedimento oportuniza a realiza¸c˜ao de debates sobre o ajuste de curvas a dados experimentais, destacando aos estudantes que n˜ao s˜ao os dados experimentais que apontam o tipo de curva que deve ser ajustada, como sustentam empiristas ingˆenuos, mas sim que a escolha da curva a ser ajustada ´e norteada pelo modelo te´orico de referˆencia do experimento. O objetivo b´asico deste epis´odio de modelagem ´e possibilitar que os estudantes construam aparatos experimentais que os permitam investigar o funcio- namento de sistemas de amortecimento automotivos em laborat´orio. Para isso, ´e previsto que, primeira- mente, os pr´oprios estudantes proponham arranjos experimentais an´alogos ao sistema de amortecimento de autom´oveis. Debatendo sobre os arranjos expe- rimentais propostos, o professor sugere ent˜ao que os estudantes investiguem o comportamento de um corpo suspenso por uma mola que oscila imerso em um ﬂuido para aprofundar seus conhecimentos so- bre o amortecimento de osciladores mecˆanicos para, em uma etapa futura, terem melhores condi¸c˜oes de compreender sistemas automotivos. Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 4. Sistema de amortecimento automotivo O enunciado do problema compartilhado com os estudantes na dis- cuss˜ao inicial do epis´odio de modelagem ´e exposto abaixo. Outra possibilidade de investiga¸c˜ao sugerida no guia da atividade ´e a realiza¸c˜ao de um experimento com o intuito de avaliar a inﬂuˆencia da constante el´astica da mola na qual o corpo est´a preso no seu movimento no interior de um ﬂuido. Para isso, ampa- rados no modelo de sistema massa-mola amortecido, os estudantes podem medir os per´ıodos dos movi- mentos de um corpo quando ele oscila imerso em um mesmo ﬂuido suspenso por diferentes molas com constantes el´asticas conhecidas. Aspectos relacio- nados com o controle de vari´aveis tamb´em podem ser debatidos aqui. Por exemplo, ´e importante que os estudantes utilizem molas que tenham massas Que caracter´ısticas devem ter os compo- nentes do sistema de amortecimento de um autom´ovel? Como essas caracter´ısticas inﬂuenciam nesse sistema? Quais os prin- cipais fatores que inﬂuenciam no amorteci- mento da oscila¸c˜ao de um corpo suspenso DOI: http://dx.doi.org/10.1590/S1806-11173812080 1504-10 1504-10 Atividades experimentais com enfoque no processo de modelagem cient´ıﬁca Atividades experimentais com enfoque no processo de modelagem cient´ıﬁca que os modelos tˆem o mundo como referencial ime- diato, sendo c´opias especulares da realidade [38-39]. O epis´odio de modelagem “Arquimedes e a coroa do rei” foi delineado com o objetivo de salientar os motivos pelos quais diferen¸cas entre predi¸c˜oes e dados coletados experimentalmente s˜ao inevit´aveis. Procura-se destacar que as predi¸c˜oes de um mo- delo te´orico s˜ao aproximadas, pois os modelos s˜ao apenas representa¸c˜oes simpliﬁcadas da realidade, e n˜ao c´opias exatas. Al´em disso, busca-se lev´a-los a compreender que a imprecis˜ao de um instrumento de medida est´a relacionada com o modelo te´orico que fundamenta o seu funcionamento. Para isso, ´e solicitado que os estudantes me¸cam a composi¸c˜ao de uma amostra de chumbo e alum´ınio por meio de dois m´etodos experimentais fundamentados em diferentes modelos te´oricos e que demandam o uso de distintos instrumentos de medida. muito menores do que a massa do corpo suspenso, pois, como o modelo de sistema massa-mola amorte- cido pressup˜oe que a mola que sustenta o corpo tem massa nula, n˜ao ser´a poss´ıvel detectar se varia¸c˜oes no per´ıodo de oscila¸c˜ao do corpo investigado s˜ao decorrentes de varia¸c˜oes na massa ou na constante el´astica das molas utilizadas. 4. Sistema de amortecimento automotivo “A lenda aﬁrma que Arquimedes teria no- tado que transbordava uma quantidade de ´agua da banheira, correspondente ao seu pr´oprio volume, quando entrava nela e que, utilizando um m´etodo semelhante, pode- ria comparar o volume da coroa com os volumes de iguais pesos de prata e ouro: bastava coloc´a-los em um recipiente cheio de ´agua, e medir a quantidade de l´ıquido derramado. Feliz com essa fant´astica des- coberta, Arquimedes teria sa´ıdo correndo nu pelas ruas gritando ‘eur´eka’! (em grego: ‘evidentemente!’)”. Historiadores da ciˆencia usam uma s´erie de argu- mentos para questionar profundamente a veracidade desse relato [40]. De maneira distinta, Giancoli [41] sugere que Arquimedes investigou a coroa do rei Hieron II utilizando outro m´etodo. O cientista teria suspendido a coroa em um dinamˆometro e compa- rado a leitura do instrumento quando a coroa estava no ar com a sua leitura quando a coroa estava imersa na ´agua. 4. Sistema de amortecimento automotivo Com base nos experimentos realizados, os estudan- tes podem avaliar a adequa¸c˜ao do modelo de sistema massa-mola amortecido para representar arranjos experimentais constitu´ıdos por corpos oscilando pre- sos em molas imersos em ﬂuidos. A partir disso, os estudantes podem aprofundar seus entendimentos sobre autom´oveis, podendo estimar o tipo de sis- tema de amortecimento mais adequado para eles, as propriedades que possibilitam que eles amorte¸cam de forma apropriada, e os problemas que ocorrem quando eles n˜ao possuem alguma das propriedades preconizadas. O mote desta atividade ´e a lenda de que Arqui- medes, a mando do rei Hieron II de Siracusa, teria conclu´ıdo que a coroa real, supostamente produzida apenas com ouro puro, continha uma parcela de prata em sua composi¸c˜ao. Martins [40] sintetiza tal lenda dizendo: p O epis´odio de modelagem “Sistema de Amorteci- mento Automotivo” evidencia aos estudantes que os experimentos podem ser delineados com o intuito de aprofundar o entendimento de problemas mais complexos do que os estudados em laborat´orio. Evi- dencia tamb´em que os experimentos s˜ao delineados, executados e analisados com base em um modelo te´orico de referˆencia. Al´em disso, o epis´odio de mo- delagem p˜oe os estudantes frente a situa¸c˜oes que podem dar sentido a conceitos da f´ısica como, por exemplo, de densidade, viscosidade e constante de tempo, assim como a conceitos relacionados com a modelagem cient´ıﬁca como os de experimento e de controle de vari´aveis. Os experimentos tamb´em envolvem o uso de conhecimentos de f´ısica como, por exemplo, de que o amortecimento do movimento de um corpo oscila preso por uma mola imerso em um ﬂuido depende da densidade e da viscosidade do ﬂuido. A atividade oportuniza ainda a realiza¸c˜ao de debates sobre as diferen¸cas entre for¸cas de arrasto inercial e de arrasto viscoso. “A lenda aﬁrma que Arquimedes teria no- tado que transbordava uma quantidade de ´agua da banheira, correspondente ao seu pr´oprio volume, quando entrava nela e que, utilizando um m´etodo semelhante, pode- ria comparar o volume da coroa com os volumes de iguais pesos de prata e ouro: bastava coloc´a-los em um recipiente cheio de ´agua, e medir a quantidade de l´ıquido derramado. Feliz com essa fant´astica des- coberta, Arquimedes teria sa´ıdo correndo nu pelas ruas gritando ‘eur´eka’! (em grego: ‘evidentemente!’)”. Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 DOI: http://dx.doi.org/10.1590/S1806-11173812080 5. Arquimedes e a coroa do rei Os processos de coleta e an´alise de dados experi- mentais s˜ao fundamentais no trabalho experimental. Desse modo, ´e necess´ario que os estudantes compre- endam o signiﬁcado das incertezas experimentais, en- tendendo os motivos pelos quais elas s˜ao intr´ınsecas ao processo de medi¸c˜ao e interpretando-as adequa- damente com base em teorias de incertezas. No entanto, pesquisas tˆem mostrado que os estudantes tˆem diﬁculdades para interpretar a dispers˜ao de con- juntos de dados experimentais [36-37]. Al´em disso, signiﬁcativa parcela dos estudantes, quando analisa os resultados de seus experimentos, considera que as predi¸c˜oes contrastadas nos seus experimentos s˜ao referˆencias absolutas, evidenciando que concebem Procurando propor uma investiga¸c˜ao sobre a plau- sibilidade da lenda de Arquimedes, ´e solicitado que os estudantes, partindo de medidas da composi¸c˜ao de amostras de chumbo e alum´ınio realizadas com dois m´etodos experimentais distintos, analisem a conﬁabilidade das conclus˜oes obtidas por meio de uma avalia¸c˜ao das incertezas das composi¸c˜oes men- suradas. Essa an´alise fornece subs´ıdios para que os estudantes avaliem tamb´em a possibilidade de Ar- quimedes ter utilizado ou n˜ao algum desses m´etodos. DOI: http://dx.doi.org/10.1590/S1806-11173812080 1504-11 Heidemann et al. Heidemann et al. Al´em de promover um debate sobre aspectos re- lacionados com a hist´oria da ciˆencia, esta atividade tem potencial para enfatizar aos estudantes que dis- tintos m´etodos podem ser utilizados com o intuito de medir uma mesma grandeza f´ısica, e que cada um desses m´etodos envolve diferentes instrumentos de medida cujos funcionamentos s˜ao suportados por modelos ou hip´oteses auxiliares. Por exemplo, um anemˆometro de Robinson tem seu funcionamento fundamentado em um modelo te´orico que relaciona a frequˆencia angular das p´as do aparelho com a velocidade do ar que o circunda. Outro exemplo ´e identiﬁcado em Aguiar e Souza [42] quando, ampa- rados em um modelo te´orico que vincula a tempera- tura de um corpo com suas dimens˜oes, os autores contrastam experimentalmente a lei de resfriamento de Newton realizando medidas da dilata¸c˜ao t´ermica de uma barra de alum´ınio. • avaliar a composi¸c˜ao de um objeto que cont´em chumbo e alum´ınio por meio de dois m´etodos experimentais distintos buscando comparar a pre- cis˜ao dos resultados obtidos com cada um deles; • analisar quais as idealiza¸c˜oes e apro- xima¸c˜oes que foram consideradas no modelo te´orico que utilizar´as; • buscar avaliar a plausibilidade f´ısica da lenda de Arquimedes. 5. Arquimedes e a coroa do rei Para auxiliar os estudantes na busca por uma solu¸c˜ao para o problema proposto, o professor pode sugerir que eles deduzam uma equa¸c˜ao matem´atica que relaciona a massa de chumbo na amostra mPb com a massa total mt da amostra, o volume total Vt da amostra e as densidades do Chumbo ρPb e do Alum´ınio ρAl. Essa equa¸c˜ao ´e exposta a seguir (a dedu¸c˜ao dessa equa¸c˜ao est´a dispon´ıvel na Ref. [43]). Em modelos te´oricos auxiliares, assim como em qualquer modelo cient´ıﬁco, considera-se uma s´erie de idealiza¸c˜oes que, por sua vez, implicam impre- cis˜oes nas medidas realizadas com os instrumentos de medida que se sustentam neles. Por exemplo, um termˆometro de bulbo tem seu funcionamento base- ado em um modelo que relaciona o volume do l´ıquido conﬁnado com a sua temperatura. Parte-se do pres- suposto de que a altura desse l´ıquido no bulbo do termˆometro ´e diretamente proporcional `a sua tem- peratura, o que nada mais ´e do que uma idealiza¸c˜ao, pois o coeﬁciente de dilata¸c˜ao de um l´ıquido sofre pequenas varia¸c˜oes quando a sua temperatura se altera. Desse modo, a temperatura mensurada com um termˆometro de bulbo n˜ao ´e incerta somente em fun¸c˜ao da imprecis˜ao da leitura do indiv´ıduo que a realiza, mas tamb´em porque a temperatura medida ´e inferida a partir de um modelo te´orico auxiliar que cont´em simpliﬁca¸c˜oes da realidade. mPb = mt −ρAl.Vt 1 −ρAl ρP b . (2) (2) Analisando a Eq. (2), pode-se concluir que, de posse das densidades do Chumbo e do Alum´ınio, a inferˆencia da composi¸c˜ao solicitada na atividade de- pende apenas de medidas da massa e do volume da amostra. A primeira dessas medidas pode ser facil- mente realizada com o uso de uma balan¸ca. J´a para medir o volume da amostra, ainda que os estudantes possam utilizar diferentes m´etodos, o professor deve sugerir que eles escolham usar os dois m´etodos expe- rimentais j´a citados nesta se¸c˜ao. No primeiro deles, o objeto investigado ´e inserido em um recipiente com ´agua e ´e medida a varia¸c˜ao do n´ıvel da ´agua no mesmo, que ´e igual ao volume da amostra. No segundo, o objeto investigado ´e suspenso em um dinamˆometro e ´e registrada a leitura do instrumento quando o objeto est´a no ar e quando ele ´e imerso na ´agua. No segundo, o volume da amostra ´e inferido a partir da mensura¸c˜ao do empuxo que ela sofreu quando imersa em ´agua. Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 6. Resfriamento de sistemas A constru¸c˜ao de um modelo cient´ıﬁco envolve a es- colha de uma s´erie de idealiza¸c˜oes e aproxima¸c˜oes, assim como de uma teoria geral. Desse modo, depen- dendo das escolhas realizadas, diferentes modelos podem ser produzidos para representar um mesmo evento real. Apesar disso, pesquisas tˆem identiﬁ- cado evidˆencias de que uma signiﬁcativa parcela dos estudantes entende que apenas um modelo pode representar corretamente um evento real [26-27]. O epis´odio de modelagem “Resfriamento de sistemas” foi delineado com o objetivo de evidenciar aos estu- dantes que diferentes modelos cient´ıﬁcos podem ser constru´ıdos com o intuito de representar o mesmo evento real, e que um modelo pode ser expandido com o objetivo de ampliar o seu dom´ınio de validade. Especiﬁcamente, ´e solicitado que os estudantes in- vestiguem o resfriamento de sistemas em eventos que extrapolam o dom´ınio de validade da lei de resfria- mento de Newton, reﬂetindo sobre modiﬁca¸c˜oes que precisam ser promovidas nessa lei para que ela repre- sente o resfriamento desses sistemas com satisfat´oria precis˜ao. tos ´e explorado: o resfriamento de uma por¸c˜ao de ´agua exposta ao ambiente n˜ao saturado de vapor d’´agua. Esse evento n˜ao ´e bem representado pela lei de resfriamento de Newton, pois, nesse caso, devido a perdas de energia por evapora¸c˜ao, a temperatura da ´agua atinge um valor menor do que a tempera- tura do ambiente que a circunda. O enunciado do problema compartilhado com os estudantes na dis- cuss˜ao inicial do epis´odio de modelagem ´e exposto abaixo. Os dados representados na Fig. 15 apresen- tam medidas de temperatura de dois pratos cheios de ´agua (um deles aberto e outro fechado) em fun¸c˜ao do tempo. Tamb´em ´e apresentada a temperatura ambiente do local onde o experimento foi realizado. No dia em que foi realizado o experimento (in´ıcio de janeiro em Porto Alegre/RS) a umidade relativa era de 70%. O local onde os dois pratos ﬁcavam era pr´oximo de uma janela que estava aberta. Assim sendo, tamb´em havia uma pequena cor- rente de ar no local. Nesta atividade vocˆe dever´a: p A lei de resfriamento de Newton tem como pressu- posto b´asico a hip´otese de que as trocas de energia por condu¸c˜ao, convec¸c˜ao e irradia¸c˜ao de um ob- jeto com o meio que o circunda s˜ao proporcionais `a diferen¸ca entre a temperatura do objeto e a tempe- ratura do ambiente [45]. 5. Arquimedes e a coroa do rei As medidas realizadas com os dois m´etodos podem ser comparadas, oportuni- zando uma discuss˜ao sobre incertezas de medidas uma vez que ´e importante se avaliar se os resultados obtidos com os dois m´etodos s˜ao condizentes. Al´em disso, a an´alise das incertezas dos dados obtidos pro- piciar´a uma reﬂex˜ao sobre a plausibilidade de que Arquimedes tenha utilizado algum dos dois m´etodos para medir a composi¸c˜ao da coroa do rei Hieron II. Outro debate que pode ser suscitado na atividade O enunciado do problema compartilhado com os estudantes na discuss˜ao inicial do epis´odio de modelagem ´e exposto abaixo. Diz a lenda que Arquimedes, a mando do rei Hieron II de Siracusa, teria conclu´ıdo que a coroa real, supostamente produzida apenas com ouro puro, continha uma par- cela de prata em sua composi¸c˜ao. Arqui- medes teria descoberto o princ´ıpio que leva seu nome durante um banho, no qual te- ria visto o n´ıvel da ´agua subir enquanto mergulhava seu corpo nela. Apesar da ve- racidade de tal relato ser profundamente questionada por historiadores da ciˆencia, o m´etodo que Arquimedes supostamente utilizou para avaliar a composi¸c˜ao da co- roa do rei ´e utilizado at´e hoje em bons laborat´orios de f´ısica. Outro debate que pode ser suscitado na atividade envolve uma an´alise das idealiza¸c˜oes consideradas nos modelos te´oricos auxiliares que amparam os dois m´etodos experimentais explorados. Por exem- plo, efeitos relacionados com a presen¸ca de pequenas bolhas de ar na superf´ıcie da amostra s˜ao conside- rados desprez´ıveis nas medidas realizadas. Al´em Nesta tarefa, vocˆe dever´a: DOI: http://dx.doi.org/10.1590/S1806-11173812080 Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 1504-12 1504-12 Atividades experimentais com enfoque no processo de modelagem cient´ıﬁca Figura 1: Representac¸˜ao gr´aﬁca da evoluc¸˜ao temporal da temperatura da ´agua contida em dois pratos (um aberto e um fechado) assim como da temperatura ambiente do local onde tais pratos resfriavam. A ´agua contida no prato aberto atinge uma temperatura menor que a temperatura ambiente. disso, caso seja usado um dinamˆometro de mola no segundo m´etodo experimental utilizado, ´e tomado como pressuposto que a elonga¸c˜ao da mola do instru- mento ´e diretamente proporcional `a for¸ca aplicada nela, o que n˜ao passa de uma idealiza¸c˜ao e implica em imprecis˜oes nas medidas realizadas. A investiga¸c˜ao proposta neste epis´odio de mode- lagem evidencia aos estudantes que as idealiza¸c˜oes consideradas nos modelos te´oricos que amparam os procedimentos experimentais inﬂuenciam direta- mente na precis˜ao dos dados coletados. 5. Arquimedes e a coroa do rei Evidencia-se ainda que a for¸ca de empuxo que age sobre um corpo imerso em um ﬂuido ´e igual ao peso de um volume de ﬂuido equivalente ao volume do corpo imerso no ﬂuido [44], o que ´e um conhecimento importante no campo da est´atica de ﬂuidos. A atividade d´a oportunidade ainda para que os estudantes deem sentido aos conceitos de empuxo e de densidade, por exemplo. Figura 1: Representac¸˜ao gr´aﬁca da evoluc¸˜ao temporal da temperatura da ´agua contida em dois pratos (um aberto e um fechado) assim como da temperatura ambiente do local onde tais pratos resfriavam. A ´agua contida no prato aberto atinge uma temperatura menor que a temperatura ambiente. 5Dados gentilmente cedidos pelo prof. Fernando Lang da Silveira (IF-UFRGS). Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 6. Resfriamento de sistemas Partindo disso, ela prediz que a temperatura de um corpo que resfria decai exponencialmente, se igualando `a temperatura do meio circundante ap´os um intervalo de tempo suﬁ- cientemente longo. As predi¸c˜oes decorrentes da lei de resfriamento de Newton, que para alguns podem parecer um tanto intuitivas, n˜ao s˜ao v´alidas para alguns eventos. Nesta atividade, um desses even- • analisar as diferen¸cas ocorridas entre a evolu¸c˜ao temporal da temperatura da ´agua contida no prato aberto e no prato fechado do experimento re- presentado na Fig. 1, identiﬁcando os 5Dados gentilmente cedidos pelo prof. Fernando Lang da Silveira (IF-UFRGS). DOI: http://dx.doi.org/10.1590/S1806-11173812080 Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 1504-13 1504-13 Heidemann et al. dade, assim como planilhas eletrˆonicas podem ser utilizadas para se analisar os dados coletados. fatores que explicam essas diferen¸cas e avaliando as modiﬁca¸c˜oes que pre- cisam ser efetuadas na lei de resfri- amento de Newton para que ela re- presente com maior precis˜ao o resfri- amento da ´agua. O enfrentamento do problema proposto nesta ati- vidade procura mostrar aos estudantes que um mo- delo te´orico pode ser expandido por meio de modi- ﬁca¸c˜oes nas simpliﬁca¸c˜oes da realidade inicialmente realizadas, ou podem alterar o grau de precis˜ao dos resultados te´oricos obtidos atrav´es do modelo. Al´em disso, essa atividade torna expl´ıcito que di- ferentes modelos podem ser usados para represen- tar um mesmo evento real. A atividade oportuniza ainda que os estudantes: a) identiﬁquem fatores que inﬂuenciam na taxa de evapora¸c˜ao da ´agua e, conse- quentemente, na sua taxa de resfriamento; b) deem sentido a conceitos como umidade relativa, tempera- tura e transferˆencia de energia, fundamentais para a compreens˜ao dos experimentos realizados. • delinear e realizar um experimento para avaliar a inﬂuˆencia dos fatores identiﬁcados no resfriamento da ´agua. • delinear e realizar um experimento para avaliar a inﬂuˆencia dos fatores identiﬁcados no resfriamento da ´agua. Mais do que construir um modelo matem´atico para representar o evento investigado, espera-se que os estudantes reﬂitam sobre poss´ıveis modiﬁca¸c˜oes que podem ser promovidas na lei de resfriamento de Newton com o objetivo de torn´a-la mais precisa em suas predi¸c˜oes sobre o resfriamento de por¸c˜oes de ´agua. 7. Considera¸c˜oes ﬁnais Ainda que a experimenta¸c˜ao tenha um papel pro- eminente no fazer cient´ıﬁco, o desenvolvimento de aulas de laborat´orio n˜ao se justiﬁca por si s´o [3-4]. ´E fundamental que o delineamento das atividades experimentais seja acompanhado de reﬂex˜oes sobre os motivos pelos quais elas podem auxiliar os estu- dantes a alcan¸carem os objetivos de aprendizagem estabelecidos. Al´em disso, ´e necess´ario ainda que a experimenta¸c˜ao seja fundamentada em referen- ciais epistemol´ogicos bem estabelecidos, de modo que as atividades n˜ao favore¸cam a constru¸c˜ao de concep¸c˜oes epistemol´ogicas ingˆenuas por parte dos estudantes. Ainda que a experimenta¸c˜ao tenha um papel pro- eminente no fazer cient´ıﬁco, o desenvolvimento de aulas de laborat´orio n˜ao se justiﬁca por si s´o [3-4]. ´ [ ]) Entre os experimentos que os estudantes podem realizar, est´a o uso de um aparato no qual por¸c˜oes de ´agua de mesma massa e mesma temperatura inicial resfriam com diferentes ´areas expostas ao ambiente. Comparando a evolu¸c˜ao temporal da temperatura e da massa dessas por¸c˜oes, a serem mensuradas no experimento, os estudantes poder˜ao identiﬁcar rela¸c˜oes entre a ´area da por¸c˜ao de ´agua exposta ao ambiente, sua taxa de evapora¸c˜ao e sua taxa de resfriamento. Outra possibilidade ´e avaliar a in- ﬂuˆencia da umidade relativa do ar no resfriamento da ´agua. Para isso, os estudantes podem realizar medidas de temperatura e massa de duas por¸c˜oes de ´agua inicialmente idˆenticas quando elas s˜ao ex- postas a meios com diferentes umidades relativas. Esses ambientes podem ser preparados com o uso de estufas, (des)umidiﬁcadores e/ou aparelhos de ar-condicionado. Os resultados desse experimento possibilitar˜ao que os estudantes compreendam que a taxa de evapora¸c˜ao da ´agua e, consequentemente, a sua taxa de resfriamento s˜ao inversamente propor- cionais `a umidade relativa do meio que a circunda. Nesta atividade o uso de recursos tecnol´ogicos pode novamente ser explorado. Como ´e demons- trado na Ref. [45], expans˜oes da lei de resfriamento de Newton que consideram trocas de energia por eva- pora¸c˜ao podem envolver equa¸c˜oes diferenciais que s´o podem ser resolvidas numericamente. Tais modelos podem ser simulados sem grandes conhecimentos de inform´atica com o uso do software Modellus. Al´em disso, como ´e demonstrado na Ref. [46], a placa Ardu´ıno pode ser utilizada para a aquisi¸c˜ao autom´atica de dados nos experimentos desta ativi- Entre os experimentos que os estudantes podem realizar, est´a o uso de um aparato no qual por¸c˜oes de ´agua de mesma massa e mesma temperatura inicial resfriam com diferentes ´areas expostas ao ambiente. 6. Resfriamento de sistemas Almeja-se que os alunos tornem-se capa- zes de apontar fatores que inﬂuenciam na taxa de resfriamento da ´agua como, por exemplo, a umi- dade relativa e a velocidade do ar circundante, a temperatura do l´ıquido e a ´area do l´ıquido exposta ao ambiente (detalhes sobre um modelo te´orico ex- pandido sobre o resfriamento da ´agua podem ser consultados na Ref. [45]). Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 DOI: http://dx.doi.org/10.1590/S1806-11173812080 7. Considera¸c˜oes ﬁnais 38, nº 1, 1504, 2016 Atividades experimentais com enfoque no processo de modelagem cient´ıﬁca Atividades experimentais com enfoque no processo de modelagem cient´ıﬁca Atividades experimentais com enfoque no processo de modelagem cient´ıﬁca 1504-14 Atividades experimentais com enfoque no processo de modelagem cient´ıﬁca 1504-14 [2] H. Kaya and U. Boyuk, European Journal of Physics Education 2, 38 (2011). ver o engajamento dos estudantes na formula¸c˜ao de quest˜oes de pesquisa, no delineamento experimental e na an´alise de dados, que entendemos ser funda- mental para que eles potencializem a aprendizagem durante as aulas de laborat´orio. Ademais, procura- mos com as discuss˜oes ﬁnais propiciar aos estudantes situa¸c˜oes para que eles desenvolvam competˆencias para se comunicar de forma oral, argumentando com base em conhecimentos cient´ıﬁcos e evidˆencias experimentais, o que ´e imprescind´ıvel na forma¸c˜ao de um bom f´ısico. [3] D. Hodson, Ense˜nanza de las Ciencias 12, 541 (1994). [4] A.T. Borges, Caderno Brasileiro de Ensino de F´ısica 24, 9 (2002). [5] A. Hofstein and V.N. Lunetta, Science Education 88, 28 (2004). [6] J. Carrascosa, D. Pe`ırez, A. Vilches e P. Valde`ıs, Ca- derno Brasileiro de Ensino de F´ısica 23, 157 (2006). [7] R.P. Feynman, Surely You’re Joking, Mr. Feynman! (W.W. Norton & Company, New York, 1997). Para favorecer esse engajamento, propomos uma metodologia de ensino que coloca os estudantes como protagonistas de suas aprendizagens, possibilitando que eles tenham, em algum n´ıvel, liberdade para direcionarem suas a¸c˜oes com o intuito de sanar suas curiosidades. A realiza¸c˜ao de uma etapa de inves- tiga¸c˜ao em que os estudantes procuram resolver um problema que cont´em um grau de abertura adequado ao n´ıvel de ensino dos alunos, como ´e sugerido nos epis´odios de modelagem, ´e uma alternativa para incentivar os estudantes a se engajarem nas ativida- des, possibilitando que eles sejam ativos nas aulas de laborat´orio, reﬂetindo sobre como os conhecimentos cient´ıﬁcos podem ser mobilizados no enfrentamento de problemas sobre eventos reais. [8] A.M.P. de Carvalho (org.), Calor e Temperatura: Um Ensino por Investiga¸c˜ao (Editora Livraria da F´ısica, S˜ao Paulo, 2003). [9] A.M.P. de Carvalho (org.), Ensino de Ciˆencias por Investiga¸c˜ao: Condi¸c˜oes para Implementa¸c˜ao em Sala de Aula (Cengage Learning, S˜ao Paulo, (2013). [10] L.A. Heidemann, I.S. Araujo e E.A. Veit, Caderno Brasileiro de Ensino de F´ısica, no prelo (2016). [11] J. Gilbert, International Journal of Science and Mathematics Education 2, 115 (2004). [12] I.T. Koponen, Science & Education 16, 751 (2007). [13] P.S. Oh and S.J. 7. Considera¸c˜oes ﬁnais Oh, International Journal of Sci- ence Education 33, 1109 (2011). [14] I.S. Araujo e E. Mazur, Caderno Brasileiro de Ensino de F´ısica 30, 362 (2013). Foge ao escopo deste artigo apresentar resultados de pesquisa relacionados com o uso dos epis´odios de modelagem em disciplinas experimentais. No en- tanto, podemos destacar que nossas investiga¸c˜oes [23, 34, 47] evidenciam que as atividades aqui des- critas d˜ao oportunidade para que os estudantes: i) construam atitudes positivas em rela¸c˜ao `as aulas de laborat´orio e ii) evoluam em suas concep¸c˜oes sobre a natureza da Ciˆencia. Em linhas gerais, os resultados mostram tamb´em que o desenvolvimento das competˆencias dos estudantes relacionadas com o processo de modelagem cient´ıﬁca proporcionado por apenas quatro epis´odios de modelagem ´e bas- tante t´ımido. Esse resultado refor¸ca a concep¸c˜ao de que atividades que coloquem os estudantes frente a situa¸c˜oes de modelagem precisam permear muitas das disciplinas do curso de f´ısica. [15] C.E. Heiner, A.I. Banet and C. Wieman, American Journal of Physics 82, 989 (2014). [16] L.A. Heidemann, A.M. Oliveira e E.A. Veit, F´ısica na Escola 11, 30 (2010). [17] J. Jackson, L. Dukerich and D. Hestenes, Science Educator 17, 10 (2008). [18] L.A. Heidemann, I.S. Araujo e E.A. Veit, Caderno Brasileiro de Ensino de F´ısica 29, 965 (2012). [19] D. Halliday, R. Resnick e J. Walker, Fundamentos de F´ısica - Vol. 2 (Editora LTC, Rio de Janeiro, 2009), 8a ed., p. 95. [20] L.A. Heidemann, I.S. Araujo e E.A. Veit, Epis´odios de Modelagem sobre Oscila¸c˜oes Mecˆanicas, Fluidos e Termodinˆamica (Hiperm´ıdia, Porto Alegre, 2015). Dispon´ıvel em http://www.if.ufrgs.br/gpef/ modelagem/hipermidia/ (acesso em 02/08/2015). [21] J. Valadares e M. Gra¸ca, Avaliando para Melhorar a Aprendizagem (Pl´atano Edi¸c˜oes T´ecnicas, Lisboa, 1998). Agradecimento Agradecemos aos professores Fernando Lang da Sil- veira e Mario Norberto Baibich pelas importantes contribui¸c˜oes que tiveram no delineamento das ativi- dades descritas neste artigo. Agradecemos tamb´em ao ´arbitro da RBEF pelas cr´ıticas dirigidas ao artigo que em muito contribu´ıram para a sua melhoria. [22] E. Panadero and A. Jonsson, Educational Research Review 9, 129 (2013). [23] L.A. Heidemann, Ressigniﬁca¸c˜ao das Atividades Ex- perimentais no Ensino de F´ısica por meio do En- foque no Processo de Modelagem Cient´ıﬁca. Tese de Doutorado, UFRGS, Porto Alegre, 2015. Dis- pon´ıvel em http://www.lume.ufrgs.br/handle/ 10183/117767 (acesso em 20/7/2015). Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 7. Considera¸c˜oes ﬁnais Comparando a evolu¸c˜ao temporal da temperatura e da massa dessas por¸c˜oes, a serem mensuradas no experimento, os estudantes poder˜ao identiﬁcar rela¸c˜oes entre a ´area da por¸c˜ao de ´agua exposta ao ambiente, sua taxa de evapora¸c˜ao e sua taxa de resfriamento. Outra possibilidade ´e avaliar a in- ﬂuˆencia da umidade relativa do ar no resfriamento da ´agua. Para isso, os estudantes podem realizar medidas de temperatura e massa de duas por¸c˜oes de ´agua inicialmente idˆenticas quando elas s˜ao ex- postas a meios com diferentes umidades relativas. Esses ambientes podem ser preparados com o uso de estufas, (des)umidiﬁcadores e/ou aparelhos de ar-condicionado. Os resultados desse experimento possibilitar˜ao que os estudantes compreendam que a taxa de evapora¸c˜ao da ´agua e, consequentemente, a sua taxa de resfriamento s˜ao inversamente propor- cionais `a umidade relativa do meio que a circunda. O enfoque inadequado das aulas de laborat´orio tradicionalmente desenvolvidas no ensino de ciˆencias tˆem suscitado uma s´erie de cr´ıticas `as atividades experimentais [3-6]. Mais do que apresentar possi- bilidades de atividades experimentais que podem ser promovidas em disciplinas de gradua¸c˜ao, visa- mos neste artigo inspirar professores e pesquisadores para o delineamento de atividades fundamentadas em concep¸c˜oes adequadas do ponto de vista epis- temol´ogico e que possibilitem que teoria e pr´atica sejam tratadas de forma integrada, demandando a reﬂex˜ao por parte dos estudantes sobre os fun- damentos te´oricos que amparam as investiga¸c˜oes realizadas. Nesta atividade o uso de recursos tecnol´ogicos pode novamente ser explorado. Como ´e demons- trado na Ref. [45], expans˜oes da lei de resfriamento de Newton que consideram trocas de energia por eva- pora¸c˜ao podem envolver equa¸c˜oes diferenciais que s´o podem ser resolvidas numericamente. Tais modelos podem ser simulados sem grandes conhecimentos de inform´atica com o uso do software Modellus. Al´em disso, como ´e demonstrado na Ref. [46], a placa Ardu´ıno pode ser utilizada para a aquisi¸c˜ao autom´atica de dados nos experimentos desta ativi- Procuramos demonstrar ainda que ´e poss´ıvel ino- var no delineamento e na condu¸c˜ao de atividades experimentais sem a necessidade de equipamentos modernos ou incomuns. Ao contr´ario, os epis´odios de modelagem descritos neste artigo envolvem o uso de arranjos experimentais e instrumentos de medida comuns em laborat´orios de f´ısica. A inova¸c˜ao est´a no tipo de problema que ´e proposto aos alunos e na forma como eles s˜ao enfrentados. Buscamos promo- DOI: http://dx.doi.org/10.1590/S1806-11173812080 Revista Brasileira de Ensino de F´ısica, vol. Referˆencias [24] K.R. Popper, Conjecturas e Refuta¸c˜oes (Editora Universidade de Bras´ılia, Bras´ılia, 1982). [1] scitation.aip.org, acessado em 20/7/2015. DOI: http://dx.doi.org/10.1590/S1806-11173812080 Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 1504-15 Heidemann et al. Arduino. Trabalho de Conclus˜ao de Curso de Licenciatura em F´ısica, IFRS (2013). Dispon´ıvel em http://200.132.6.238:8080/pergamumweb/ vinculos/00001d/00001d50.pdf (acesso em 20/7/2015). Arduino. Trabalho de Conclus˜ao de Curso de Licenciatura em F´ısica, IFRS (2013). Dispon´ıvel em http://200.132.6.238:8080/pergamumweb/ vinculos/00001d/00001d50.pdf (acesso em 20/7/2015). [25] P.K. Feyerabend, Contra o M´etodo (Francisco Alves, Rio de Janeiro, 1989). [26] S.M. Islas y M.A. Pesa, Ciencia, Docencia y Tecno- log´ıa 29, 117 (2004). [27] A. Raviolo, A. Aguilar, P. Ram´ırez y E. L´opez, Revista Electr´onica de Investigaci´on en Educaci´on en Ciencias 6, 61 (2011). [47] [47] L.A. Heidemann, I.S. Araujo e E.A. Veit, Revista Alexandria, no prelo (2015). [28] M. Bunge, Teoria e Realidade (Editora Perspectiva, S˜ao Paulo, (1974). ( ) [29] F.L. da Silveira e L.O.Q. Peduzzi, Caderno Brasi- leiro de Ensino de F´ısica 23, 26 (2006). [30] M.A. Moreira e F. Ostermann, Caderno Brasileiro de Ensino de F´ısica 10, 108 (1993). [31] S.M. Arruda e C.E. Labur´u, in: Educa¸c˜ao em Ciˆencias: Da Pesquisa `a Pr´atica Docente (Editora Escrituras, S˜ao Paulo, 1998), cap. 6, p. 53. [32] M.M. Andr´es, M.A. Pesa e J. Meneses, Parad´ıgma 27, 37 (2006). [33] A. Guillon and M. S´er´e, in: Teaching and Learning in Science Education (Kluwer Academic Publishers, New York, 2002), p. 121. [34] L.A. Heidemann, I.S. Araujo y E.A. Veit, Revista Electr´onica de Ense˜nanza de las Ciencias, submetido (2016). ( ) [35] D. Halliday, R. Resnick e J. Walker, op. cit., p. 101. [36] A. Buﬄer, F. Lubben and B. Ibrahim, International Journal of Science Education 31, 1137 (2009). [37] T.S. Volkwyn, S. Allie, A. Buﬄer and F. Lubben, Physical Review Special Topics – Physics Education Research 4, 010108 (2008). [38] F. Marineli e J. Pacca, Revista Brasileira de Ensino de F´ısica 28, 497 (2006). [39] F. Marineli y J. Pacca, Revista Electr´onica de In- vestigaci´on en Educaci´on en Ciencias 9, 13 (2014). [40] R.A. Martins, Caderno Brasileiro de Ensino de F´ısica 17, 115 (2000), p. 115. [41] D.C. Giancoli, Physics: Principles and Applications (Pearson Prentice Hall, Upper Saddle River, 2005), p. 265. [42] C.E. Aguiar e L.F. de Souza, in: Atas do XVIII Simp´osio Nacional de Ensino de F´ısica (Vit´oria, 2009). Dispon´ıvel em http://www.sbf1.sbfisica.org.br/eventos/ snef/xviii/sys/resumos/T0402-2.pdf (acesso em 20/7/2015). [43] http://www.if.ufrgs.br/gpef/modelagem/ composicao_amostra.pdf, acessado em 20/7/2015. Revista Brasileira de Ensino de F´ısica, vol. 38, nº 1, 1504, 2016 Referˆencias [44] F.L. da Silveira e A. Medeiros, Caderno Brasileiro de Ensino de F´ısica 26, 273 (2009). [45] L.A. Heidemann, I.S. Araujo, E.A. Veit e F.L. da Silveira, in: Atas do XX Simp´osio Nacional de Ensino de F´ısica (SBF, S˜ao Paulo, 2013). Dispon´ıvel em http://www.sbf1.sbfisica.org. br/eventos/snef/xx/sys/resumos/T0179-1.pdf (acesso em 20/7/2015). [46] T. Salvatori, Avalia¸c˜ao de um Modelo sobre o Resfriamento de L´ıquidos com o uso da Placa DOI: http://dx.doi.org/10.1590/S1806-11173812080
https://openalex.org/W2357763069	https://europepmc.org/articles/pmc4893159?pdf=render	English	null	Multiomic Analysis of the UV-Induced DNA Damage Response	Cell reports	2,016	cc-by	15,458	Resource Resource Multiomic Analysis of the UV-Induced DNA Damage Response Correspondence jesper.svejstrup@crick.ac.uk Multiomic Analysis of the UV-Induced DNA Damage Response Multiomic Analysis of the UV-Induced DNA Damage Response Authors Stefan Boeing, Laura Williamson, Vesela Encheva, ..., Michael Howell, Ambrosius P. Snijders, Jesper Q. Svejstrup Correspondence jesper.svejstrup@crick.ac.uk In Brief Boeing et al. investigate the UV-induced Authors Stefan Boeing, Laura Williamson, Vesela Encheva, ..., Michael Howell, Ambrosius P. Snijders, Jesper Q. Svejstrup Correspondence jesper.svejstrup@crick.ac.uk In Brief Boeing et al. investigate the UV-induced DNA damage response by combining a range of proteomic and genomic screens. A function in this response for the melanoma driver STK19 as well as a number of other factors are uncovered. Boeing et al., 2016, Cell Reports 15, 1597–1610 May 17, 2016 ª 2016 The Author(s) http://dx.doi.org/10.1016/j.celrep.2016.04.047 SUMMARY sures that RNAPII is ubiquitylated and degraded by the protea- some, enabling repair by other mechanisms (Wilson et al., 2013). sures that RNAPII is ubiquitylated and degraded by the protea- some, enabling repair by other mechanisms (Wilson et al., 2013). Importantly, bulky DNA lesions not only block RNAPII prog- ress, but also affect transcription genome-wide so that even un-damaged genes temporarily cease to be expressed (Mayne and Lehmann, 1982; Rockx et al., 2000; Proietti-De-Santis et al., 2006). The mechanisms and factors that underlie TC- NER and the more general DNA-damage-induced repression of gene expression are still poorly understood. In order to facilitate the identiﬁcation of factors and pathways in the cellular response to UV-induced DNA damage, several descriptive proteomic screens and a functional genomics screen were performed in parallel. Numerous factors could be identiﬁed with high conﬁdence when the screen results were super- imposed and interpreted together, incorporating bio- logical knowledge. A searchable database, bioLOGIC, which provides access to relevant information about a protein or process of interest, was established to host the results and facilitate data mining. Besides uncov- ering roles in the DNA damage response for numerous proteins and complexes, including Integrator, Cohe- sin, PHF3, ASC-1, SCAF4, SCAF8, and SCAF11, we uncovered a role for the poorly studied, melanoma- associated serine/threonine kinase 19 (STK19). Be- sides effectively uncovering relevant factors, the multiomic approach also provides a systems-wide overview of the diverse cellular processes connected to the transcription-related DNA damage response. Importantly, bulky DNA lesions not only block RNAPII prog- ress, but also affect transcription genome-wide so that even un-damaged genes temporarily cease to be expressed (Mayne and Lehmann, 1982; Rockx et al., 2000; Proietti-De-Santis et al., 2006). The mechanisms and factors that underlie TC- NER and the more general DNA-damage-induced repression of gene expression are still poorly understood. Cockayne syndrome B protein (CSB, also named ERCC6) plays a key role in both TC-NER and the global transcription response to DNA damage (Vermeulen and Fousteri, 2013). CSB is recruited to damage-stalled RNAPII, allowing assembly of the core NER machinery around it (Fousteri et al., 2006). CSB is also required for the subsequent DNA incisions, permit- ting lesion removal (Anindya et al., 2010). Importantly, CSB addi- tionally helps regulate global RNAPII-mediated transcription. In Brief Boeing et al. investigate the UV-induced DNA damage response by combining a range of proteomic and genomic screens. A function in this response for the melanoma driver STK19 as well as a number of other factors are uncovered. Highlights Highlights d A multiomic screening approach examines the UV-induced DNA damage response d Multiple factors are connected to the transcription-related DNA damage response d Melanoma gene STK19 is required for a normal DNA damage response d Multiple factors are connected to the transcription-related DNA damage response d Melanoma gene STK19 is required for a normal DNA damage response Boeing et al., 2016, Cell Reports 15, 1597–1610 May 17, 2016 ª 2016 The Author(s) http://dx.doi.org/10.1016/j.celrep.2016.04.047 Cell Reports Resource Multiomic Analysis of the UV-Induced DNA Damage Response Stefan Boeing,1,5 Laura Williamson,1 Vesela Encheva,2 Ilaria Gori,3 Rebecca E. Saunders,3 Rachael Instrell,3 Ozan Ayg€un,1,7 Marta Rodriguez-Martinez,1 Juston C. Weems,4 Gavin P. Kelly,5 Joan W. Conaway,4,6 Ronald C. Conaway,4,6 Aengus Stewart,5 Michael Howell,3 Ambrosius P. Snijders,2 and Jesper Q. Svejstrup1,* 1Mechanisms of Transcription Laboratory, the Francis Crick Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK 2Protein Analysis and Proteomics Laboratory, the Francis Crick Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK 3High Throughput Screening Laboratory, the Francis Crick Institute, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK 4Stowers Institute for Medical Research, Kansas City, MO 64110, USA 5Bioinformatics and Biostatistics Laboratory, the Francis Crick Institute, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK 6Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA 7Present address: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Correspondence: jesper.svejstrup@crick.ac.uk http://dx doi org/10 1016/j celrep 2016 04 047 SUMMARY Indeed, CSB is crucial for the general recovery of transcription after DNA damage (Mayne and Lehmann, 1982), in a process that is partly independent of its role in repair (Rockx et al., 2000; Proietti-De-Santis et al., 2006). CSB contains a function- ally important ubiquitin-binding domain (Anindya et al., 2010) and is itself both ubiquitylated (Groisman et al., 2003; Groisman et al., 2006) and phosphorylated (Christiansen et al., 2003), sup- porting the idea that post-translational modiﬁcations (PTMs) are important in the DNA damage response. Some CSB ubiquityla- tion is carried out by a ubiquitin ligase complex containing CSA (Groisman et al., 2006), a TC-NER factor that transfers to chromatin only after DNA damage (Kamiuchi et al., 2002). Cell Reports 15, 1597–1610, May 17, 2016 ª 2016 The Author(s) 1597 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Cell Reports 15, 1597–1610, May 17, 2016 ª 2016 The Author(s) 1597 e under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Cell Reports 15, 1597–1610, May 17, 2016 ª 2016 The Author(s) 1597 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). INTRODUCTION The proteomic screens and the siRNA screen used to investigate the damage response are outlined. UV irradiation (30 J/m2) was used for all proteomic analysis, while 15 J/m2 was used in the RNAi screen. Figure 1. Graphical Overview of the Multiomic Approach to Charting the Transcription-Related DNA Damage Response UV-induced DNA damage has effects both at the local (‘‘repairosome’’) and the global level. The proteomic screens and the siRNA screen used to investigate the damage response are outlined. UV irradiation (30 J/m2) was used for all proteomic analysis, while 15 J/m2 was used in the RNAi screen. Figure 1. Graphical Overview of the Multiomic Approach to Charting the Transcription-Related DNA Damage Response UV-induced DNA damage has effects both at the local (‘‘repairosome’’) and the global level. The proteomic screens and the siRNA screen used to investigate the damage response are outlined. UV irradiation (30 J/m2) was used for all proteomic analysis, while 15 J/m2 was used in the RNAi screen. of DNA damage on (1) the RNAPII interactome, (2) the CSB inter- actome, (3) chromatin association dynamics, (4) the protein ubiquitylome, and (5) the phosphoproteome. This was comple- mented with (6) a functional RNAi screen. Candidates were then ranked based on their performance in the screens overall and further ﬁltered for biological relevance and technical robust- ness and are searchable using a newly established database, named bioLOGIC. The multiomic approach not only conﬁrmed the involvement of well-known TC-NER factors, but also uncov- ered numerous new factors and cellular pathways that have not previously been connected to the transcription-related DNA damage response. One example is the poorly studied melanoma gene STK19. tracted from HEK293 cells at 3 hr after UV-induced DNA dam- age. We hoped this would uncover factors across the whole DNA damage response, from early events, such as DNA damage signaling and gene expression shutdown, to late events, such as post-incision repair factors and transcription-recovery proteins (see Figure 1). The proteomic screens were performed under identical conditions and all made use of quantitative stable isotope labeling by amino acids in cell culture (SILAC) prote- omics (Ong et al., 2002), enabling us to distinguish between ‘‘constitutive’’ and UV-induced interactors and modiﬁcations. Moreover, proteasome inhibition has previously been shown to prevent dissociation of certain DNA-damage-induced protein in- teractions (Groisman et al., 2006). We therefore also carried out all proteomic experiments in the presence of proteasome inhib- itor MG132. INTRODUCTION The cellular response to bulky DNA lesions, such as those induced by UV irradiation is multi-faceted. The effect of such damage on transcription is particularly complex. Bulky DNA le- sions in the transcribed strand cause stalling of RNA polymerase II (RNAPII), resulting in a block to transcript elongation. Damage- stalled RNAPII then functions as a molecular beacon that triggers transcription-coupled nucleotide excision repair (TC-NER), the process whereby DNA damage in the transcribed strand of active genes is preferentially removed (Gaillard and Aguilera, 2013). On the other hand, if the DNA lesion for some reason cannot be removed by TC-NER, a mechanism of last resort en- With these factors and mechanisms in mind, we set out to chart the transcription-related DNA damage response. In mod- ern ‘‘global screens,’’ the characteristics of thousands of pro- teins or genes can be mapped concomitantly, but it is often problematic to recognize the important candidates in a list of hundreds of scoring proteins. In the hope of addressing this dif- ﬁculty, we developed a multiomic approach. In this approach, distinct global screens were performed under the same condi- tions and the results then overlapped and integrated. Specif- ically, we used quantitative proteomics to determine the impact Interactomes PTMs Chromatin Proteome Light Heavy untreated Chromatin extraction FLAG-IP & control IP Peptide Elution Mass Spectrometry RNAPII CSB ?? ?? UV-treated Upon UV, interaction with Data integration, bioLOGIC Network analysis, identification of individual ‘hits’ 0 Transcription Global Local ! UVSSA CSB UVSSA TFIIH RPACSA-Cul Remodeler XPF- ERCC1 RNA RNAPII Phosphorylation UV-induced Tryptic digest Enrich K-Gly-Gly peptides, Mass Spectrometry Enrich Phos-peptides Ubiquitylation or Chromatin translocation - + Chromatin extraction Mass Spectrometry Nascent transcription after UV low normal high siRNAs UV Measure nascent transcription 6 12 24 48 hrs SiRNA screen Light Heavy untreated UV-treated upon UV-irradiation Light Heavy untreated UV-treated UV-irradiation DNA XPG Figure 1. Graphical Overview of the Multiomic Approach to Charting the Transcription-Related DNA Damage Response UV-induced DNA damage has effects both at the local (‘‘repairosome’’) and the global level. The proteomic screens and the siRNA screen used to investigate the damage response are outlined. UV irradiation (30 J/m2) was used for all proteomic analysis, while 15 J/m2 was used in the RNAi screen. UV-irradiation Data integration, bioLOGIC Figure 1. Graphical Overview of the Multiomic Approach to Charting the Transcription-Related DNA Damage Response UV-induced DNA damage has effects both at the local (‘‘repairosome’’) and the global level. CSB Interactome The amount of Cohesin in the RNAPII IP, as measured by the intensity-based absolute quanti- ﬁcation (iBAQ) value (reﬂecting absolute protein abundance) (Schwanha¨ usser et al., 2011), was much larger than that of RPA or CSB, for example, suggesting that Cohesin association with RNAPII increases widely; i.e., that it is not conﬁned to actual sites of DNA damage. Several factors connected to transcript elongation, such as the RNAPII CTD-kinase CDK9, the histone H3-K36me3 methyltransferase SETD2, the PAF complex, the helicase RECQL5, and the CTD-binding proteins SCAF4 and SCAF8 were also identiﬁed as UV-induced RNAPII interactors. Excitingly, several other interesting interactions were de- tected (Figure 2A; Table S1) (see also the searchable database at http://www.biologic-db.org [username: guest, password: guest01]). Only a few interactions are highlighted here. For example, the WDR82/PPP1R10/TOX4 complex was recruited to CSB upon DNA damage. This complex recognizes DNA ad- ducts generated by platinum anticancer drugs (Bounaix Mor- and du Puch et al., 2011), but a role in the UV damage response has not previously been reported. The Integrator complex, previously linked to small nuclear RNA maturation and more generally to RNAPII transcription (Baillat and Wagner, 2015), was strongly recruited as well. Interestingly, ASUN, C7ORF26, VWA9/C15orf44, DDX26B, and NABP1/2 were recruited with strikingly similar proteomic characteristics to those of the ‘‘canonical’’ integrator complex subunits (Baillat et al., 2005), supporting the idea that they are de facto Integrator subunits (Malovannaya et al., 2010). Indeed, immu- noprecipitation (IP) of FLAG-tagged C7ORF26 brought down all these proteins, except for NABP1 (Table S2). NABP1 is part of the so-called sensor of ssDNA (SOSS) complex, which participates in ATM kinase activation and repair of DSBs and contains the Integrator subunits INTS6, DDX26B, and INTS3 (Zhang et al., 2013 and references therein). Our data thus raise the interesting possibility that a complete, NABP1-con- taining Integrator ‘‘super-complex’’ is recruited to CSB upon UV irradiation. We note that it remains unknown how damage-stalled RNAPII is initially recognized. Among proteins with a TFIIS-like RNAPII- binding domain (TFS2M), TCEA1 and TCEA2 (encoding TFIIS), PHF3, and DIDO1 were detected as RNAPII interactors, but only PHF3 was recruited in response to DNA damage. The PHRF1 protein was also recruited; it contains a PHD domain, which binds methylated histone H3K36, possibly put in place by the co-recruited SETD2 protein. We also note that several proteins were lost from RNAPII upon DNA damage (Figure 2C; Table S3). Chromatin Proteome TC-NER factors such as CSA associate tightly with chromatin only upon DNA damage (Kamiuchi et al., 2002), prompting us to identify proteins that are associated with chromatin before and after DNA damage (Figure 3A; Table S4). At the same time, the chromatin proteome served as a reference proteome (input sample) for the interactomes described above. In the cases where it was tested, IP-western experiments conﬁrmed these results (Figure 2B). Proteins with a markedly increased presence in chromatin after DNA damage, such as EMC8 and the dehydrogenase HIBADH were observed, irrespective of treatment with MG132. Conversely, other proteins appeared to be depleted from chro- matin upon UV irradiation. Some of these might be candidates for UV-induced proteasomal degradation. More than 150 pro- teins were markedly lost from chromatin in response to UV irradiation in the absence of MG132, with 50 of these failing to disappear in the presence of the proteasome inhibitor. As expected, RNAPII was among the latter proteins, but other inter- esting factors, such as interacts with SPT6 1 (IWS1), also disap- peared upon DNA damage unless the proteasome was inhibited. The abundance of its partner, SPT6 (SUPT6H), was reduced after UV treatment in the absence of MG132, but not in its pres- ence (Figure 3A; Table S4). CSB Interactome For example, interactions with transcription initiation factors such as TFIIF (GTF2F), the on- coprotein MYC, mRNA capping protein CMTR1, and termination factor XRN2 were markedly reduced. Although further mecha- nistic studies will be required, these changes might help ex- plain the DNA-damage-induced, global transcription shutdown observed after UV irradiation. Surprisingly, TERF2 (otherwise known as TRF2) and the TERF2-interacting protein TERF2IP (otherwise known as RAP1) were also recruited upon UV irradiation. Although predominantly studied as telomeric proteins, TERF2 and TERF2IP have previ- ously been implicated in a general response to DNA double- strand breaks (Bradshaw et al., 2005; Williams et al., 2007), but a connection to the UV damage response has not been reported. Several other factors, such as PHF3, SETD2, PCF11, CDK9, SCAF4/SCAF8, the CTD phosphatase regulator and human ho- molog of yeast Rtt103, RPRD1B (Morales et al., 2014; Ni et al., 2014), as well as TCEB3/Elongin A1, were markedly recruited to CSB after UV irradiation as well (Table S1). CSB Interactome To uncover factors with a role in the transcription-related DNA damage response, we carried out a combination of proteomic and genomic screens. The UV-induced DNA damage response has typically been studied at early time-points (30 min to 1 hr af- ter UV exposure), but in order to also gain insight at the recovery phase, we performed all proteomics screens with material ex- CSB is the central transcription-repair coupling factor and is spe- ciﬁcally recruited to damage-stalled RNAPII. The UV-induced CSB interactome was evaluated, starting from chromatin (Ayg€un et al., 2008). Numerous proteins became recruited to CSB in response to UV irradiation, with the identiﬁcation of TC-NER 1598 Cell Reports 15, 1597–1610, May 17, 2016 and RNF168—a ubiquitin ligase involved in amplifying ubiquitin signals at sites of DNA damage (Doil et al., 2009; Marteijn et al., 2009)—were also detected. Potential components of the damage response were also uncovered. For example, the Cohe- sin complex interacted much more with RNAPII upon DNA dam- age. Cohesin has multiple functions (Dorsett and Merkenschl- ager, 2013), including a role in the response to UV damage in yeast (Nagao et al., 2004). The amount of Cohesin in the RNAPII IP, as measured by the intensity-based absolute quanti- ﬁcation (iBAQ) value (reﬂecting absolute protein abundance) (Schwanha¨ usser et al., 2011), was much larger than that of RPA or CSB, for example, suggesting that Cohesin association with RNAPII increases widely; i.e., that it is not conﬁned to actual sites of DNA damage. Several factors connected to transcript elongation, such as the RNAPII CTD-kinase CDK9, the histone H3-K36me3 methyltransferase SETD2, the PAF complex, the helicase RECQL5, and the CTD-binding proteins SCAF4 and SCAF8 were also identiﬁed as UV-induced RNAPII interactors. factors such as UVSSA, the CSA-ubiquitin ligase complex (CUL4/DDB1/CSA), and the core transcription factor II H (TFIIH) complex validating the screen. and RNF168—a ubiquitin ligase involved in amplifying ubiquitin signals at sites of DNA damage (Doil et al., 2009; Marteijn et al., 2009)—were also detected. Potential components of the damage response were also uncovered. For example, the Cohe- sin complex interacted much more with RNAPII upon DNA dam- age. Cohesin has multiple functions (Dorsett and Merkenschl- ager, 2013), including a role in the response to UV damage in yeast (Nagao et al., 2004). RNAPII Interactome Other proteins can be searched at http://www.biologic also Tables S1 and S2. RNAPII Interactome We similarly examined the changes in the RNAPII interactome upon UV irradiation (Figure 2C; Table S3). RNAPII is ubiquity- lated and degraded upon DNA damage, so for this screen we only cultured cells in the presence of proteasome inhibitor MG132. All RNAPII interactors previously detected by this approach in the absence of DNA damage (Ayg€un et al., 2008) were detected, attesting to the reproducibility of the technique. More than 70 proteins quantiﬁed in the RNAPII IP became preferentially asso- ciated with the polymerase after DNA damage. The well-known UV-induced interaction between RNAPII and CSB that takes place at the site of DNA damage was detected, validating the screen. As additional validations, the RPA complex Cell Reports 15, 1597–1610, May 17, 2016 1599 RNAPII-IP (MG132) 4 5 6 7 8 9 10 Z-score - - UV - - + - - Input IP (FLAG) CSB-FLAG Ser2p Ser7p RECQL5 C7Orf26 RPB1 INTS4 SCAF4 SCAF8 Ser5p Thr4p Tyr1p GTF2H1 B CSB-FLAG UVSSA IgG IgG IgG IgG F F F F + + + + + - - UV - - + - MG132 - Input IP (FLAG) IgG IgG IgG IgG F F F F + + + + + MG132 SILAC z-score CSB-IP SILAC z-score CSB-IP (MG132) -4 -2 0 2 4 -4 -2 0 2 4 0 4 1 2 3 1 0 2 3 4 UVSSA CUL4A/B GTF2H1 NABP1/2 TFIIH.com CSA.com RNAPII Integrator.com PAF.com PHF3 GTF2H2 INTS10 SCAF4 INTS12 INTS3 PCF11 INTS8 INTS9 ERCC8 C7ORF26 INTS4 INTS5 INTS7 INTS2 DDX26B TERF2 CPSF3L ASUN INTS6 INTS1 PPP1R10 VWA9 TERF2IP A UVSSA MYC CMTR1 GTF2F1 GTF2F2 XRN2 RNAPII Cohesin.com PHRF1 RPA1 RPA2 SCAF8 SCAF4 RECQL5 SETD2 CSB/ USP24 RNF168 PHF3 iBAQ/protein amount -4 -2 0 2 4 CSB-AP VS CSB-AP (MG132) PAF1 NEDD8 CDK9 TCEB3 RPRD1B RPRD1A ERCC3/XPB PAF.com ERCC6 C ure 2. Effect of UV-Induced DNA Damage on the CSB and RNAPII Interactomes eft: UV-induced CSB interactome, in the presence and absence of MG132 as indicated. Right: enlargement of section indicated by box on the left. F a few interesting proteins are indicated. Integrator subunits are labeled in yellow. Western blots of CSB-Flag immunoprecipitation. The CSB-FLAG panel is duplicated to indicate that the panel rows belong to the same experiment. B does not seem to enrich a speciﬁc, phosphorylated form of RNAPII (left panel). The RNAPII interactome, in the presence of MG132. Some interesting proteins are indicated. RNAPII Interactome SILAC z-score CSB-IP SILAC z-score CSB-IP (MG132) -4 -2 0 2 4 -4 -2 0 2 4 0 4 1 2 3 1 0 2 3 4 UVSSA CUL4A/B GTF2H1 NABP1/2 TFIIH.com CSA.com RNAPII Integrator.com PAF.com PHF3 GTF2H2 INTS10 SCAF4 INTS12 INTS3 PCF11 INTS8 INTS9 ERCC8 C7ORF26 INTS4 INTS5 INTS7 INTS2 DDX26B TERF2 CPSF3L ASUN INTS6 INTS1 PPP1R10 VWA9 TERF2IP A UVSSA CSB-AP VS CSB-AP (MG132) PAF1 NEDD8 CDK9 TCEB3 RPRD1B RPRD1A ERCC3/XPB - - UV - - + - - Input IP (FLAG) CSB-FLAG Ser2p Ser7p RECQL5 C7Orf26 RPB1 INTS4 SCAF4 SCAF8 Ser5p Thr4p Tyr1p GTF2H1 B CSB-FLAG UVSSA IgG IgG IgG IgG F F F F + + + + + - - UV - - + - MG132 - Input IP (FLAG) IgG IgG IgG IgG F F F F + + + + + MG132 SILAC z-score CSB-IP SILAC z-score CSB-IP (MG132) -4 -2 0 2 4 -4 -2 0 2 4 0 4 1 2 3 1 0 2 3 4 UVSSA CUL4A/B GTF2H1 NABP1/2 TFIIH.com CSA.com RNAPII Integrator.com PAF.com PHF3 GTF2H2 INTS10 SCAF4 INTS12 INTS3 PCF11 INTS8 INTS9 ERCC8 C7ORF26 INTS4 INTS5 INTS7 INTS2 DDX26B TERF2 CPSF3L ASUN INTS6 INTS1 PPP1R10 VWA9 TERF2IP A UVSSA CSB-AP VS CSB-AP (MG132) PAF1 NEDD8 CDK9 TCEB3 RPRD1B RPRD1A ERCC3/XPB SILAC z-score CSB-IP SILAC z-score CSB-IP (MG132) -4 -2 0 2 4 -4 -2 0 2 4 UVSSA TFIIH.com CSA.com RNAPII Integrator.com PAF.com A CSB-AP VS CSB-AP (MG132) CSB-AP VS CSB-AP (MG132) A - - UV - - + - - Input IP (FLAG) CSB-FLAG Ser2p Ser7p RECQL5 C7Orf26 RPB1 INTS4 SCAF4 SCAF8 Ser5p Thr4p Tyr1p GTF2H1 B CSB-FLAG UVSSA IgG IgG IgG IgG F F F F + + + + + - - UV - - + - MG132 - Input IP (FLAG) IgG IgG IgG IgG F F F F + + + + + MG132 - - UV - - + - - Input IP (FLAG) CSB-FLAG Ser2p Ser7p RPB1 Ser5p Thr4p Tyr1p B IgG IgG IgG IgG F F F F + + + + + - - Inp + MG132 B RNAPII-IP (MG132) 4 5 6 7 8 9 10 Z-score MYC CMTR1 GTF2F1 GTF2F2 XRN2 RNAPII Cohesin.com PHRF1 RPA1 RPA2 SCAF8 SCAF4 RECQL5 SETD2 CSB/ USP24 RNF168 PHF3 iBAQ/protein amount -4 -2 0 2 4 PAF.com ERCC6 C RNAPII-IP (MG132) 4 5 6 7 8 9 10 Z-score MYC CMTR1 GTF2F1 GTF2F2 XRN2 RNAPII Cohesin.com PHRF1 RPA1 RPA2 SCAF8 SCAF4 RECQL5 SETD2 CSB/ USP24 RNF168 PHF3 iBAQ/protein amount -4 -2 0 2 4 PAF.com ERCC6 C C Figure 2. RNAPII Interactome (MG132) Z-score Chromatin LDLRAP1 RPL12 RPS10 TMPO RPL7 NAP1L1 ERCC2 CDT1 CSPF3L SETD3 SNW1 CSB SUPT6H IWS1 XPC CUEDC2 USP32 PCNA DDB2 RPA1 YBX1/2 MARK2 FOXO3 HTATSF1 SMC1A SMC3 BCLAF1 BRCA1 TP53BP1 MDC1 FANCD2 FANCI THRAP3 NBN NBN RAD50 ATR consensus mot we detected 396 S/T were not described i (http://www.phospho phorylation in respon be found in Tables S Intriguingly, the Co several sites in a UV- essential part of th induced ATM/ATR p wide variety of other cated in the DNA dou phorylated after UV CHEK1, CHEK2, CLS TP53BP1 d XRCC Chr. vs. Chr. (MG132) A -10 -5 0 5 10 Z-score Chrom. (MG132) -10 -5 0 5 Z-score Chromatin MBNL2 RNAPII 0 1 2 3 4 5 0 1 2 3 4 5 EMC8 HIBADH Z-score Chrom. (MG132) Z-score Chromatin CSPF3L SETD3 SNW1 CSB SUPT6H IWS1 XPC Chr. vs. Chr. (MG132) Figure 3. Effect of UV-Induced DNA Damage on the Chromatin Proteome, Ubiquitylome, and the Phosphoproteome A (A) Left: effect of UV irradiation on the chromatin proteome in the presence and absence of MG132, as indicated. Right: enlargement of section indi- catedbyboxontheleft.Afew proteinsareindicated. (B) As in (A), but for ubiquitylation. (C) As in (A) and (B), but phosphorylation. Other proteins can be searched at http://www.biologic- db.org. See also Tables S3, S4, and S5. Z-score Ubiq. (MG132) B Z-score Ubiquitylation -5 0 5 10 -5 0 5 10 0 2 4 6 8 10 0 2 4 6 8 10 Z-score Ubiquitylation Z-score Ubiq. (MG132) Ubi. vs. Ubi. (MG132) LDLRAP1 RPL12 RPS10 TMPO RPL7 NAP1L1 ERCC2 CDT1 CUEDC2 PCNA DDB2 RPA1 YBX1/2 FANCD2 FANCI Ubi. vs. Ubi. (MG132) B after UV irradiation as well. A connection between YBX proteins and the DNA dam- age response has not previously been re- ported, but the fact that elevated levels of these proteins occur in a number of human malignancies and is associated with poor prognosis and disease recurrence (Kos- nopfel et al., 2014), is potentially signiﬁcant in this connection. Interestingly, however, the group of proteins that appeared to have the most marked increase in site- speciﬁc ubiquitylation comprised ribo- some proteins and included RPS10, RPL7, and RPL12 (Figure 3B; Table S5). Phos. vs. Phos. RNAPII Interactome (MG132) Z-socre Phos (MG132) C Z-score Phosphorylation -5 0 5 -5 0 5 10 -5 0 5 -5 0 5 -10 -5 0 5 10 Z-score Phosphorylation Z-socre Phos (MG132) 0 2 4 6 8 10 0 2 4 6 8 10 USP32 MARK2 FOXO3 HTATSF1 SMC1A SMC3 BCLAF1 BRCA1 TP53BP1 MDC1 THRAP3 NBN NBN RAD50 Phos. vs. Phos. (MG132) C UV Phosphoproteome We also recorded the UV-induced phos- phoproteome (Figure 3C; Table S6A). Serine, threonine, and tyrosine phosphor- ylation sites were detected. Of these, 543 serines, 91 threonines, and 1 tyrosine (MAPK9 Y185) were markedly more phos- phorylated in response to UV irradiation. As expected, damage-induced phos- phorylation of H2AX (H2AFX) at serine 140 was detected (gH2AX). hosphorylation By analyzing the sequence motifs that increase in phosphorylation status after UV irradiation, we found that the ATM/ ATR consensus motif S/T-Q was generally enriched. In total, we detected 396 S/TQ phosphorylation sites. Forty-ﬁve of these were not described in the phosphosite plus reference database (http://www.phosphosite.org) and 14 of these increased in phos- phorylation in response to UV irradiation. Lists of these sites can be found in Tables S6B and S6C. Z-score Phosphorylation RNAPII Interactome Effect of UV-Induced DNA Damage on the CSB and RNAPII Interactomes g g (A) Left: UV-induced CSB interactome, in the presence and absence of MG132 as indicated. Right: enlargement of section indicated by box on the left. For clarity, only a few interesting proteins are indicated. Integrator subunits are labeled in yellow. (B) Western blots of CSB-Flag immunoprecipitation. The CSB-FLAG panel is duplicated to indicate that the panel rows belong to the same experiment. Note that CSB does not seem to enrich a speciﬁc, phosphorylated form of RNAPII (left panel). (C) The RNAPII interactome, in the presence of MG132. Some interesting proteins are indicated. Other proteins can be searched at http://www.biologic-db.org. See also Tables S1 and S2. 1600 Cell Reports 15, 1597–1610, May 17, 2016 UV Ubiquitylome We next used SILAC proteomics in combination with afﬁnity puriﬁcation of ubiquitin remnants to identify >10,000 ubiquityla- tion sites, proteome-wide. Of these, 900 were affected by DNA damage. As a positive control, and consistent with prior work by others (Povlsen et al., 2012; Elia et al., 2015b), we de- tected markedly increased levels of RPA1- (K163, K167, K331), PCNAK164-, FANCIK523-, and FANCD2K561 ubiquitylation upon UV treatment (Figure 3B; Table S5). Ubiquitylation of Cohesin subunits changed markedly upon DNA damage: RAD21K573 became ubiquitylated, while SMC1A appears to become de-ubiquitylated at several sites, further rein- forcing the connection suggested by the RNAPII interactome. The YBX proteins, involved in both transcriptional and translational t l (M t t d B 2005) b h il bi it l t d Z-score Ubiq. (MG132) B Phos. vs. Phos. (MG132) Z-socre Phos (MG132) C Z-score Ubiquitylation -5 0 5 10 Z-score Phosphorylation -5 0 5 -5 0 5 10 Chr. vs. Chr. (MG132) A -10 -5 0 5 10 Z-score Chrom. (MG132) -10 -5 0 5 Z-score Chromatin MBNL2 RNAPII 0 1 2 3 4 5 0 1 2 3 4 5 -5 0 5 10 0 2 4 6 8 10 0 2 4 6 8 10 Z-score Ubiquitylation Z-score Ubiq. (MG132) -5 0 5 -5 0 5 -10 -5 0 5 10 Z-score Phosphorylation Z-socre Phos (MG132) 0 2 4 6 8 10 0 2 4 6 8 10 EMC8 HIBADH Ubi. vs. Ubi. (MG132) Z-score Chrom. UV Ubiquitylome We next used SILAC proteomics in combination with afﬁnity puriﬁcation of ubiquitin remnants to identify >10,000 ubiquityla- tion sites, proteome-wide. Of these, 900 were affected by DNA damage. As a positive control, and consistent with prior work by others (Povlsen et al., 2012; Elia et al., 2015b), we de- tected markedly increased levels of RPA1- (K163, K167, K331), PCNAK164-, FANCIK523-, and FANCD2K561 ubiquitylation upon UV treatment (Figure 3B; Table S5). Intriguingly, the Cohesin complex was also phosphorylated at several sites in a UV-induced manner. Nipped-B-like (NIPBL), an essential part of the Cohesin loading-complex, had a UV- induced ATM/ATR phosphorylation site as well. Moreover, a wide variety of other proteins, which have primarily been impli- cated in the DNA double strand break response were also phos- phorylated after UV irradiation. These included ATRIP, BRCA1, CHEK1, CHEK2, CLSPN, FANCD2, MDC1, NBN, RAD50, TIPIN, TP53BP1, and XRCC4, BCLAF1, and THRAP3. Ubiquitylation of Cohesin subunits changed markedly upon DNA damage: RAD21K573 became ubiquitylated, while SMC1A appears to become de-ubiquitylated at several sites, further rein- forcing the connection suggested by the RNAPII interactome. The YBX proteins, involved in both transcriptional and translational control (Matsumoto and Bay, 2005), became heavily ubiquitylated Cell Reports 15, 1597–1610, May 17, 2016 1601 RNAi Screen A genome-wide small interfering RNA (siRNA) screen, surveying gene products that affect transcription after UV irradiation, com- plemented the proteomic screens above. Brieﬂy, Dharmacon siRNA SMARTpools were used to induce knockdown. Upon UV irradiation, cells were allowed to recover for 18 hr before nascent transcription was measured (Figures 4A and 4B). siRNA pools targeting CSB and RNAPII, which should both decrease nascent RNA synthesis (CSB knockdown speciﬁcally so after UV irradiation), were included as positive controls, while non-tar- geting siRNAs, and siRNAs that are not taken up by the RNA- induced silencing (RISC) complex and thus do not lead to knock- down of any gene (RISC-free siRNA), were included as negative controls. In the absence of UV irradiation, average nascent tran- scription per nucleus (as measured by 50 ethynyl uridine [EU] incorporation) followed a normal distribution (Figure 4C, No UV). However, in response to UV, a distinct population of lowly transcribing cells was clearly detectable, even after 18–20 hr (Figure 4C, Control). siRNAs giving rise to low transcription mark- edly increased the percentage of such cells (Figure 4C, Low). UV Ubiquitylome Other siRNAs resulted in a signiﬁcant shift of the proﬁle toward the right, suggesting high levels of transcription in these cells (Figure 4C, High). The results from the genome-wide screen are summarized in Figure 4D (see also Table S7). siRNAs target- ing NER- or TC-NER-related gene products such as ERCC1, XAB2, HIRA, ERCC5 (XPG), TTDA, and ERCC4 (XPF) resulted in low transcription, and the known NER factors were generally Transfection of a 21,120 pool siRNA library into MRC5VA cells 48 hrs UV-irradiation Labelling of nascent RNA by 5’ethyl uridine (EU) pulse Covalent attachment of flurophore to EU-labelled RNA Scanning siRNA leading to a Low transcribing phenotype Control siRNA DAPI DAPI DAPI EU EU EU Incomplete repair No transcription recovery Lower overall transcription No transcription shut-down Strong transcription recovery Higher overall transcription siRNA leading to a High transcribing phenotype 19.1% 69.7% 6.5% Control siRNA Low High A 18 hrs 2 hrs B C D 4.2% No UV UV, 18 hrs N=1005 N=539 18111 genes -2 0 2 4 6 RNAi High Transcription score SAMD4B INTS12 INTS2 ACIN1 HTATSF1 SNW1 ERCC5 CSB MED20 LTN1 PKP1 C6orf52 -4 -2 0 2 4 RNAi Low Transcription score TCERG1 HIRA Although a signiﬁ missed due to RNAi the results from the important as they in teins uncovered in t Data Integration: Pathways In all proteomic and gene hits are genera which of the ‘‘hits’’ a approach, informati signiﬁcantly enrich i greatly increasing c addition, such data i mation about the p involvement of a giv ples of this in the d teome to illustrate th It was immediate previously been co damage response m itive transcription elo ing CDK9 kinase). UV Ubiquitylome 4 6 SAMD4B S12 cription score TCERG1 ACIN1, HTATSF1, STK19, SAMD4B, LARP7L, HNRNPCL1, NAE1, NOP58, PRPF31, EXOSC3, FIP1L1, MOV10, PAXIP1, ISY1, SMU1, and SNW1 (low transcribers), as well as MED20, LTN1, and PKP1 (high transcribers). Although a signiﬁcant number of genuine RNAi hits are likely missed due to RNAi-induced lethality or insufﬁcient knockdown, the results from the functional genomics screen are particularly important as they indicate the functional signiﬁcance of the pro- teins uncovered in the descriptive proteomic screens. UV Ubiquitylome Fi more with both RNA suggesting a dama Moreover, 21 know RNAi Screen A genome-wide small interfering RNA (siRNA) screen surveying Transfection of a 21,120 pool siRNA library into MRC5VA cells 48 hrs UV-irradiation Labelling of nascent RNA by 5’ethyl uridine (EU) pulse Covalent attachment of flurophore to EU-labelled RNA Scanning siRNA leading to a Low transcribing phenotype Control siRNA DAPI DAPI DAPI EU EU EU Incomplete repair No transcription recovery Lower overall transcription No transcription shut-down Strong transcription recovery Higher overall transcription siRNA leading to a High transcribing phenotype 19.1% 69.7% 6.5% Control siRNA Low High A 18 hrs 2 hrs B C D 4.2% No UV UV, 18 hrs N=1005 N=539 18111 genes -2 0 2 4 6 RNAi High Transcription score SAMD4B INTS12 INTS2 ACIN1 HTATSF1 SNW1 ERCC5 CSB MED20 LTN1 PKP1 C6orf52 -4 -2 0 2 4 RNAi Low Transcription score TCERG1 HIRA Although a signiﬁ missed due to RNA Figure 4. An siRNA Screen for Genes Affecting Transcription upon UV Irradiation (A) The experimental approach. (B) Typical examples of siRNAs that result in either (left) ‘‘low transcription,’’ or (right) ‘‘high transcrip- tion,’’ relative to the controls (middle). Different putative causes (not necessarily mutually exclu- sive) of the outcome are listed below arrows. (C) Nascent transcription proﬁles across a cell population in the absence of UV irradiation, and in the examples from (B), used to identify siRNAs giving rise to low and high transcription, respec- tively. EU intensity (y axis) across the population of cells in an individual plate well (x axis) is shown. (D) Graphical representation of the screen result. High transcribers are labeled green, and low tran- scribers are red. Speciﬁc genes are indicated. Other proteins can be searched at http://www. biologic-db.org. Transfection of a 21,120 pool siRNA library into MRC5VA cells 48 hrs UV-irradiation Labelling of nascent RNA by 5’ethyl uridine (EU) pulse Covalent attachment of flurophore to EU-labelled RNA Scanning A 18 hrs 2 hrs siRNA leading to a Low transcribing phenotype Control siRNA DAPI DAPI DAPI EU EU EU Incomplete repair No transcription recovery Lower overall transcription No transcription shut-down Strong transcription recovery Higher overall transcription siRNA leading to a High transcribing phenotype B A B Figure 4. An siRNA Screen for Genes Affecting Transcription upon UV Irradiation (A) The experimental approach. RNAi Screen However, in response to UV, a distinct population of lowly transcribing cells was clearly detectable, even after 18–20 hr (Figure 4C, Control). siRNAs giving rise to low transcription mark- edly increased the percentage of such cells (Figure 4C, Low). Other siRNAs resulted in a signiﬁcant shift of the proﬁle toward the right, suggesting high levels of transcription in these cells (Figure 4C, High). The results from the genome-wide screen are summarized in Figure 4D (see also Table S7). siRNAs target- ing NER- or TC-NER-related gene products such as ERCC1, XAB2, HIRA, ERCC5 (XPG), TTDA, and ERCC4 (XPF) resulted in low transcription, and the known NER factors were generally UV Ubiquitylome siRNA leading to a High transcribing phenotype Transfection of a 21,120 pool siRNA library into MRC5VA cells A Control siRNA (B) Typical examples of siRNAs that result in either (left) ‘‘low transcription,’’ or (right) ‘‘high transcrip- tion,’’ relative to the controls (middle). Different putative causes (not necessarily mutually exclu- sive) of the outcome are listed below arrows. (C) Nascent transcription proﬁles across a cell population in the absence of UV irradiation, and in the examples from (B), used to identify siRNAs giving rise to low and high transcription, respec- tively. EU intensity (y axis) across the population of cells in an individual plate well (x axis) is shown. (D) Graphical representation of the screen result. High transcribers are labeled green, and low tran- scribers are red. Speciﬁc genes are indicated. Other proteins can be searched at http://www. biologic-db.org. Incomplete repair No transcription recovery Lower overall transcription 19.1% 69.7% 6.5% Control siRNA Low High C 4.2% No UV UV, 18 hrs D N=1005 N=539 18111 genes -2 0 2 4 6 RNAi High Transcription score SAMD4B INTS12 INTS2 ACIN1 HTATSF1 SNW1 ERCC5 CSB MED20 LTN1 PKP1 C6orf52 -4 -2 0 2 4 RNAi Low Transcription score TCERG1 HIRA C D signiﬁcantly represented in the list of siRNA targets that reduced transcription after UV irradiation (p = 0.01142), vali- dating the approach. A number of inter- esting factors affected transcription in this screen, including INTS2, INTS12, ACIN1, HTATSF1, STK19, SAMD4B, LARP7L, HNRNPCL1, NAE1, NOP58, PRPF31, EXOSC3, FIP1L1, MOV10, PAXIP1, ISY1, SMU1, and SNW1 (low transcribers), as well as MED20, LTN1, and PKP1 (high transcribers). N=1005 4 6 SAMD4B S12 ACIN1 HTATSF1 SNW1 ERCC5 CSB cription score TCERG1 HIRA signiﬁcantly represented in the list of siRNA targets that reduced transcription after UV irradiation (p = 0.01142), vali- dating the approach. A number of inter- esting factors affected transcription in this screen, including INTS2, INTS12, ACIN1, HTATSF1, STK19, SAMD4B, LARP7L, HNRNPCL1, NAE1, NOP58, PRPF31, EXOSC3, FIP1L1, MOV10, PAXIP1, ISY1, SMU1, and SNW1 (low transcribers), as well as MED20, LTN1, and PKP1 (high transcribers). Although a signiﬁcant number of genuine RNAi hits are likely missed due to RNAi-induced lethality or insufﬁcient knockdown, the results from the functional genomics screen are particularly important as they indicate the functional signiﬁcance of the pro- teins uncovered in the descriptive proteomic screens. RNAi Screen RNAi Screen A genome-wide small interfering RNA (siRNA) screen, surveying gene products that affect transcription after UV irradiation, com- plemented the proteomic screens above. Brieﬂy, Dharmacon siRNA SMARTpools were used to induce knockdown. Upon UV irradiation, cells were allowed to recover for 18 hr before nascent transcription was measured (Figures 4A and 4B). siRNA pools targeting CSB and RNAPII, which should both decrease nascent RNA synthesis (CSB knockdown speciﬁcally so after UV irradiation), were included as positive controls, while non-tar- geting siRNAs, and siRNAs that are not taken up by the RNA- induced silencing (RISC) complex and thus do not lead to knock- down of any gene (RISC-free siRNA), were included as negative controls. In the absence of UV irradiation, average nascent tran- scription per nucleus (as measured by 50 ethynyl uridine [EU] incorporation) followed a normal distribution (Figure 4C, No UV). However, in response to UV, a distinct population of lowly transcribing cells was clearly detectable, even after 18–20 hr (Figure 4C, Control). siRNAs giving rise to low transcription mark- edly increased the percentage of such cells (Figure 4C, Low). Other siRNAs resulted in a signiﬁcant shift of the proﬁle toward the right, suggesting high levels of transcription in these cells (Figure 4C, High). The results from the genome-wide screen are summarized in Figure 4D (see also Table S7). siRNAs target- ing NER- or TC-NER-related gene products such as ERCC1, XAB2, HIRA, ERCC5 (XPG), TTDA, and ERCC4 (XPF) resulted in low transcription, and the known NER factors were generally A genome-wide small interfering RNA (siRNA) screen, surveying gene products that affect transcription after UV irradiation, com- plemented the proteomic screens above. Brieﬂy, Dharmacon siRNA SMARTpools were used to induce knockdown. Upon UV irradiation, cells were allowed to recover for 18 hr before nascent transcription was measured (Figures 4A and 4B). siRNA pools targeting CSB and RNAPII, which should both decrease nascent RNA synthesis (CSB knockdown speciﬁcally so after UV irradiation), were included as positive controls, while non-tar- geting siRNAs, and siRNAs that are not taken up by the RNA- induced silencing (RISC) complex and thus do not lead to knock- down of any gene (RISC-free siRNA), were included as negative controls. In the absence of UV irradiation, average nascent tran- scription per nucleus (as measured by 50 ethynyl uridine [EU] incorporation) followed a normal distribution (Figure 4C, No UV). g Pathways In all proteomic and genomic approaches, long lists of protein/ gene hits are generated, but it can often be difﬁcult to determine which of the ‘‘hits’’ are biologically meaningful. In the multiomic approach, information from the other screens can be used to signiﬁcantly enrich information gained from a screen in question, greatly increasing conﬁdence in the relevance of the result. In addition, such data integration can often provide important infor- mation about the potential molecular mechanisms behind the involvement of a given gene/protein. There are numerous exam- ples of this in the data, but here we focus on the phosphopro- teome to illustrate the point. It was immediately apparent that some kinases that had not previously been connected to the transcription-related DNA damage response must play an important role, such as the pos- itive transcription elongation factor b (pTEFb) complex (contain- ing CDK9 kinase). First, we found that CDK9 itself interacts much more with both RNAPII and CSB upon DNA damage, strongly suggesting a damage-induced role in transcription or repair. Moreover, 21 known CDK9 partners and interactors featured a 1602 Cell Reports 15, 1597–1610, May 17, 2016 A B SRPK1 BCLAF1 THRAP3 HNRNPM SRRM2 STAU1 PNN ALYREF PAXIP1 FLOT1 PPIG NKAP CHEK2 NELFE HSP90AA1 USP39 MAPK14 C18ORF25 HMGN3 SRSF12 •Ub EIF4A3 ACIN1 •Ub •Ub HNRNPA1 OXSR1 SKIV2L •Ub PLEC TRAP1 SMAD1 TRIP12 RUVBL1 RUVBL2 SETD1B PAXIP1 TAL1 TRIM33 UBE2A SNW1 CASK CCNT2 MED28 ATRIP CLSPN SUPT5H MED1 BRD4 BCL10 FKBP5 MATR3 BRCA1 MEPCE LARP7 TRIM28 MDFIC CCT3 UBR5 TAF7 MED26 AFF4 MYBL2 HSP90AA1 •Ub •Ub •Ub •Ub •Ub CDK9 HTATSF1 Figure 5. Enriching the Phosphoproteome with Results from the Other Screens (A) Proteins that interact with CDK9 (pTEFb) and that become phosphorylated upon UV irradiation. Proteins are labeled increasingly blue with increasing phosphorylation. Proteins that scored in the RNAi screen, (squares with red border), interacted with RNAPII (small green spheres under name), interacted with CSB (red spheres), or became ubiquitylated upon UV irradiation (yellow ‘‘dUb’’) are indicated. Examples of CSB or RNAPII interactions that increased (black circle around spheres) or decreased (yellow circle around spheres) upon UV irradiation are also speciﬁed. (B) As in (A), but for proteins that interact with SRPK1. g Pathways A HNRNPA1 OXSR1 SKIV2L •Ub PLEC TRAP1 SMAD1 TRIP12 RUVBL1 RUVBL2 SETD1B PAXIP1 TAL1 TRIM33 UBE2A SNW1 CASK CCNT2 MED28 ATRIP CLSPN SUPT5H MED1 BRD4 BCL10 FKBP5 MATR3 BRCA1 MEPCE LARP7 TRIM28 MDFIC CCT3 UBR5 TAF7 MED26 AFF4 MYBL2 HSP90AA1 •Ub •Ub •Ub •Ub •Ub CDK9 HTATSF1 pTEFb in a protein phosphatase (PP2B/PPP3CA and PP1a/ PPP1CA)-dependent manner (Chen et al., 2008). Interestingly, PPP1CA scored in the siRNA screen, and both PPP1CA and its regulatory subunit PPP1R10 interacted with RNAPII and CSB. Indeed, PPP1R10 was markedly recruited to CSB upon UV irradiation, like CDK9. PPP1R10 contains a domain, TFIIS-N, which is also found in transcription proteins such as MED26, Elongin A, IWS1, and TFIIS, raising the intriguing possi- bility that this domain is important for PPP1R10’s proposed role in transcription and in the transcription-related DNA damage response in particular. Together, these results place PTEFb (CDK9 and its cyclin partners) at the core of the transcription- related DNA damage response. A Several well-known DNA-damage kinases were associated with a large number of increasingly phosphorylated proteins. For example, ATM kinase has 26 known interactors that were increasingly phosphorylated (22.8%), and ATR (16; 19.5%) and CHEK2 (10; 18.9%) had many as well (see Table S8). These ki- nases are all best known for their role in signaling double-strand breaks, reinforcing the connection to this pathway observed in several of our screens. B SRPK1 BCLAF1 THRAP3 HNRNPM SRRM2 STAU1 PNN ALYREF PAXIP1 FLOT1 PPIG NKAP CHEK2 NELFE HSP90AA1 USP39 MAPK14 C18ORF25 HMGN3 SRSF12 •Ub EIF4A3 ACIN1 •Ub •Ub Fi 5 E i hi th Ph h t ith R lt f th B We also further examined potential transcription-related DNA damage-signaling kinases by focusing on those that scored in the RNAi screen and were associated with a large number of UV-induced phosphorylation events. These were SRPK1 (asso- ciated with 20 proteins showing UV-induced phosphorylation [9.5% of its interactors]), CSNK2A2 and ILK (both 15; 13.1% and 7.8%, respectively), CLK2 (13; 20.6%), and CDK8 (12; 16.4%). Forty-ﬁve other protein kinases scored in the RNAi screen, and 35 of these were associated with at least one UV- induced phosphorylation event. p p y A network analysis of SRPK1-associated proteins revealed that the two most highly phosphorylated SRPK1-interacting proteins are the tumor-associated genes BCLAF1 and THRAP3, both of which interact with both RNAPII and CSB (Figure 5B, blue spheres; Table S8). Data Integration—Intersecting and Weighting Individual Screens Data Integration—Intersecting and Weighting Individual Screens g Pathways Another SRPK1-interacting phosphoprotein, apoptotic chromatin condensation inducer 1 (ACIN1), is particu- larly interesting as it also interacts with both RNAPII and CSB and scores as a low transcriber in the siRNA screen. SRPK1 has been reported as a cisplatin sensitivity factor (Schenk et al., 2001), providing an intriguing link to NER. In general, SRPK1, BCLAF1, THRAP3, and ACIN1 are associated with several net- worked proteins (PAXIP1, HNRNPM, SRRM2, EIF4A3, PNN, ALYREF, and STAU1) that also scored in many of our screens, including the RNAi screen (Figure 5B). Together, these data sug- gest a previously unrecognized role for the splicing-related kinase SRPK1 and its network partners in the transcription-related DNA damage response. Numerous other examples of affected path- ways can be analyzed by the use of bioLOGIC. Figure 5. Enriching the Phosphoproteome with Results from the Other Screens Enriching the Phosphoproteome with Results from th (A) Proteins that interact with CDK9 (pTEFb) and that become phosphorylated upon UV irradiation. Proteins are labeled increasingly blue with increasing phosphorylation. Proteins that scored in the RNAi screen, (squares with red border), interacted with RNAPII (small green spheres under name), interacted with CSB (red spheres), or became ubiquitylated upon UV irradiation (yellow ‘‘dUb’’) are indicated. Examples of CSB or RNAPII interactions that increased (black circle around spheres) or decreased (yellow circle around spheres) upon UV irradiation are also speciﬁed. (B) As in (A) but for proteins that interact with SRPK1 (B) As in (A), but for proteins that interact with SRPK1. UV-induced phosphorylation event (12.5%) (Figure 5A; Table S8). Among the CDK9 interactors, numerous scored in the RNAi screen and many interacted with RNAPII, CSB, or both. Importantly, one of the CDK9 cyclins, CCNT2, scored in the RNAi screen. HTATSF1, a pTEFb partner (Zhou et al., 1998), was strongly phosphorylated at several sites upon UV irradiation and scored as a low-transcriber in the RNAi screen as well. In addition, LARP7 regulates pTEFb activity by binding to and sta- bilizing 7SK RNA (He et al., 2008), which is in turn released from Screens gg g •TCEB2 •HIRA •ISY1 •COPS7B •ERCC1 •EAF1 •THOC1 •POLR2L •TCEA1 •POLR2H •POLR2D •PAF1 •SUPT16H •CTR9 •SUPT4H •CNOT3 •PCNA •ERCC2 •POLR2A •THOC2 •LEO1 •POLR2B •CUL4B •ERCC8 •GTF2H2 •GPS1 •BRCA1 •TCEB3 •DOT1L •HMGN1 •SUPT5H •RPA1 •ERCC6 •PPIE •CUL2 •COPS8 •ERCC5 •CNOT8 •XAB2 •GTF2H4 •DDB1 •WDR61 •POLR2G •POLR2C •CDC73 •SSRP1 •TCEA2 •POLE2 •VCP •PCNA •PARP1 •POLR2E •POLR2J •ATR •CUL4A •UVSSA •ERCC3 •GTF2H1 •COPS2 •TP53BP1 •RFC1 •MDC1 •ALYREF •UBE2N •RPA3 •RPA2 Ubi CSB AP RNAi-High RNAi-Low RNAP AP (MG132) CSB AP (MG132) Phospho Phospho (MG132) Ubi (MG132) Chromatin Chromatin (MG132) E E It is important to emphasize that there is no single ‘‘correct way’’ of compiling score lists. However, if a factor scores highly no matter which method is used, this obviously increases conﬁdence. Nevertheless, even factors that only scored highly by one or two methods might still be interesting and included with high conﬁdence after an assessment of the un- derlying core data. A non-exhaustive list of high-scoring pro- teins, which we thought to be of particular interest and of high conﬁdence, is shown in Figure 6D. It is important to emphasize that there is no single ‘‘correct way’’ of compiling score lists. However, if a factor scores highly no matter which method is used, this obviously increases conﬁdence. from almost 2,200 proteins scoring in one screen, to only two genes, RPA1 and ASCC3, scoring in six (Figure 6A; Table S9). Realizing that setting arbitrary Z score threshold for inclusion might not be ideal, we also ranked candidates based on aggre- gate Z scores (Figure 6B; Table S9). None of these approaches take into account the possibility that some screens might be much better at uncovering relevant factors than others. To address this, we created a comprehensive list of ‘‘transcription- repair coupling factors’’ (Table S10), based on an authoritative recent review (Gaillard and Aguilera, 2013) and our own knowl- edge of the published literature. This category was then used as a ‘‘training category’’ to benchmark the individual experiments Next, we determined which cellular pathways are enriched in the list of high-scoring proteins (Table S11). For simplicity, this analysis was performed with the data obtained by weighted scoring (Figure 6C), but similar results were achieved using the other scoring approaches (data not shown). Screens Finding new factors with a role in the transcription-related DNA damage response was the main initial motivation for this work. To uncover such factors, we used several different approaches to create ranked score lists. Initially, we simply awarded a point to each gene/protein scoring above the Z score threshold in an individual experiment. This yielded a distribution of scores, Cell Reports 15, 1597–1610, May 17, 2016 1603 ASCC3 CSB SMC3 A TCR ALL 1.00 355 80 7 3 2 SMC3 0 1 2 3 4 5 6 ASCC3 RPA1 RPS6 RPL19 FANCD2 EIF3A TRIM28 INTS2 NUMA1 -2 -1 0 1 2 0.17 Equal weight -2 -1 0 1 2 3 2187 CSB/ERCC6 Weighted 0.41 RNAPII- and transcription-related: ASC complex Integrator super-complex PCF11 SCAF4 SCAF8 SCAF11 PHF3 HTATSF1 TCERG1 RPRD1A/B RPRD2 Splicing: HNRNPCL1 SNW1/SNW1-complex SMU1 ACIN1 (Acinus) Chromatin: Cohesin complex SETD2 MeCP1 complex Connections to other complexes and pathways: Nop56p-associated pre- rRNA complex ANAPC2 (APC) UBD (FAT10, ubiquitin-like) WDR82/PPP1R10/TOX4 complex Interesting enzymes of unknown connection: SRPK1 HIBADH PPP1CA PPP1R10 PPP1R12A PARD3 HERC1 STK19 PDIA3 P4HB Miscellanous: YBX1 and 2 CACYBP HYLS1 (Hydroethalus syndrome) TMPO (nuclear lamina) D HERC1 CSB TMPO CACYBP ERCC2 B C TCR ALL TCR ALL PCF11 Number of proteins Number of proteins Number of proteins Point score Av. aggregate z-score/screen Av. aggregate z-score/screen •TCEB2 •HIRA •ISY1 •COPS7B •ERCC1 •EAF1 •THOC1 •POLR2L •TCEA1 •POLR2H •POLR2D •PAF1 •SUPT16H •CTR9 •SUPT4H •CNOT3 •PCNA •ERCC2 •POLR2A •THOC2 •LEO1 •POLR2B •CUL4B •ERCC8 •GTF2H2 •GPS1 •BRCA1 •TCEB3 •DOT1L •HMGN1 •SUPT5H •RPA1 •ERCC6 •PPIE •CUL2 •COPS8 •ERCC5 •CNOT8 •XAB2 •GTF2H4 •DDB1 •WDR61 •POLR2G •POLR2C •CDC73 •SSRP1 •TCEA2 •POLE2 •VCP •PCNA •PARP1 •POLR2E •POLR2J •ATR •CUL4A •UVSSA •ERCC3 •GTF2H1 •COPS2 •TP53BP1 •RFC1 •MDC1 •ALYREF •UBE2N •RPA3 •RPA2 Ubi CSB AP RNAi-High RNAi-Low NAP AP (MG132) CSB AP (MG132) Phospho Phospho (MG132) Ubi (MG132) Chromatin hromatin (MG132) E SCAF11 SCAF4 PPP1R12A PPP1R10 ANAPC2 YBX1/2 SMU1 HYLS HIBADH PDIA3 P4HB SCAF8 PHF3 HTATSF1 RPDR2 RPRD1A HNRNPCL1 ACIN1 SETD2 GATAD2B STK19 SMC3 RPA1 SMC3 A TCR ALL 1.00 355 80 7 3 2 SMC3 0 1 2 3 4 5 6 ASCC3 RPA1 RPS6 RPL19 FANCD2 EIF3A TRIM28 INTS2 NUMA1 -2 -1 0 1 2 0.17 Equal weight 2187 CSB/ERCC6 HERC1 CSB TMPO CACYBP B C TCR ALL Number of proteins Number of proteins Point score Av. Screens aggregate z-score/screen SCAF11 SCAF4 PPP1R12A PPP1R10 ANAPC2 HIBADH SCAF8 HTATSF1 RPDR2 SETD2 GATAD2B SMC3 RPA1 SMC3 SMC3 -2 -1 0 1 2 0.17 Equal weight D HERC1 CSB TMPO CACYBP B TCR ALL Number of proteins Av. aggregate z-score/screen SCAF11 HTATSF1 RPDR2 SETD2 SMC3 RPA1 A TCR ALL 1.00 355 80 7 3 2 0 1 2 3 4 5 6 ASCC3 RPA1 RPS6 RPL19 FANCD2 EIF3A TRIM28 INTS2 NUMA1 2187 CSB/ERCC6 Number of proteins Point score SCAF4 PPP1R12A PPP1R10 ANAPC2 HIBADH SCAF8 GATAD2B A Figure 6. Proteins and Processes that Score Highly in the Multiomic Screening Approach (A) Bar graph showing the number of proteins that scored above the Z score threshold in one or more screen. B (B) Distribution of hits using aggregated Z scores. (C) As in (B), but with TC-NER score weighting. In (B) and (C), the colored wheels indicate relative weighting of scores from individual screens (see Figure S1A). Names and dots in red are examples from the TC-NER training category (‘‘TCR’’). Please note that, for clarity, even if a candidate scores highly in several scoring schemes, it is typically only indicated once. ASCC3 CSB -2 -1 0 1 2 3 Weighted 0.41 ERCC2 C TCR ALL PCF11 Number of proteins Point score Av. aggregate z-score/screen YBX1/2 SMU1 HYLS PDIA3 P4HB PHF3 RPRD1A HNRNPCL1 ACIN1 STK19 C D (D) Biased list of interesting proteins and protein complexes that scored highly. (E) Proteins from the TC-NER training category that scored above the Z score threshold (indicated by red bar) in screens across the multiomic approach (see Figure S1B). See also Figures S2, S3, and S4. See also Figures S2, S3, and S4. for information value regarding the known TC-NER factors. The underlying assump- tion is that unknown factors in the tran- scription-related DNA damage response will often (although obviously not invari- ably) follow the same pattern in the data as the known TC-NER factors. As ex- pected, the screens had varying abilities to capture proteins from this training category, with the CSB interactome, dam- age-induced ubiquitylation, and RNAi low transcription particularly effective in un- covering such factors (Figure S1A). Weighting the individual screening experi- ments according to their performance in this respect and applying it to score all pro- teins increased the median score of known TC-NER protein from 0.17 to 0.41 (Fig- ure 6C; Table S9). STK19 Is Important for the Transcription-Related DNA Damage Response To further characterize STK19, we investigated the effect of its knockdown on global transcription both in the presence and absence of DNA damage. STK19 knockdown had little effect on transcription in the absence of UV irradiation or on the global shutdown of transcription immediately after DNA dam- age (2 hr) (Figure 7A, left and middle panels). However, cells depleted for STK19 were clearly deﬁcient in the recovery of transcription after DNA damage (Figures 7A, right panels, and 7B), similar to what is observed in CSB knockdown cells (see Figure S5). To investigate how this correlated with cell viability after DNA damage, we also performed a clonogenic UV sensitivity assay (Figure 7C). Gratifyingly, cells lacking STK19 were indeed UV- sensitive, and this held true with any of the individual siRNAs from the Dharmacon pool that knocked down STK19 (Figures 7D and 7E). We also investigated whether STK19 might work at least partly via being recruited to DNA damage. For this pur- pose, GFP-tagged STK19 was expressed in HEK293 cells, and the localization of the protein tested after local laser-induced DNA damage. STK19, indeed, accumulated in areas of such DNA damage (Figure 7F). Enrichment analysis of the results from multiomic screening thus enables discovery of new systems-wide connections in the DNA damage response. Screens Gene set enrichment analysis (GSEA), originally developed for interpreting gene expression data (Subramanian et al., 2005), illustrates the enrichment of NER factors in our datasets: the proteins uncovered from this category (no less than 66 out of the 125 proteins in it) scored widely across the screens (Fig- ure 6E; see also Figures S1B and S2). This was in contrast to some other gene ontology categories showing high overall enrichment, such as ‘‘ribosome-related’’ categories, where enrichment was based primarily on very high scores in the ubiq- uitylation screens (Figure S3). We also queried the screen results against the Corum data- base of protein complexes (http://mips.helmholtz-muenchen. de/genre/proj/corum/). Indeed, the importance of a single sub- unit of a protein complex scoring in a single screen might be considered doubtful, but if several subunits score in several screens, then the involvement of that complex can be stated with greater conﬁdence. A list of Corum complexes and their as- sociation with the transcription-related DNA damage response can be found in Table S12. Besides detecting repair-associated complexes such as the ubiquitin ligase complex containing CSA, this analysis uncovered protein complexes that have not previ- ously been connected to the UV damage response, such as Integrator, MeCP1 histone deacetylase complex, and several others. Screens Gratifyingly, several pathways related to ‘‘nucleotide excision repair’’ were enriched (adjusted p values of 2.04 3 105 to 9.78 3 103), but other 1604 Cell Reports 15, 1597–1610, May 17, 2016 pathways such as ‘‘mRNA translation and ribosomes,’’ ‘‘virus lifecycle, -transcription, and -translation,’’ and ‘‘mRNA splicing’’ were highly enriched in our data as well. We also noted a broad-based connection to ‘‘double-strand break repair,’’ which supports the idea that the response to UV-induced DNA damage is both multi-pronged and extensive. Importantly, most of the pathways were not highly enriched in the individual experiments (Table S11), consistent with the idea that the triggered pathways can be detected with higher conﬁdence when taking several independent experimental approaches into consideration. pathways such as ‘‘mRNA translation and ribosomes,’’ ‘‘virus lifecycle, -transcription, and -translation,’’ and ‘‘mRNA splicing’’ were highly enriched in our data as well. We also noted a broad-based connection to ‘‘double-strand break repair,’’ which supports the idea that the response to UV-induced DNA damage is both multi-pronged and extensive. Importantly, most of the pathways were not highly enriched in the individual experiments (Table S11), consistent with the idea that the triggered pathways can be detected with higher conﬁdence when taking several independent experimental approaches into consideration. pointing to a direct effect on transcription and/or repair. ASCC3 also becomes highly ubiquitylated and phosphorylated in response to UV irradiation, suggesting regulation via post-trans- lational modiﬁcation. Other results showing an involvement of ASCC3 in the transcription-related DNA damage response will be described separately (L.W., A.S., J.S., S.B., G.P.K., M.H., M. Saponaro, P. East, R. Mitter, A. Lobley, J. Walker, and B. Spencer-Dene, unpublished data), but as a further illustration of the power of the multiomic approach, we describe the initial ﬁndings on serine-threonine kinase 19 (STK19). STK19, a poorly studied protein, would be unlikely to be pur- sued based on the results from any of the individual screens, but it scored in the top 25 of hits ranked by the weighted scoring approach (Figure 6C; Table S9). Speciﬁcally, STK19 interacts with CSB after DNA damage, and its knockdown affects transcription recovery after DNA damage. Using bioLOGIC to cross-reference all high-scoring proteins with cancer databases made it clear that STK19 is potentially of great interest. Indeed, it is mutated in melanoma (Hodis et al., 2012) and listed among the Broad Institute cancer driver genes (Lawrence et al., 2014), yet its function has remained undetermined. Integrating with Other Databases To enable easy interrogation of the screen results, we devel- oped a new database interface, named bioLOGIC (http:// www.biologic-db.org). Besides allowing visualization and su- perimposition of results from different selected screens, bioLOGIC also enables easy integration with public databases, such as those detailing common cancer drivers, or prior DNA damage-focused screens. Furthermore, it allows rapid assessment of other information about a factor of interest, via one-click links to information in databases such as UniProt, CORUM, BioGrid, etc. These data expose the melanoma gene STK19 as a factor in the transcription-related DNA damage response. DISCUSSION It is crucial to emphasize that per- forming several screens side-by-side also often bypasses the pressing need for independent conﬁrmation that typiﬁes single-screen approaches; the conﬁrma- tion for a hit in, for example, the siRNA ded when the same factor also scores as APII and/or CSB, and/or by transferring to becoming ubiquitylated or phosphorylated As a consequence, easy cross-referencing sults and with other published screens is mpting to make sense of datasets that are, omplete. For this and other purposes, a rface, bioLOGIC, was developed. 10 15 2) 10 15 J/m2) approach should thus be distinguished from ‘‘cataloging approaches’’ where detailed information about one particular aspect of a process/reaction is obtained. While screens cataloguing phosphoryla- tion, ubiquitylation, and transfer to chro- matin in response to UV-mediated DNA damage have previously been performed (although in other cell lines and under other conditions; e.g., see Chou et al., 2010; Povlsen et al., 2012; Elia et al., 2015a), screens to map damage-induced RNAPII- and CSB-interactors have not previously been reported and neither have genome-wide screens for transcrip- tion recovery after UV irradiation. In any case, an integration of multiple screen re- sults such as that described here is best based on screens performed under the same conditions and in the same cell lines. It is crucial to emphasize that per- forming several screens side-by-side also often bypasses the pressing need for independent conﬁrmation that typiﬁes single-screen approaches; the conﬁrma- tion for a hit in, for example, the siRNA screen is thus provided when the same factor also scores as an interactor of RNAPII and/or CSB, and/or by transferring to chromatin, and/or becoming ubiquitylated or phosphorylated upon DNA damage. As a consequence, easy cross-referencing between screen results and with other published screens is important when attempting to make sense of datasets that are, by their nature, incomplete. For this and other purposes, a searchable web interface, bioLOGIC, was developed. DISCUSSION UV (J/m2) 0 5 10 15 0.001 0.01 0.1 1 UV (J/m2) Surviving fraction pool E CSB NT STK19-2 STK19-3 STK19-4 UV-treated UV (J/m2) 0 5 10 15 0.001 0.01 0.1 1 UV (J/m2) Surviving fraction D NT 1 2 3 4 pool 0.0 0.2 0.4 0.6 0.8 1.0 Relative expression STK19 siRNA E CSB NT STK19-2 STK19-3 STK19-4 UV treated D NT 1 2 3 4 pool 0.0 0.2 0.4 0.6 0.8 1.0 Relative expression STK19 siRNA D E F ( ) F Before DNA damage After DNA damage cells either genetically or biochemically. The enormous amount of data from such screens is, however, typically accompanied by a fundamental problem—a very low signal-to-noise ratio. Even though experimental variation can sometimes be reduced by employing a sufﬁcient number of replicates (typically at very substantial effort and cost), this does not eliminate principal blind spots in individual experimental techniques. In the multiomic approach described here, several independent approaches are used to characterize the same cellular response pathway. As it explores the same process from different angles, it might be viewed as the biological screening equivalent of the statistical chain rule (or general product rule) of probability: it places less emphasis on hits from any individual screen and instead focuses primarily on factors and pathways that score in several screens. Its primary aim is to discover new pathways/factors, and the 1606 Cell Reports 15, 1597–1610, May 17, 2016 DISCUSSION (F) Recruitment of GFP-tagged STK19 to DNA damage induced by laser micro-irradiation in a diffraction-limited spot (blue arrows) or stripe (yel- low arrows). Cells were imaged immediately before and 2 hr after micro-irradiation. See also Figures S4 and S5 and the Supplemental Experimental Procedures for details. approach should thus be distinguished from ‘‘cataloging approaches’’ where detailed information about one particular aspect of a process/reaction is obtained. While screens cataloguing phosphoryla- tion, ubiquitylation, and transfer to chro- matin in response to UV-mediated DNA damage have previously been performed (although in other cell lines and under other conditions; e.g., see Chou et al., 2010; Povlsen et al., 2012; Elia et al., 2015a), screens to map damage-induced RNAPII- and CSB-interactors have not previously been reported and neither have genome-wide screens for transcrip- tion recovery after UV irradiation. In any case, an integration of multiple screen re- sults such as that described here is best based on screens performed under the same conditions and in the same cell lines. It is crucial to emphasize that per- forming several screens side-by-side also often bypasses the pressing need for independent conﬁrmation that typiﬁes single-screen approaches; the conﬁrma- tion for a hit in, for example, the siRNA screen is thus provided when the same factor also scores as an interactor of RNAPII and/or CSB, and/or by transferring to chromatin, and/or becoming ubiquitylated or phosphorylated upon DNA damage. As a consequence, easy cross-referencing between screen results and with other published screens is important when attempting to make sense of datasets that are, by their nature, incomplete. For this and other purposes, a searchable web interface, bioLOGIC, was developed. Untreated 2 hr 20 hr UV-treated NT STK19 Untreated 2 hr 20 hr 0 20 40 60 80 100 120 % low transcribing cells Non-targeting siRNA STK19 siRNA UV-treated n.s n.s A B 0 5 10 15 0.001 0.01 0.10 1 Surviving fraction UV (J/m2) * CSB NT STK19 pool 0 5 10 15 0.001 0.01 0.1 1 UV (J/m2) Surviving fraction C D NT 1 2 3 4 pool 0.0 0.2 0.4 0.6 0.8 1.0 Relative expression STK19 siRNA E CSB NT STK19-2 STK19-3 STK19-4 16.7% 63.9% F Before DNA damage After DNA damage Untreated 2 hr 20 hr UV-treated NT STK19 A 16.7% 63.9% Figure 7. DISCUSSION Involvement of STK19 in the Tran- scription-Related DNA Damage Response (A and B) Lack of normal transcription recovery in cells lacking STK19. Figure 7. Involvement of STK19 in the Tran- scription-Related DNA Damage Response (A and B) Lack of normal transcription recovery in cells lacking STK19. A (C–E) Cells lacking STK19 are UV-sensitive. The individual siRNAs that knock down STK19 (D) also give rise to UV sensitivity (E). The result of CSB knockdown is shown for comparison. NT, non- targeting siRNA. NT STK19 Untreated 2 hr 20 hr 0 20 40 60 80 100 120 % low transcribing cells Non-targeting siRNA STK19 siRNA UV-treated n.s n.s * B 0 5 10 15 0.001 0.01 0.10 1 Surviving fraction UV (J/m2) * CSB NT STK19 pool C 63.9% (F) Recruitment of GFP-tagged STK19 to DNA damage induced by laser micro-irradiation in a diffraction-limited spot (blue arrows) or stripe (yel- low arrows). Cells were imaged immediately before and 2 hr after micro-irradiation. Untreated 2 hr 20 hr 0 20 40 60 80 100 120 % low transcribing cells Non-targeting siRNA STK19 siRNA UV-treated n.s n.s * B 0 5 10 15 0.001 0.01 0.10 1 Surviving fraction UV (J/m2) * CSB NT STK19 pool C C B See also Figures S4 and S5 and the Supplemental Experimental Procedures for details. approach should thus be distinguished from ‘‘cataloging approaches’’ where detailed information about one particular aspect of a process/reaction is obtained. While screens cataloguing phosphoryla- tion, ubiquitylation, and transfer to chro- matin in response to UV-mediated DNA damage have previously been performed (although in other cell lines and under other conditions; e.g., see Chou et al., 2010; Povlsen et al., 2012; Elia et al., 2015a), screens to map damage-induced RNAPII- and CSB-interactors have not previously been reported and neither have genome-wide screens for transcrip- tion recovery after UV irradiation. In any case, an integration of multiple screen re- sults such as that described here is best based on screens performed under the same conditions and in the same cell lines. DISCUSSION During evolution, cells have developed sophisticated responses to genomic insult, ranging from delaying progression through the cell cycle to ﬁrst repairing DNA damage where it matters most, namely in active genes. In this report, we describe a systems approach to discovering new DNA damage response factors, with particular emphasis on transcription. Although the multiomic approach does not integrate all pub- licly available information and cannot substitute for expert knowledge, it provides a powerful way of ranking genes/proteins by their strength of candidacy of being involved in the transcrip- tion-related DNA damage response. For example, the highest scoring gene/protein in the multiomic screening approach, ASCC3, was found to interact with both RNAPII and CSB, which was independently conﬁrmed by IP-western blotting (Figure S4), The advent of sensitive techniques for genome- and prote- ome-wide analysis now enables the screening of mammalian Cell Reports 15, 1597–1610, May 17, 2016 1605 cells either genetically or biochemically. The enormous amount of data from such screens is, however, typically accompanied by a fundamental problem—a very low signal-to-noise ratio. Even though experimental variation can sometimes be reduced by employing a sufﬁcient number of replicates (typically at very substantial effort and cost), this does not eliminate principal blind spots in individual experimental techniques. In the multiomic approach described here, several independent approaches are used to characterize the same cellular response pathway As it Untreated 2 hr 20 hr UV-treated NT STK19 Untreated 2 hr 20 hr 0 20 40 60 80 100 120 % low transcribing cells Non-targeting siRNA STK19 siRNA UV-treated n.s n.s * A B 0 5 10 15 0.001 0.01 0.10 1 Surviving fraction UV (J/m2) * CSB NT STK19 pool 0 5 10 15 0.001 0.01 0.1 1 UV (J/m2) Surviving fraction C D NT 1 2 3 4 pool 0.0 0.2 0.4 0.6 0.8 1.0 Relative expression STK19 siRNA E CSB NT STK19-2 STK19-3 STK19-4 16.7% 63.9% F Before DNA damage After DNA damage Figure 7. Involvement of STK19 in the Tran- scription-Related DNA Damage Response (A and B) Lack of normal transcription recovery in cells lacking STK19. (C–E) Cells lacking STK19 are UV-sensitive. The individual siRNAs that knock down STK19 (D) also give rise to UV sensitivity (E). The result of CSB knockdown is shown for comparison. NT, non- targeting siRNA. bioLOGIC Biological datasets are becoming ever more complex so it is of vital importance to enable quick and intuitive access. We there- fore put great emphasis on an interactive database, named bio- LOGIC (http://www.biologic-db.org), which makes it straightfor- ward to retrieve data on any individual human gene or protein, 1606 Cell Reports 15, 1597–1610, May 17, 2016 whether it has scored in our screens or not (‘‘bioLOGIC Data’’). Importantly, bioLOGIC allows one-click access to basic informa- tion about individual candidates in different public databases, as well as immediate access to information about the protein’s functional domains and about the protein complexes it belongs to. It is possible, for example, to directly learn how other subunits of the same complex score in the screens (‘‘Category mem- bers’’). Finally, bioLOGIC ‘‘Categoryview’’ makes it possible to quickly sample, for example, how all proteins within a particular gene ontology category scored in the screens. The bioLOGIC interface thus permits quick judgment of the relevance and importance in the DNA damage response of any protein or pro- cess of interest. in the siRNA screen. We noted with interest that ribosomal pro- teins, such as RPS6, RPS9, and RPS15A, previously also scored in a genomic screen performed by the Paulsen et al. (2009) lab- oratory for genes whose knockdown result in elevated gH2AX levels in response to ionizing radiation. Together, these intriguing results point to an unexplored connection between translation and the transcription-related DNA damage response that merits further investigation. Intriguing connections to viral infection, interferon signaling, and the immune system are also worth mentioning. The inter- feron system is a powerful antiviral response capable of control- ling virus infections in the absence of adaptive immunity (Randall and Goodbourn, 2008). The connection to interferon signaling was mostly uncovered via the RNAi screen. First, DNA-pattern receptors of the innate immune response scored in the RNAi screen (Toll-like receptor 2 (TLR2, TLR7, and TLR9). Further downstream in this cascade, components of the NF-kappa B pathway, such as CHUK, ERC1, NFKBIB, TICAM1, and NR2C2 (also known as TAK1) scored as well. Other components of the innate immune response, such as TRIM56 (linked to TLRs), also scored. The connection to virus biology is even more wide-ranging, with cellular proteins linked to HIV-Tat scoring highly in almost all screens. Individual Factors and Complexes p A large number of individual proteins and protein complexes scored highly across our screens. Given our interest in transcrip- tion and transcript elongation in particular, we are especially interested in the unexplored role in the DNA damage response of factors such as ASCC3 (L.W., A.S., J.S., S.B., G.P.K., M.H., M. Saponaro, P. East, R. Mitter, A. Lobley, J. Walker, and B. Spencer-Dene, unpublished data), the SCAF proteins, PCF11, PHF3, and Integrator complex. Interestingly, our experiments point to the existence of a large Integrator super-complex, including ASUN, C7ORF26, DDX26B, and VWA9/C15ORF44, as well as NABP1, which might well be a specialized form of Inte- grator for the DNA damage response. Indeed, Integrator super- complex not only interacted with CSB upon DNA damage, but subunits such as INTS2 and INTS12 also scored in the functional RNAi screen, while others changed their level of post-transla- tional modiﬁcation in response to UV irradiation. Similarly, the SCAF proteins, PCF11 and PHF3, also scored in several of our screens, while PCF11 and Integrator subunits also previously scored in an siRNA screen for increased damage signaling (Paul- sen et al., 2009), all in all providing a solid starting point for further detailed functional analysis of their physiological role. In general, an involvement in the DNA damage response of these basal mRNA processing/termination factors might potentially help explain the dramatic downregulation of transcription occurring upon DNA damage. However, the role in the DNA damage It is worth noting that DNA damage response pathways, such as the ATM pathway, can also be activated by replication stress, which occurs as a consequence of UV-induced DNA damage at later time points (Zeman and Cimprich, 2014). Although we believe that replication stress is not a major factor in our results, a follow-up study speciﬁcally targeted at this pathway would be required to tease apart the responses to DNA damage and repli- cation stress. Besides the systems level overlaps described above, unex- pected connections were also uncovered. The high scores of ri- bosomal subunits is a particularly striking example. This score is at least partly due to a dramatic increase in phosphorylation, and especially ubiquitylation, at one or more sites of a very large number of ribosomal proteins in response to UV irradiation. How- ever, ribosomal subunit genes such as RPS27A, RPL19, and RPS6 also scored in the RNAi screen. bioLOGIC Again, the mechanism and signiﬁ- cance of these results remain to be established, but we note that our own gene expression data (not shown), as well as those of others (Zaidi et al., 2011; Shen et al., 2015), also suggest an overlap between interferon signaling and the UV damage response. It is an intriguing possibility that the sophisticated and complex response of higher cells to viruses and infection might have evolved from and/or adopted aspects of a more ancient DNA damage response. Pathways, Processes, and Complexes At the systems level, the multiomic approach and bioLOGIC pro- vide a birds-eye view of the cellular responses triggered by UV irradiation. As expected, DNA/chromatin- and RNA-related pro- cesses dominate the list, including processes such as mRNA splicing, RNAPII transcript elongation, chromosome mainte- nance, and DNA repair, and protein complexes such as Spliceo- some, PAF complex, and MeCP1, for example. A multi-level overlap between genome instability and mRNA splicing has become apparent over the last few years (Lenzken et al., 2013; Chan et al., 2014), and this connection is obvious in our data as well. Interestingly, our data also corroborate anec- dotal evidence for an overlap between the response to UV irradi- ation and double-strand DNA breaks. This overlap might be based on a re-use of response proteins and signaling pathways for different kinds of DNA damage. However, it is equally possible that transcription-impeding damage caused by UV irra- diation results in double-strand breaks more frequently than pre- viously assumed, for example through the formation of R loops and their faulty processing, as suggested by Sollier and Cimprich (2015). Finally, it is not impossible that the doses of UV irradiation used in our study are high enough to directly cause some dou- ble-strand breaks or inter-strand DNA crosslinks, which could also help explain some of the observations. ACKNOWLEDGMENTS This work was supported by the Francis Crick Institute (grant FCI01), which re- ceives its core funding from Cancer Research UK, the UK Medical Research Council, and the Wellcome Trust. The work was also supported by grants from the European Research Council, and Worldwide Cancer Research (formerly known as Association for International Cancer Research to J.Q.S.). We thank the Francis Crick institute’s Cell Service facility for assistance and Barbara Dirac Svejstrup and other members of the J.Q.S. lab for comments on the manuscript. We also thank the members of the Bioinformatics and Biostatistics Laboratory for helpful discussions and Professor Dirk Eick, Uni- versity of Munich for the kind gift of RNAPII CTD antibodies. In conclusion, by investigating a complex cellular process from a number of distinct angles, the data presented here pro- vides an unprecedented systems level view at the transcrip- tion-related DNA damage and at the same time uncovers numerous factors involved in it. The detailed study of the many connections revealed will be a major undertaking. Received: November 24, 2015 Revised: February 25, 2016 Accepted: April 10, 2016 Published: May 12, 2016 Received: November 24, 2015 Revised: February 25, 2016 Accepted: April 10, 2016 Published: May 12, 2016 Individual Factors and Complexes Translation initiation factor EIF3A scored in this screen as well and is ubiquitylated and inter- acts more strongly with RNAPII upon UV irradiation. Intriguingly, LTN1 (Listerin), a ubiquitin ligase and component of the Ribo- some Quality Control complex (Wolff et al., 2014), also scored Cell Reports 15, 1597–1610, May 17, 2016 1607 Cell Lines Anindya, R., Mari, P.O., Kristensen, U., Kool, H., Giglia-Mari, G., Mullenders, L.H., Fousteri, M., Vermeulen, W., Egly, J.M., and Svejstrup, J.Q. (2010). A ubiquitin-binding domain in Cockayne syndrome B required for transcrip- tion-coupled nucleotide excision repair. Mol. Cell 38, 637–648. HEK293 cell lines expressing FLAG-tagged proteins were used for proteomic analysis. Cells were cultured in light or heavy SILAC medium for at least seven generations, UV-irradiated, and allowed to recover at 37C for 3 hr. Micro-irra- diation and imaging was performed with a PerkinElmer UltraVIEW VoX spin- ning disk microscope. Ayg€un, O., Svejstrup, J., and Liu, Y. (2008). A RECQ5-RNA polymerase II as- sociation identiﬁed by targeted proteomic analysis of human chromatin. Proc. Natl. Acad. Sci. USA 105, 8580–8584. Extract Preparation Cells were lysed by dounce homogenization and nuclei pelleted by centrifuga- tion. Following nucleoplasmic extraction, the chromatin pellet was treated with Benzonase. FLAG-M2 beads (Sigma-Aldrich) were employed for afﬁnity puri- ﬁcation. Elution was with 3xFLAG peptide. Antibodies were purchased from Bethyl Laboratories, Abcam, and Santa Cruz Biotechnology. Baillat, D., and Wagner, E.J. (2015). Integrator: surprisingly diverse functions in gene expression. Trends Biochem. Sci. 40, 257–264. Baillat, D., Hakimi, M.A., Na¨ a¨ r, A.M., Shilatifard, A., Cooch, N., and Shiekhat- tar, R. (2005). Integrator, a multiprotein mediator of small nuclear RNA pro- cessing, associates with the C-terminal repeat of RNA polymerase II. Cell 123, 265–276. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, ﬁve ﬁgures, and twelve tables and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2016.04.047. AUTHOR CONTRIBUTIONS S.B. performed proteomic experiments (except for the C7ORF26 interactome, which was analyzed by O.A.). V.E. and B.S. were responsible for mass spec- trometry analysis. L.W. performed the functional genomics screen, with help from I.G., R.E.S., R.I., and M.H. M.R., J.C.W., J.W.C., and R.C.C. were respon- sible for the experiments with STK19. S.B., G.P.K., and A.S. performed bioin- formatics analysis. J.Q.S. supervised the work and wrote the paper, with input from all authors. RNAi Screen response of other high-scoring proteins with an often poorly un- derstood role in transcription, such as HTATSF1, TCERG1, RPRD1A/B, RPRD2, and SND1 certainly also merits further study. The siRNA screen was performed in MRC5VA cells with the Dharmacon Hu- man siGENOME library. Plates were exposed to short-wavelength UV (UV- C) light and then incubated for a further 18 hr before labeling nascent RNA with 50-ethynyl uridine. Automated image acquisition was performed (Cello- mics Array Scan VTI) using a 103 objective. Image analysis was performed us- ing HCS Studio (Thermo Scientiﬁc). Obviously, the ultimate goal of any screening approach is to uncover new factors without prior connections to the process of interest. We believe that the multiomic approach achieved this goal; numerous proteins of unknown function, including several enzymes, were uncovered that are connected to the transcription-related DNA damage response. As an example, we followed up on two proteins, ASCC3 and STK19. Our data on ASCC3 will be described elsewhere (L.W., A.S., J.S., S.B., G.P.K., M.H., M. Saponaro, P. East, R. Mitter, A. Lobley, J. Walker, and B. Spencer-Dene, unpublished data). In the case of STK19, there is some evidence that it is a kinase (Gomez- Escobar et al., 1998), but no cellular function had been assigned to the protein. Our data now place this extremely poorly studied protein in the DNA-damage response. Indeed, the multiomic analysis showed that STK19 interaction with CSB increases dramatically after DNA damage in the presence of MG132. Moreover, in the absence of STK19, transcription fails to recover upon UV irradiation. Crucially, our follow-up experiments showed that STK19 deﬁciency also results in UV sensitivity, and the protein is recruited to sites of DNA damage. 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https://openalex.org/W4213292600	https://hal.science/hal-03739942/document	English	null	Topological Sigma-Semiring Separation and Ordered Measures in Noetherian Hyperconvexes	Symmetry	2,022	cc-by	6,918	To cite this version: Susmit Bagchi. Topological Sigma-Semiring Separation and Ordered Measures in Noetherian Hyper- convexes. Symmetry, 2022, 14 (2), pp.422. 10.3390/sym14020422. hal-03739942 Citation: Bagchi, S. Topological Sigma-Semiring Separation and Ordered Measures in Noetherian Hyperconvexes. Symmetry 2022, 14, 422. https://doi.org/10.3390/ sym14020422 1. Introduction The interplay between topological spaces, Borel sets, Baire categorization and mea- surability in a σ −semiring structure is interesting as well as complex. The interactions between topology and measure theory are generally formulated by forming the smallest σ −f ield, where the compactness of a subspace facilitates the computation of measure [1]. It is known that a metrizable space may be separable or may not be separable, affecting the measures in the σ −semiring structures within the spaces. This effectively gives rise to the formation of a Borel hierarchy in metrizable spaces [2]. Note that the Borel sets as well as Baire sets are members of σ−algebra generated by a set of subspaces in a topological space X. The existence of scattering in a topological space affects Borel classiﬁcation as well as measures. A topological space X is called a scattered space if ∀A ⊂X, ∃a ∈A such that A ∩(U ⊂X) = {a} where U = Uo (here, Uo represents the interior of a respective open set). If we consider a continuous function f : X →Y from a topological space X to a Hausdorff topological space Y, then an interesting question arises: what is the condition to form a function f (.) in Baire ﬁrst class? The answer is mainly two-fold in view of topology and convexity: (1) if X is a metric space and Y is a convex subspace of a Banach space, and (2) if X is a normal topological space and Y = R, where R is a set of real numbers. Interestingly, the connectedness of a topological space has a role in this case. For example, the properties of Baire ﬁrst category are preserved by f : X →Y if the topological space X is normal and the topological space Y is arc-connected [2]. If a space is metrizable, then one can ﬁnd σ−discrete bases within the space affecting the measurability. A function f : X →Y induces the co−σ−discrete bases in Y, given as { f (Uo) : Uo ⊂X}, if X has respective σ−discrete bases [3]. Note that the concept of σ−discrete bases in a space can be extended to the concept of hyper-Borel sets in a space [3]. Interestingly, the continuous Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2022 by the author. Licensee MDPI, Basel, Switzerland. Article Topological Sigma-Semiring Separation and Ordered Measures in Noetherian Hyperconvexes Susmit Bagchi Department of Aerospace and Software Engineering (Informatics), Gyeongsang National University, Jinju 660-701, Korea; profsbagchi@gmail.com Abstract: The interplay between topological hyperconvex spaces and sigma-ﬁnite measures in such spaces gives rise to a set of analytical observations. This paper introduces the Noetherian class of k- ﬁnite k-hyperconvex topological subspaces (NHCs) admitting countable ﬁnite covers. A sigma-ﬁnite measure is constructed in a sigma-semiring in a NHC under a topological ordering of NHCs. The topological ordering relation maintains the irreﬂexive and anti-symmetric algebraic properties while retaining the homeomorphism of NHCs. The monotonic measure sequence in a NHC determines the convexity and compactness of topological subspaces. Interestingly, the topological ordering in NHCs in two isomorphic topological spaces induces the corresponding ordering of measures in sigma-semirings. Moreover, the uniform topological measure spaces of NHCs need not always preserve the pushforward measures, and a NHC semiring is functionally separable by a set of inner-measurable functions. Keywords: topological spaces; sigma-semiring; measure spaces; convex; Noetherian class MSC: 54F05; 54E15; 28C15 Citation: Bagchi, S. Topological Sigma-Semiring Separation and Ordered Measures in Noetherian Hyperconvexes. Symmetry 2022, 14, 422. https://doi.org/10.3390/ sym14020422 Academic Editor: Basil Papadopoulos Received: 31 January 2022 Accepted: 18 February 2022 Published: 20 February 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2022 by the author. 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/). HAL Id: hal-03739942 https://hal.science/hal-03739942v1 Submitted on 28 Jul 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. symmetry S S Motivation and Contributions The developments in topological measure theory are propelled by Alexandrov and Varadarajan, considering that the topological spaces are always completely regular as well as Hausdorff [6,7]. The fundamental question in measure theory and its topological variants is the extensibility of σ−algebras [8]. The approach of Alexandrov is based upon the ﬁnitely additive set-valued functions in a topological space, and the approach of Varadarajan is primarily based upon the Cb algebraic forms of bounded continuous real-valued functions in the completely regular spaces. Kirk and Crenshaw further generalized the Cb algebraic approach by introducing the concept of paving W(X) in a space X and then constructing a σ −ring based on the paving [6]. However, the concept of paving has a strong ﬂavor of general topology, and the corresponding topological measure is ﬁnitely W −regular. Moreover, the structure of σ −ring depending on W(X) is a modiﬁcation of a standard σ −semiring in a topological space. Furthermore, the algebra-based topological separa- tion of subspaces also depends on W(X). In the case of a completely regular topological space, an extremely disconnected space (i.e., closure of open set is open) exists, where the corresponding Baire sets become reduced and the zero-sets are easy to identify [7]. In other words, the topological determination of measure compactness becomes simpler in this setting. It is shown that topological measures and deﬁcient measures may not always support subadditivity and the properties of linear functionals while admitting the weak convergence of topological measures, which is a variety of Alexandrov weak conver- gence [9]. Interestingly, if we consider a ring of sets σ(A) and a topological vector space X, then the measure µ : σ(A) →X may show strong convergence to zero if µ(⟨B⟩n i=1) →0 in σ(A) where the sets in sequence ⟨B⟩n i=1 under measure are disjoint [10]. These observations are the motivation to investigate the properties of topological measure in the topologically ordered spaces under an anti-symmetric ordering relation. Moreover, it is interesting to analyze the inherent topological properties, such as invariances and measure sequences, if the topological spaces are hyperconvex Noetherian varieties. The interesting questions are as follows: (1) How do we formulate an irreﬂexive and anti-symmetric topological order- ing relation between two Noetherian classes? (2) What are the properties of topological measures in such Noetherian hyperconvex classes under topological ordering relation? (3) What are the properties of a topological measure sequence in the hyperconvex space? 1. Introduction This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/symmetry Symmetry 2022, 14, 422. https://doi.org/10.3390/sym14020422 Symmetry 2022, 14, 422 2 of 10 2 of 10 Borel measurable functions between non-separable spaces do not necessarily preserve the structures of σ−discrete bases. Similarly, the interplay between the topological homeo- morphism and Borel isomorphism shows that not all topological properties are retained in Borel isomorphic spaces. A bijection f : X →Y is called Borel isomorphic if f, f −1 are both Borel. Moreover, every Borel measurable function is analytically measurable (i.e., Souslin measurable) [4]. However, it is found that every topological homeomorphism is a Borel isomorphism, but the converse is not always true [4]. As a consequence, one cannot guarantee that the measurability can be preserved in a generalized topological space, even if the Borel isomorphism is attained by f : X →Y. Moreover, the Borel measure need not be always real valued in a topological space. For example, the complex-valued Borel measures exist in a Minkowski topological space, which allows computing densities with respect to the Lebesgue measure [5]. 2. Preliminaries In general, a real-valued measure is formulated based on the algebraic semiring struc- ture on sets. The generalized algebraic structure of the semiring on a set S is given by ⟨S, +, ·⟩where ⟨S, +⟩is a commutative semigroup, ⟨S, ·⟩is a semigroup and the multi- plication · : S2 →S distributes over + : S2 →S within the structure. The concept of the zero-set plays important roles in the inter-relationship between the algebraic semiring structure and the topological space, which is deﬁned as follows [11]. Deﬁnition 1 (zero-set in topological space). Let X be a Hausdorff topological space and f : X →R be a real-valued function. The zero-set in the topological space A ⊂X is deﬁned to be a subset such that A = f −1(0). Deﬁnition 1 (zero-set in topological space). Let X be a Hausdorff topological space and f : X →R be a real-valued function. The zero-set in the topological space A ⊂X is deﬁned to be a subset such that A = f −1(0). The co-zero set is the complement of the zero-set, which is denoted as coz(A). The set of continuous functions in a topological space can generate a σ−semiring structure. As a result, we can deﬁne the zero-set in a topological space alternatively as presented the following deﬁnition [2,12]. Definition 2 (semiring zero-set in topological space). If X is a topological space and C(X, (0, ∞]) denotes a set of continuous functions generating a semiring in the topological space X, then a closed set Cl( f ) of a function f (.) is the zero-set such that Cl( f ) = f −1(0). Definition 2 (semiring zero-set in topological space). If X is a topological space and C(X, (0, ∞]) denotes a set of continuous functions generating a semiring in the topological space X, then a closed set Cl( f ) of a function f (.) is the zero-set such that Cl( f ) = f −1(0). It is well known that a Hausdorff topological space X is a Tychonoff space if every subspace B ⊆X and a point a ∈X\B are functionally separable, where B = B ̸= φ. Note that B is the closure of the corresponding set. Suppose we consider a family of subspaces F in a Tychonoff topological space X. Hence, we can deﬁne the concept of measurability, which is given as follows [2]. Deﬁnition 3 (measurable topological subspaces). Motivation and Contributions This paper addresses these questions and presents the analytical results by combining the elements of topology and measure theory. The main contributions made in this paper can be summarized as follows. A Noethe- rian k-hyperconvex class (NHC) in a Hausdorff topological space is constructed such that every local neighborhood basis is countably coverable, and a ﬁber can be suitably attached for ﬁnite k. A topological ordering relation is introduced between two NHCs, where the ordering relation is irreﬂexive, anti-symmetric and transitive without affecting the home- omorphism of topological spaces. This paper proposes a set of analytical properties of ﬁnite measures in sigma-semirings under the topological ordering relation in NHCs. We Symmetry 2022, 14, 422 3 of 10 3 of 10 show that pushforward measures are not always maintainable, and the sigma-semiring is topologically separable by inner-measurable functions. show that pushforward measures are not always maintainable, and the sigma-semiring is topologically separable by inner-measurable functions. The rest of the paper is organized as follows. The preliminary concepts and a set of existing classical results are presented in Section 2. The proposed deﬁnitions of topological structures are presented in Section 3. The analytical results are presented in Section 4. Finally, Section 5 concludes the paper. 2. Preliminaries Let X be a set and Y be a Tychonoff space. A function f : X →Y is called F−measurable if [U ⊂Y] ⇒[ f −1(U) ∈F] where U = Uo. It is important to note that not all subspaces are measurable. For example, the Bernstein set, which is a Baire–Lindelöf variety, is not measurable [11]. In a linear space, the convexity of functionals and bounded real-valued linear functions have an interesting relationship in terms of measures. Suppose ω : X →R ∪{+∞} is an increasing functional on the linear space of real-valued functions with convexity [13]. If we consider two functions f and g in the space X, then the convexity of ω : X →R ∪{+∞} satisﬁes the condition given by [ f ≥g] ⇒[ω( f ) ≥ω(g)]. Let us consider that X is a family of continuous real-valued functions on a topological space A represented as f : A →R. If the measure µ : σ(X) →R is ﬁnite, then it results in the following theorem [13]. Theorem 1. Every ﬁnite measure µ : σ(X) →R is regular and closed. Moreover, if ⟨Bn⟩m n=1 is a sequence of compact sets in σ(X) such that the measure preserves µ(⟨Bn⟩m n=1) →µ(A) then the measure is regular in the corresponding topological measure space. The inter-relationship between the measurability and Baire categorization of a topo- logical space X is presented in the following theorem where zer(A) denotes a respective zero-set A within the topological space [11]. Symmetry 2022, 14, 422 4 of 10 Theorem 2. A real-valued function f : X →R in a topological space X is Baire ﬁrst category if, and only if, f (.) is zer(A)−measurable. There is a relationship between the homeomorphism in topological spaces and the multiplicative isomorphism of a semiring structure under the mapping, which is presented in the following theorem [12]. Theorem 3. In a topological space X the function f (x) = 1/x induces the homeomorphism between the topological spaces (0, ∞], [0, ∞) and also induces the multiplicative isomorphism between semirings in (0, ∞], [0, ∞). The interplay between the convexity of a topological subspace and homeomorphism is illustrated in the following theorem [11]. Theorem 4. Any completely regular topological space X is homeomorphic to a closed subspace A ⊂X if X is convex compact, where A is a set of extreme points of the respective topological space. 2. Preliminaries Note that if the set of extreme points of a topological space A ⊂X is Lindelöf, then the Baire ﬁrst category measurable function f (.) exists in A ⊂X, and it can be extended to X, which is also Baire ﬁrst category measurable. Moreover, it is important to note that the Zariski topological space can be established within the Noetherian space, admitting a ﬁnite as well as signed Borel measure [14]. 3. Deﬁnitions: Hyperconvexity and Measures Note that the condition given by ∃HNp ⊂ ∩ ∀i∈ΛAi such that ∃k ∈Λ, Np(k) ∈NBp and HNp ⊂HNp(k) is maintained in SX ≡Sp, where k < +∞. The neighborhood ﬁber µp×I is a symmetrically compact ﬁber in SX if ∃(a ∈R) ̸= 0, I = [−a, a]. Remark 2. Note that the condition given by ∃HNp ⊂ ∩ ∀i∈ΛAi such that ∃k ∈Λ, Np(k) ∈NBp and HNp ⊂HNp(k) is maintained in SX ≡Sp, where k < +∞. The neighborhood ﬁber µp×I is a symmetrically compact ﬁber in SX if ∃(a ∈R) ̸= 0, I = [−a, a]. If we consider that (X, τX) and (Y, τY) are two ﬁrst-countable Hausdorff topological spaces with respective Noetherian hyperconvex class SX and the Noetherian class SY then it is possible to establish a topological ordering relation < f between the spaces under the function f : (X, τX) →(Y, τY) by considering the closure of subspaces. The deﬁnition of topological ordering is deﬁned as follows. Deﬁnition 7 (topological ordering). The Noetherian hyperconvex class SX and the Noetherian class SY in the respective ﬁrst-countable Hausdorff topological spaces are topologically ordered if ∀Ai ∈SX, ∃Bi ∈SY such that f −1(E ⊂Bi) ⊂(F ⊂Ai). The topological ordering is represented as SX < f SY. It can be observed that the topological ordering relation preserves the concept of continuity of f : (X, τX) →(Y, τY). Later, we will show that SX < f SY enforces Noetherian hyperconvexity in the codomain of f : (X, τX) →(Y, τY) under homeomorphism. Interest- ingly, if ∀Ai ∈SX one can ﬁnd that Ai = ∪ k∈[1,n]Fk, Fk = Fo k such that n ∈Λ, n < +∞then SX ∼= σsr(X), where σsr(X) is a σ −semiring in SX. As a result, one can consider the corre- sponding topological space as a topological measure space (X, σsr(X), µX) incorporating a consistent topological measure as deﬁned next. Deﬁnition 8 (NHC measure). A ﬁnite measure µX : σsr(X) →[0, +∞) is a topological NHC measure in SX if the following conditions are maintained. ∀Fk ∈σsr(X), µX(Fk) < µX(Fk), µX({φ}) = 0, ∀m, n ∈Λ, [Am ⊆An] ⇒[µX(Am) ≤µX(An)], [Am ⊂An] ⇒[µX(Am ∪An) = µX(An)]. (2) (2) Note that the NHC topological measure in a σ −semiring is measure consistent in local subspaces and also in the global subspaces within the corresponding topological space. 3. Deﬁnitions: Hyperconvexity and Measures In this paper, Λ ⊆Z+ denotes an index set, and the topological spaces are Hausdorff as well as ﬁrst countable. If two topological spaces, A, B are isomorphic, then it is denoted by the algebraic relation A ∼=isom B. Deﬁnition 4 (topological k-hyperconvexity). Let (X, τX) be a Hausdorff topological space and xp ∈X be a point. An open neighborhood of xp given by Np ⊂X is called topologically hyperconvex if Np ⊂∩ i∈ΛAi where each Ai is convex in X and i < +∞. A hyperconvex open neighborhood of xp ∈τX in Hausdorff space is denoted by HNp. A HNp is called k −hyperconvex if i ∈[1, k]. In this paper, we write the hyperconvex subspace to indicate a k −hyperconvex sub- space for k > 1. Note that the topological hyperconvexity maintains the countable and ﬁnitely boundedness property such that if i ∈I ⊂Λ then sup(I) < +∞and \|I\|> 1, in general. However, in this case, the ﬁnite intersection property excludes the possibilities of attaining HNp = φ as well as xp = HNp where xp ∈τX. As a result, the concept of hyperconvex Noetherian class within the topological space (X, τX) can be established, which is deﬁned as follows. Definition 5 (hyperconvex Noetherian class). Let xp ∈X be a point in Hausdorff first countable topo- logical space (X, τX) with a hyperconvex open neighborhood basis NBp = n Np(k) ⊂X : k ∈Λ, HNp(k) ∼= Np(k) o within the space. An open convex collection Sp = Ai ⊂X : i ∈Λ, xp ∈Ai is called a Noethe- rian hyperconvex class (NHC) at xp ∈τX if the following properties are satisﬁed. ∀Ai ∈Sp, Ai = Ao i , ∀Ai ∈Sp, ∃HNp(i)∃HNp(k), HNp(i) ⊂Ao i ⊂HNp(k), [i ≤k] ⇒[Ai ⊆Ak]. (1) (1) A Noetherian hyperconvex class Sp is a relaxed variety such that HNp(i) ̸= Ai. In other words, HNp(i) need not be locally dense in subspaces in Sp. The Noetherian hyperconvex class Sp is called ﬁnite if i ∈I ⊂Λ. A Noetherian hyperconvex class Sp is a relaxed variety such that HNp(i) ̸= Ai. In other words, HNp(i) need not be locally dense in subspaces in Sp. The Noetherian hyperconvex class Sp is called ﬁnite if i ∈I ⊂Λ. Symmetry 2022, 14, 422 5 of 10 Remark 1. 3. Deﬁnitions: Hyperconvexity and Measures Note that, in general, Sp is not a proper neighborhood basis of xp ∈τX although Sp is countable. The reason is that if we consider that X is not compact and I = Λ, then ∃k ∈Λ such that ∀i > k, Ai = Ak in (X, τX) admitting a ﬁnite Noetherian class. In an alternative view, it is possible that ∪ ∀i∈Λ(Ai ∈Sp) ⊂X, where (X, τX) is a compact topological space. In summary, the compactness of a topological space does not inﬂuence the nature of ﬁnite Sp. Remark 1. Note that, in general, Sp is not a proper neighborhood basis of xp ∈τX although Sp is countable. The reason is that if we consider that X is not compact and I = Λ, then ∃k ∈Λ such that ∀i > k, Ai = Ak in (X, τX) admitting a ﬁnite Noetherian class. In an alternative view, it is possible that ∪ ∀i∈Λ(Ai ∈Sp) ⊂X, where (X, τX) is a compact topological space. In summary, the compactness of a topological space does not inﬂuence the nature of ﬁnite Sp. Remark 1. Note that, in general, Sp is not a proper neighborhood basis of xp ∈τX although Sp is countable. The reason is that if we consider that X is not compact and I = Λ, then ∃k ∈Λ such that ∀i > k, Ai = Ak in (X, τX) admitting a ﬁnite Noetherian class. In an alternative view, it is possible that ∪ ∀i∈Λ(Ai ∈Sp) ⊂X, where (X, τX) is a compact topological space. In summary, the compactness of a topological space does not inﬂuence the nature of ﬁnite Sp. Note that from now on, if we consider two Hausdorff ﬁrst-countable topological spaces (X, τX) and (Y, τY), then the corresponding Noetherian hyperconvex classes at any arbitrary points in two spaces are denoted as SX and SY, respectively. The formation of a neighborhood ﬁber in a hyperconvex topological subspace at a point xp ∈X in the corresponding Noetherian hyperconvex classes SX ≡Sp in (X, τX) is deﬁned as follows. Deﬁnition 6 (neighborhood ﬁber). Let (X, τX) be a ﬁrst-countable topological space and ∪ ∀i∈ΛAi ⊆X such that Ai ∈SX. A ﬁber I ⊆R, µp×I = xp × I at xp ∈τX is a neighborhood ﬁber if HNp ⊂ ∩ ∀i∈ΛAi is a hyperconvex neighborhood of xp. Remark 2. 4. Main Results The analytical results are presented in two parts as follows. First, we illustrate the topological and measure theoretic properties of sigma-semiring measures in NHC in Section 4.1. The topological separability of sigma-semiring structures in a NHC and the properties of measures are presented in Section 4.2. 3. Deﬁnitions: Hyperconvexity and Measures The NHC topological measure µae : σsr(X∪Y) →[0, +∞) is called almost-everywhere in two topological spaces X, Y if ∀xp ∈X, ∃yp ∈Y it is true that ∃Nx ∈σsr(X), ∃Ny ∈σsr(Y) such that µae(Nx) = µae(Ny), where xp ∈Nx, yp ∈Ny are two respective open neighborhoods and X ∩Y = φ. Note that the NHC topological measure in a σ −semiring is measure consistent in local subspaces and also in the global subspaces within the corresponding topological space. The NHC topological measure µae : σsr(X∪Y) →[0, +∞) is called almost-everywhere in two topological spaces X, Y if ∀xp ∈X, ∃yp ∈Y it is true that ∃Nx ∈σsr(X), ∃Ny ∈σsr(Y) such that µae(Nx) = µae(Ny), where xp ∈Nx, yp ∈Ny are two respective open neighborhoods and X ∩Y = φ. Symmetry 2022, 14, 422 6 of 10 6 of 10 Remark 3. A topological NHC measure in SX generates a non-zero monotone sequence Φ(SX) = µ(Ai) n i=1 determining the compactness as well as convexity of (X, τX). For ex- ample, if (X, τX) is a compact and convex topological space, then n < +∞, ∃l ∈R+ such that ∪ ∀i∈ΛAi = X and Φ(SX) →l > 0. As a result, Φ(SX) is bounded and strongly convergent. Otherwise, the sequence Φ(SX) is divergent in nature, where n →+∞. ∀i∈Λ Otherwise, the sequence Φ(SX) is divergent in nature, where n →+∞. Theorem 6. If (X, τX) and (Y, τY) are ﬁrst-countable topological spaces with hyperconvex E ⊂X and E < f (F ⊂Y) , then F is also hyperconvex in Y. 4.1. Properties of Topological NHC Measures There is a relationship between the k-hyperconvex topological subspaces and the ﬁrst- countable property of a Hausdorff topological space. This interrelationship is presented in the following theorem. Theorem 5. In a topological space (X, τX) if NBp is a ﬁnite hyperconvex neighborhood system at xp ∈X then it is a Noetherian hyperconvex class if (X, τX) is a ﬁrst-countable non-compact topological space. Proof. Let (X, τX)be a first-countable topological space, where xp ∈X is an arbitrary point. A local hyperconvex neighborhood system at xp ∈X is given by NBp = n Np(k) ⊂X : k ∈Λ, HNp(k) ∼= Np(k) o such that one can find a bijection f : Z+ →NBp . The corresponding Noetherian hyperconvex class is SX at xp ∈X. If we consider that k ∈[1, n < +∞] then we can find a corresponding l ∈[1, m < n] such that ∪ l (Al ∈SX) ⊂X if, and only if, (X, τX) is non-compact and X\∪ l Al ̸= φ is open. Moreover, according to the definition ∀Np(k) ∈NBp, ∃Al ∈SX such that Np(k) ⊂Al in (X, τX). Inductively, it can be concluded that [(a ∈Λ) < (b ∈Λ)] ⇒[Np(a) ⊂Np(b)] and ∃l ∈[1, m] such that Al ⊂Np(b) in non-compact (X, τX). Hence, the local neigh- borhood system NBp is a Noetherian hyperconvex class where m < b ≤n and f (.) is ﬁnitely countable. □ Proof. Let (X, τX)be a first-countable topological space, where xp ∈X is an arbitrary point. A local hyperconvex neighborhood system at xp ∈X is given by NBp = n Np(k) ⊂X : k ∈Λ, HNp(k) ∼= Np(k) o such that one can find a bijection f : Z+ →NBp . The corresponding Noetherian hyperconvex class is SX at xp ∈X. If we consider that k ∈[1, n < +∞] then we can find a corresponding l ∈[1, m < n] such that ∪ l (Al ∈SX) ⊂X if, and only if, (X, τX) is non-compact and X\∪ l Al ̸= φ is open. Moreover, according to the definition ∀Np(k) ∈NBp, ∃Al ∈SX such that X\∪ l Al ̸= φ is open. Moreover, according to the definition ∀Np(k) ∈NBp, ∃Al ∈SX such that Np(k) ⊂Al in (X, τX). Inductively, it can be concluded that [(a ∈Λ) < (b ∈Λ)] ⇒[Np(a) ⊂Np(b)] and ∃l ∈[1, m] such that Al ⊂Np(b) in non-compact (X, τX). 4.1. Properties of Topological NHC Measures Hence, it can be concluded that E ⊂ ∩ i∈[1,k](Nq(i) ⊂Y) where k < +∞such that Bi ⊂Nq(k). As a result, E ⊂Y is also hyperconvex under SX < f SY. □ Note that the converse of Theorem 6 may not always be satisﬁed under the anti- symmetric topological ordering relation, and additional conditions are required. The following lemma is a natural extension of the topological ordering property. Lemma 1. The topological ordering SX < f SY preserves homeomorphism of f : (X, τX) →(Y, τY) Lemma 1. The topological ordering SX < f SY preserves homeomorphism of f : (X, τX) →(Y, τY). There is an interplay between the isomorphisms of the two topological subspaces, topological ordering between the respective NHCs and the corresponding topological measures of the NHCs. The topological ordering in the two NHCs induces an algebraic order between the topological measures in the corresponding NHCs. This property is presented in the following theorem. Theorem 7. If SX < f SY is preserved in topological spaces X ∼=isom Y then (µae ◦f −1)(F) < µae(E) where F ⊂Y and E ⊂X. Proof. Let (X, τX) and (Y, τY) be two ﬁrst-countable Hausdorff topological spaces with respective NHCs SX, SY. Note that the topological spaces are separated as X ∩Y = φ. Suppose we consider Ai ∈SX and Bi ∈SY preserving SX < f SY, which results in Ai < f Bi. If the topological measure µae : σsr(X∪Y) →[0, +∞) is an almost-everywhere variety and X ∼=isom Y then the µae(Ai ∈σsr(X)) = µae(Bi ∈σsr(Y)) condition is maintained. However, due to the topological ordering (E = Eo) < f (F = Fo) between E ⊂Ai and F ⊂Bi one can conclude that [ f −1(F) ⊂E] ⇒[(µae ◦f −1)(F) < µae(E)]. □ The above theorem inﬂuences the Baire categorization of topological subspaces as illustrated in the following corollary. Corollary 2. In SX < f SY and SX ∼=isom SY if (µae ◦f −1)(F) < µae(E) then E ⊂X and F ⊂Y need not be locally dense in Ai and Bi. Proof. The proof is relatively straightforward because µae is a measure consistently main- taining algebraic ordering < under topological ordering < f even if (E ∪∂E) ⊂Ai and (F ∪∂F) ⊂Bi. □ g g (F ∪∂F) ⊂Bi. □ There is an interplay between the topological ordering and pushforward measure in the two NHCs. 4.1. Properties of Topological NHC Measures Hence, the local neigh- borhood system NBp is a Noetherian hyperconvex class where m < b ≤n and f (.) is ﬁnitely countable. □ Remark 4. Note that a ﬁrst-countable topological space may admit a k-ﬁnite k-hyperconvex class. It is important to note that a non-convex Hausdorff topological space (X, τX) need not always admit a Noetherian hyperconvex class of NBp for k ∈Λ at any arbitrary xp ∈τX within the space irrespective of the compactness of X. The reason is that if Bp(k) ⊂X is not a convex neighborhood of xp ∈X in the compact non-convex (X, τX) then ∪ k∈ΛBp(k) = X; otherwise if (X, τX) is non-convex as well as non-compact, then ∪ k∈ΛBp(k) ⊂X. This results in the following corollary, which is a stronger property. Corollary 1. A Noetherian n Bp(n) ⊆X, n ∈[1, k] o admits hyperconvex NBp in a compact Hausdorff and ﬁrst-countable (X, τX) if, and only if, Cov(X) = {X ⊂Ei, i ∈[1, m]} is a countable ﬁnite cover of X , where each Fi ⊂Ei is a convex subcover of X. The topological ordering relation SX < f SY between the two spaces maintains the respective NHC structures. However, the relation < f also preserves the hyperconvexity in the NHC in the codomain of continuous f (.). The following theorem presents this property. Theorem 6. If (X, τX) and (Y, τY) are ﬁrst-countable topological spaces with hyperconvex E ⊂X and E < f (F ⊂Y) , then F is also hyperconvex in Y. 7 of 10 7 of 10 Symmetry 2022, 14, 422 Proof. Let (X, τX) and (Y, τY) be two first-countable topological spaces such that X ∩Y = φ. Suppose xp ∈X is an arbitrary point with the corresponding hyperconvex neighborhood basis NBp. If SX is a NHC in (X, τX) such that ∀Ai ∈SX, xp ∈Ai then ∃Np(n), Np(m) ∈NBp within the topological space, maintaining the property that [n < m] ⇒[Np(n) ⊂Ai ⊂Np(m)] in (X, τX). If we consider a continuous function f : (X, τX) →(Y, τY), then ∀Ai ∈SX, ∃Bi ∈SY such that [ f −1(Bi) ⊂Ai] ⇒[ f −1(Bi) ⊂Ai]. However, if Ai < f Bi topological ordering is preserved by f : SX →SY in the two respective topological spaces, then [E ⊂Bi] ⇒[ f −1(E) ⊂(F ⊂Ai)] where E = Eo and F = Fo. 4.1. Properties of Topological NHC Measures Suppose the function g : (X, σsr(X), µX) →(Y, σsr(Y), µY) is a uniformly measurable function in two isomorphic topological measure spaces. It is interesting to note that the topological ordering < f does not preserve the pushforward measure in NHC under composition with the measurable function g : (X, σsr(X), µX) →(Y, σsr(Y), µY). This property is presented in the following theorem, where f −1 is the inverse of the corresponding function under the topological ordering relation. Symmetry 2022, 14, 422 8 of 10 Theorem 8. If g : (X, σsr(X), µX) →(Y, σsr(Y), µY) is uniformly measurable in X ∼=isom Y then ( f −1 ◦g) is not a pushforward measure in SX < f SY. Theorem 8. If g : (X, σsr(X), µX) →(Y, σsr(Y), µY) is uniformly measurable in X ∼=isom Y then ( f −1 ◦g) is not a pushforward measure in SX < f SY. Proof. Let (X, σsr(X), µX), (Y, σsr(Y), µY) be two measure spaces in respective topologi- cal spaces, where the SX < f SY condition is maintained between two NHCs. Suppose g : (X, σsr(X), µX) →(Y, σsr(Y), µY) is a uniformly measurable function with X ∼=isom Y such that µX(Ai ∈σsr(X)) = µX(g−1(Bi ∈σsr(Y))). However, the topological ordering SX < f SY induces an inequality in measures under composition ( f −1 ◦g) which is given by µY(Bi ∈σsr(Y)) > µX(( f −1 ◦g)(Ai ∈σsr(X))). Hence, the condition of the pushforward measure is not preserved by ( f −1 ◦g) under < f between the two NHCs. □ Although the pushforward measure is not preserved by < f topological ordering between multiple NHCs, the hyperconvex neighborhood system is ﬁnitely measurable in each topological measure space, and the topological ordering induces an order in the corresponding measures. This observation is illustrated in the following lemma. Lemma 2. In every ﬁrst-countable (X, τX) the topological measure space (X, σsr(X), µX) admits ﬁnite measures of hyperconvex neighborhood basis and the topological ordering SX < f SY between NHCs induces a corresponding order in the neighborhood measures. Proof. Let(X,τX)beafirst-countabletopologicalspace,where NBp = n Np(k) ⊂X : k ∈Λ, HNp(k) ∼= Np(k) o is an open hyperconvex neighborhood basis. Clearly, NBp is countable under the bijection h : Z+ →NBp where NBp = n Np(k) o . 4.1. Properties of Topological NHC Measures As a result, the measure (µX ◦h) ∈(0, +∞) is ﬁnite in the corresponding measure space (X, σsr(X), µX) where 0 < µX(Np(k)) < µX(Np(k)) by the deﬁnition of topological NHC measure. Moreover, if (Y, τY) is another ﬁrst-countable topological space with NBq for some yq ∈Y then (µX ◦f −1)(Nq(k)) < µY(Nq(k)) under < f between the topological measure spaces (X, σsr(X), µX), (Y, σsr(Y), µY). □ 4.2. Topological Separation of Sigma-Semiring and Measurability It is noted earlier in this paper that the increasing convex functional ω : X →R ∪{+∞} can be formulated in a linear function space X, where ω is convex. However, the measure of the convex bounded measurable functions in a linear function space is ﬁnitely additive with the assumption that the sequential semicontinuity of Borel measurable functions is preserved. Note that the convex functional measure can be extended to be inﬁnite. The relationship between the measures and the hyperconvex topological space presented in this paper consider ﬁnite measures under the topological decomposition and separation of measure spaces while at least preserving subadditivity. The Hausdorff topological measure space admitting a NHC is considered to be continuous and simply connected in nature. p g p y Let Ak, Ak−1 ∈SX be the k −hyperconvex and (k −1) −hyperconvex subspaces, respectively, in a NHC in (X, τX). Suppose we consider E((k−1),n) ⊂X such that E((k−1),n) = (Ak ∪Ak−1)\Ak where Ak = Ak−1 and n ∈Λ. If we take the collection E((k−1),n) = ∪ i∈[1,n]D((k−1),i) such that D((k−1),i) = Do ((k−1),i), then a topological separation of the corresponding σX −semiring is given by the following equation. m, n, u ∈Λ, i ≤u, j ≤u, ∀E((k−m),u), [i ̸= j] ⇒[D((k−m),i) ∩D((k−m),j) = φ], Ω(σX) = {Ak} ∪ n D((k−m),u) o , ∪ m,uE((k−m),u) ⊂SX. (3) (3) This immediately leads to the following lemma. This immediately leads to the following lemma. Symmetry 2022, 14, 422 9 of 10 9 of 10 Lemma 3. A (k −i) −hyperconvex subspace is locally dense in the respective component in Ω(σX) if, and only if, i = 0. Lemma 3. A (k −i) −hyperconvex subspace is locally dense in the respective component in Ω(σX) if, and only if, i = 0. The proof of the lemma is directly derivable from the structure of the topologically separated σX −semiring. However, it further results in the following theorem. Theorem 9. A topologically separated Ω(σX) is functionally separable by {gv : Ω(σX) →R, v ∈Λ} such that ∩v gv = φ and every gv(.) is inner-measurable. Proof. Let a topologically separated Ω(σX) be in (X, τX) and a set of real-valued functions be given by {gv : Ω(σX) →R, v ∈Λ} such that ∩v gv = φ. 4.2. Topological Separation of Sigma-Semiring and Measurability Suppose that the functions in the set maintain the property of local continuity in the topological space by open map- ping as ∀v ∈Λ, gv(Wv) ⊂Nv such that Nv = No v and ∀B ⊂Nv, g−1 v (B) ⊂Wv with B = Bo. As a result, it can be concluded that [v ̸= (u ∈Λ)] ⇒[Nv ∩(Nu ⊂R) = φ]. More- over, as Nv = No v and Wv = Wo v , every gv(.) is pushforward inner-measurable due to (µX ◦g−1 v )(Nv) < µX(Wv). □ Example 1. Let us consider a topological space in 1D such that Ak = (−a, a), Ak−i = (−(a + δ(i)), (a + δ(i))) where δ(i) > 0, i ∈Λ. In this case, the topological separation of the σX −semiring is given by i ∈Λ, n ∈Z+ ∪{0}, Ω(σX) = {(−a, a)}∪ {(−(a + (n + 1).δ(i)), −(a + n.δ(i))), ((a + n.δ(i)), (a + (n + 1).δ(i)))}. i ∈Λ, n ∈Z+ ∪{0}, i ∈Λ, n ∈Z+ ∪{0}, Ω(σX) = {(−a, a)}∪ (4) { } Ω(σX) = {(−a, a)}∪ As a result, the topological separation Ω(σX) is also separated by gv : Ω(σX) →R if, and only if, the open neighborhoods under locally continuous mappings are disjoint as [i ̸= j] ⇒[(Ni ⊂R) ∩(Nj ⊂R) = φ] where the ∀(B = Bo) ⊂Ni, g−1 i (B) ⊂Wi condition is preserved. Moreover, every topological separation in Ω(σX) is inner-measurable because (µR ◦gi)(Wi) < µR(Ni) where µR is a ﬁnite positive measure in reals. 5. Conclusions In a Hausdorff ﬁrst-countable topological space, the Noetherian hyperconvex class is a generalization of a neighborhood basis without preserving the open property of the singleton element under the intersection of corresponding neighborhoods of that element. The k-ﬁnite convex intersection generates a k-hyperconvex topological subspace admitting a sigma-semiring, which is ﬁnitely measurable. The irreﬂexive, anti-symmetric and transitive topological ordering between two Noetherian k-hyperconvex classes retains the homeomorphism of respective topological spaces and induces the ordering in measures in corresponding sigma-semirings. The measure sequence in a Noetherian k-hyperconvex class helps in determining compactness of the topological subspace. The measures under the topological ordering do not always preserve the pushforward property, and the sigma- semirings are topologically separable by a set of inner-measurable functions. The concepts presented in this paper may ﬁnd applications in the topological analysis of dynamics. Funding: This research is funded by Gyeongsang National University, Jinju, Korea. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: The author would like to thank the anonymous reviewers and editors for their valuable comments and suggestions during the peer-review process. Conﬂicts of Interest: The author declares no conﬂict of interest. 10 of 10 Symmetry 2022, 14, 422 References Ash, R. The interplay between measure theory and topology. Meas. Integr. Funct. Anal. 1972, 4, 168–200. 2. Spurny, J. Borel sets and functions in topological spaces. Acta Math. Hung. 2010, 129, 47–69. [CrossRef] . Spu y, J. o e sets a d u ct o s topo og ca spaces. cta Math. ung. 0 0, 9, 69. [C oss e 3. Hansell, R.W. On the nonseparable theory of Borel and Souslin sets. Bull. Am. Math. Soc. 1972, 78, 236 p y 4. Stone, A.H. Non-separable Borel sets II. Gen. Topol. Appl. 1972, 2, 249–270. [CrossRef] 4. Stone, A.H. Non-separable Borel sets II. Gen. Topol. Appl. 1972, 2, 249–270. 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https://openalex.org/W4284714047	https://ojs.zrc-sazu.si/av/article/download/10964/10148	Slovene	null	Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja	Arheološki vestnik/Arheološki vestnik	2,022	cc-by-sa	63,568	Izvleček Prostor posoške skupnosti iz starejše železne dobe (8.–4. st. pr. n. št.), znane kot svetolucijska halštatska kulturna skupina, zaznamujejo različne geomorfološke značilnosti in naravne danosti. Najstarejše jedro stalne poselitve se je izoblikovalo ob naravnih poteh, ki vodijo ob Nadiži in Soči iz Furlanske nižine oz. iz zaledja severnega Jadrana v alpski visokogorski svet. O stalni poselitvi na začetku železne dobe pričajo predvsem grobišča, medtem ko je v naseljih zaradi skromne raziska nosti starejše obdobje (8.–6. st. pr. n. št.) slabše prepoznavno. Grobovi iz starejših faz kažejo na manjše lokalne skupnosti, grobni pridatki pa ne izražajo večjih družbenih razlik. Družbeno razslojevanje je zaznavno v mlajših fazah (6. do 4. st. pr. n. št.), ko se ob prevladujočem pokopu sežganih ostankov v preprosto grobno jamo, pokrito s kamnito ploščo, poja vijo tudi maloštevilni žarni grobovi v obliki kamnite skrinje in z bogatejšimi pridatki. V mlajšem obdobju je naselje na Mostu na Soči doseglo največji obseg in se razvilo v glavno regionalno središče z zgodnjeurbanimi značilnostmi. V 6. in 5. st. pr. n. št. je opazna tudi ekspanzija te skupnosti, zrasla so nova manjša naselja. Večinoma so bila deloma utrjena, v neutrjenih bi lahko videli naselja drugega ranga. V 4. st. pr. n. št. so sledili pretresi in spremembe, ki so privedli do dezintegracije skupnosti. Prebivalstvo se je iz starih aglomeracij umaknilo v stranske doline in bolj hribovite predele, v grobnih in daritvenih obredjih pa je v znatnejši meri prisotno orožje. Ključne besede: severozahodna Slovenija; Posočje; svetolucijska kulturna skupina; starejša železna doba; halštatska doba; poselitev; naselja; grobišča; depoji; kultna mesta; komunikacije Settlement in the Posočje/Sveta Lucija group – new sites and insights Miha MLINAR, Sneža TECCO HVALA 397 397 Arheološki vestnik 73, 2022, 397–469; DOI: https://doi.org/10.3986/AV.73.11; CC BY-NC-SA 4.0 Abstract The area that the Posočje community, also known as the Sveta Lucija/S. Lucia Hallstatt cultural group, inhabited in the Early Iron Age (8th–4th centuries BC) is marked by a diversity of geomorphology and the natural environment. The earliest core of permanent settlement formed along the natural passages along the Rivers Nadiža/Natisone and Soča/ Isonzo from the northern Adriatic to the mountainous areas of the Alps. Evidence of permanent settlement at the beginning of the Iron Age comes primarily from cemeteries, whereas the settlements are poorly investigated and not much is known of their early period (8th–6th centuries BC). The early graves indicate small local communities and the grave goods do not indicate a marked social differentiation. This does become perceptible in the later phases (6th to 4th centuries BC), when also the settlement at Most na Soči reached its maximum extent and developed into the main regional centre with early urban characteristics. On a broader scale, the 6th and 5th centuries BC brought an expansion of the Posočje community and with it new, small settlements. Most of these were partially fortified, whereas those without fortifications may be settlements of a different rank. This was followed by a turbulent 4th century BC with significant changes that finally led to the disintegration of the community. The population retreated from the old agglomerations into more remote valleys and mountainous areas, their grave goods and votive rituals now included weapons in a greater measure. Keywords: north-western Slovenia; Posočje; Sveta Lucija/S. Lucia cultural group; Early Iron Age; Hallstatt period; settlement pattern; settlements; cemeteries; hoards; cult places; communications 398 Miha MLINAR, Sneža TECCO HVALA Sl. 1: Najdišča svetolucijske kulturne skupine oz. posoške skupnosti iz starejše železne dobe ter pokrajinska sestava njenega poselitvenega prostora (prim. Teržan, sl. 1 v tej publikaciji). M. = 400.000. Fig. 1: Early Iron Age sites of the Sveta Lucija cultural group or the Posočje community and the landscape units of its habitation space (cf. Teržan, Fig. 1 in this volume). Scale = 400.000. Sl. 1: Najdišča svetolucijske kulturne skupine oz. posoške skupnosti iz starejše železne dobe ter pokrajinska sestava njenega poselitvenega prostora (prim. Teržan, sl. 1 v tej publikaciji). M. = 400.000. Fig. 1: Early Iron Age sites of the Sveta Lucija cultural group or the Posočje community and the landscape units of its habitation space (cf. Teržan, Fig. 1 in this volume). Scale = 400.000. 1 Pokrajinske značilnosti so povzete po Perko, Oro žen Adamič (ur.) 2001, 54–71. 2 Pred nastankom akumulacijskega jezera je bil izliv Idrijce v Sočo na n. v. 124 m (Rutar 1882). POKRAJINSKE ZNAČILNOSTI Lenart 4 Soča – med Idrskim in Tolminom 5 Tolmin – Pod gradom 6 Zatolmin – Planina Zavrh 7 Zadlaz-Čadrg – Kobilnik 8 Čadrg – Laze I 9 Zatolmin – Javorca 10 Tolminske Ravne – Planina Na kalu 11 Tolminske Ravne – Pod Zelenim vrhom 12 Selišče – pobočje Mrzlega vrha 13 Krn – Gradec 14 Vrsno – Strničelo 15 Smast 16 Ladra – Na Goricah 17 Koseč 18 Kobarid – Gradič, V Logu, Bizjakova hiša, T d V h k S č 11 Tolminske Ravne – Pod Zelenim vrhom 12 Selišče – pobočje Mrzlega vrha 13 Krn – Gradec 14 Vrsno – Strničelo 15 Smast 16 Ladra – Na Goricah 18 Kobarid – Gradič, V Logu, Bizjakova hiša, 53 Nova Gorica – Sv. Katarina (Kekec) Tonovcov grad, V mevcah, reka Soča 19 Magozd – Jajnkovec 20 Trnovo ob Soči – Trnovšček 21 Robič – Sv. Volar 21 Robič – Sv. Volar 22 Podbela – Sv. Helena, Berjač 22 Podbela – Sv. Helena, Berjač 23 Homec – Na Mlakah 24 Sedlo – Pod cerkvijo 25 Srpenica – Ograjenca, Lanišča 26 Bovec – Ravelnik, Na Raduljah 27 Log pod Mangartom 27 Log pod Mangartom 28 Trenta – Pod Razorci 29 Trenta – V plazeh 30 Stara Fužina – Veliki Vegl 31 Ribčev Laz – cerkev sv. Janeza Krstnika 31 Ribčev Laz – cerkev sv. Janeza Krstnika 32 Brod 32 Brod 33 Jereka – Dunaj, Na sedlu 34 Bitnje – Krašica 35 Lepence – Spodnje Gradišče, Na Kremnu 35 Lepence Spodnje Gradišče, Na Kremnu 36 Bohinjska Bistrica – Ajdovski gradec, Osnovna šola 37 Žlan – Groblje 38 Vogel – Dolga Planja 39 Rut – V trojah 40 Koritnica – Lajišče na nekaterih mestih so uravnana v pode in ravne. Relief so oblikovali ledeniki in tekoče vode. Dna dolin in kotlin so prekrita z nanosi ledeniškega, ledeniško-rečnega, ledeniško-jezerskega in po bočnega nastanka. V Bovški kotlini in Bohinju je prisoten fliš, večinoma pa podlago sestavljajo karbonatne kamnine, na katerih so nastali značilni visokogorski kraški pojavi (konte, kotliči, jame, brezna). V Bohinjsko–Tolminskih gorah se plitvo pojavlja limonitna in piritna železova ruda, ki je bila v preteklosti večinoma izčrpana. Površinske vode so v višjih legah redke ali jih sploh ni. Voda prihaja na dan večinoma v močnih kraških izvirih (Soča, Nadiža, Savica v Bohinju). Na vzhodnem na nekaterih mestih so uravnana v pode in ravne. Relief so oblikovali ledeniki in tekoče vode. POKRAJINSKE ZNAČILNOSTI Marija na Jezeru 44 Debenje – sv. Jakob 45 Goljevica – sv. Volbenk 46 Ajba - Fiščevo Banjšice 47 Tolminski Lom – Kal 48 Kanalski Lom – V Glavi 49 Bodrež – Loga 50 Levpa – Grad 51 Deskle – Gradišče 52 Grgar – Grašišče 53 Nova Gorica – Sv. Katarina (Kekec) 54 Trnovo – Kamni breg Idrijsko hribovje s Šentviško planoto 55 Gorenja Trebuša – Obenčel 56 Dolenja Trebuša – Sovodenj 57 Pečine – Vrh gradu 58 Idrija pri Bači – Na Robu 59 Ponikve – Kračice 60 Kneža – Grebljica 61 Gorski vrh – Jerovca 62 Šentviška Gora – Berlotov rob 63 Šentviška Gora – Lipce/Prevala 64 Daber – Na Dobcu 65 Police – Pri cierki Cerkljansko hribovje 66 Reka – Grad 67 Dolenje Ravne – Gastabil 68 Cerkno – Gradišče 69 Novaki – Mali Njivč 70 Železniki – Štalca 71 Žiri – Žirk 72 Godovič – Jelenšek Seznam najdišč po pokrajinskih enotah k sl. 1 / List of sites according to landscape units for Fig. 1: na nekaterih mestih so uravnana v pode in ravne. Relief so oblikovali ledeniki in tekoče vode Dna Pokrajinske enote / Povprečna n v / Povprečen naklon / Italija 41 Špeter Slovenov/S. Pietro al Natisone – Sv. Kvirin/S. Quirino 42 Gagliano pri Čedadu/Cividale del Friuli – Dernazzacco Kambreško pogorje 43 Golo brdo – sv. Marija na Jezeru 44 Debenje – sv. Jakob 45 Goljevica – sv. Volbenk 46 Ajba - Fiščevo Banjšice 47 Tolminski Lom – Kal 48 Kanalski Lom – V Glavi 49 Bodrež – Loga 50 Levpa – Grad 51 Deskle – Gradišče 52 Grgar – Grašišče 53 Nova Gorica – Sv. Katarina (Kekec) 54 Trnovo – Kamni breg Idrijsko hribovje s Šentviško planoto 55 Gorenja Trebuša – Obenčel 56 Dolenja Trebuša – Sovodenj 57 Pečine – Vrh gradu 58 Idrija pri Bači – Na Robu 59 Ponikve – Kračice 60 Kneža – Grebljica 61 Gorski vrh – Jerovca 62 Šentviška Gora – Berlotov rob 63 Šentviška Gora – Lipce/Prevala 64 Daber – Na Dobcu 65 Police – Pri cierki Cerkljansko hribovje 66 Reka – Grad 67 Dolenje Ravne – Gastabil 68 Cerkno – Gradišče 69 Novaki – Mali Njivč 70 Železniki – Štalca 71 Žiri – Žirk 72 Godovič – Jelenšek s according to landscape units for Fig. 1: Julijske Alpe 1 Most na Soči (Sv. Lucija/S. Lucia) 2 Bača pri Modreju – Senica 3 Volče – sv. POKRAJINSKE ZNAČILNOSTI nizi ozkih grebenov in slemen s priostrenimi vrhovi (sl. 1). Več kot polovica površja leži nad 1000 m nadmorske višine (sl. 2), najvišji vrhovi se pnejo nad 2000 m (npr. Kanin 2587 m, Krn 2244 m, Rombon 2208 m), najgloblje sežejo dna dolin v soškem porečju – okoli 350 m pri Bovcu in 152 m pri Mostu na Soči.2 Strma pobočja so ponekod oblikovana v prepadne stene in previse, Dežela posoške skupnosti v starejši železni dobi, imenovane svetolucijska kulturna skupina, sega na severu v Julijske Alpe. Za ta visokogorski svet,1 kamor spadajo Tolminsko, Kobariško z Breginjskim kotom, Bovško in Bohinj, so značilne globoko vrezane doline, nad katerimi se dvigajo 399 Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja Seznam najdišč po pokrajinskih enotah k sl. 1 / List of sites according to landscape units for Fig. 1: Seznam najdišč po pokrajinskih enotah k sl. 1 / List of sites according to landscape units for Fig. 1: Julijske Alpe 1 Most na Soči (Sv. Lucija/S. Lucia) 2 Bača pri Modreju – Senica 3 Volče – sv. Lenart 4 Soča – med Idrskim in Tolminom 5 Tolmin – Pod gradom 6 Zatolmin – Planina Zavrh 7 Zadlaz-Čadrg – Kobilnik 8 Čadrg – Laze I 9 Zatolmin – Javorca 10 Tolminske Ravne – Planina Na kalu 11 Tolminske Ravne – Pod Zelenim vrhom 12 Selišče – pobočje Mrzlega vrha 13 Krn – Gradec 14 Vrsno – Strničelo 15 Smast 16 Ladra – Na Goricah 17 Koseč 18 Kobarid – Gradič, V Logu, Bizjakova hiša, Tonovcov grad, V mevcah, reka Soča 19 Magozd – Jajnkovec 20 Trnovo ob Soči – Trnovšček 21 Robič – Sv. Volar 22 Podbela – Sv. Helena, Berjač 23 Homec – Na Mlakah 24 Sedlo – Pod cerkvijo 25 Srpenica – Ograjenca, Lanišča 26 Bovec – Ravelnik, Na Raduljah 27 Log pod Mangartom 28 Trenta – Pod Razorci 29 Trenta – V plazeh 30 Stara Fužina – Veliki Vegl 31 Ribčev Laz – cerkev sv. Janeza Krstnika 32 Brod 33 Jereka – Dunaj, Na sedlu 34 Bitnje – Krašica 35 Lepence – Spodnje Gradišče, Na Kremnu 36 Bohinjska Bistrica – Ajdovski gradec, Osnovna šola 37 Žlan – Groblje 38 Vogel – Dolga Planja 39 Rut – V trojah 40 Koritnica – Lajišče Italija 41 Špeter Slovenov/S. Pietro al Natisone – Sv. Kvirin/S. Quirino 42 Gagliano pri Čedadu/Cividale del Friuli – Dernazzacco Kambreško pogorje 43 Golo brdo – sv. POKRAJINSKE ZNAČILNOSTI V Idrijskem hribovju prevladujejo karbonatne kamnine (dolomiti in apnenci), ob zgornjem toku Idrijce so območja laporja in lapornih skrilavcev, okoli Idrije so nahajališča živega srebra in cina barita, na Šentviški planoti pa ležišča površinske železove rude, ki so jo ponekod v preteklosti tudi kopali.6 Na karbonatnih kraških območjih je oskrba z vodo težavna, večje količine vode pritekajo na površje v obliki stalnih ali občasnih izvirov. Za radi izredno pestrega reliefa so podnebne in talne razmere različne, prevladuje pa celinsko podnebje z obilnimi padavinami. Največ ravnega sveta je na valovitih planotah, širše aluvialne ravnice so še ob sotočjih, a je na splošno malo zemljišč primernih za kmetijsko izrabo. g Visokogorski svet Julijskih Alp prehaja proti jugovzhodu v Cerkljansko hribovje med rekama Bačo na severozahodu, Idrijco na jugu in Bukovsko grapo na zahodu, na jugovzhodu sega do Godo viča.3 Dobra polovica pokrajine leži nad 600 m nadmorske višine, šestina nad 800 m, najvišji vrh je Porezen (1630 m). Najnižje se spusti v dno doline Idrijce, kjer na stičišču z Bukovsko grapo doseže 230 m, v sotočju s Cerknico pa 210 m n. v. Reke in potoki imajo hudourniški značaj in so izdolbli ozke soteske in debri. Kamninska sestava je precej pestra, dobro četrtino prekrivajo dolomiti, slabo šestino apnenci, drugo pa neprepustne kamnine (glinovci, meljevci, peščenjaki, konglomerati). V okolici Cerknega so manjša nahajališča bakrove rude, ki so jo pod Škofjami (973 m) izkoriščali do začetka druge svetovne vojne.4 Pobočja na Cerkljanskem so strma in razčlenjena s plitvimi dolinicami, ravnega sveta je malo. Pokrajina ima zmerno celinsko podnebje z izdatnimi padavinami. j Južno od Mosta na Soči se razteza Banjška pla nota ali Banjšice7 med Idrijco in njenim pritokom Trebuščico na vzhodu ter Sočo na zahodu, na jugu meji na Trnovski gozd, ki se dviguje nad Vipavsko dolino. Banjško planoto od Trnovskega gozda lo čuje 300–400 m globoka zajeda v dolžini 16 km, imenovana Čepovanski dol, ki se na skrajnem jugozahodu zaključuje z Grgarsko kotlinico na n. v. 300 m. Na zahodu se planota strmo spušča v Soško dolino med Mostom na Soči in Solkanom z najnižjo nadmorsko višino 65 m, najvišje pa seže njen vzhodni rob (do 1071 m). Značilnost te pokrajine je prehodnost med dinarskim, alpskim in sredozemskim svetom. V površju je viden vpliv tektonskih prelomnic, ki potekajo v dinarski smeri. Skoraj polovica površja je zelo strmega z nakloni med 20o in 45o. POKRAJINSKE ZNAČILNOSTI Pietro al Natisone, kjer vstopa v Furlansko nižino in se po 60 km celotnega to ka izlije v reko Ter/Torre, ta pa v Sočo v zaledju severnega Jadrana. Ugodne podnebne razmere so v termalnem pasu na prisojnih pobočjih med 600 in 800 m n. v. delu zbirata vode, pritekajoče iz Julijskih Alp, Bohinjsko jezero v kotanji ledeniško-tektonskega nastanka in Sava Bohinjka, na zahodnem pa Soča, ki teče med Tolminom in Kobaridom v pravem tektonskem jarku. Soča ima več stalnih levih pri tokov, najdaljši je reka Idrijca z Bačo, večja sta še Tolminka s slikovitimi koriti in Koritnica. Relief je v tem alpskem svetu glavni dejavnik za nastanek obdelovalnih površin in prometnic. Glavnino ravnega sveta za obdelovanje predstavljajo terase v rečnih dolinah, za naselitev so marsikje primerne tudi uravnane višje prisojne lege. Podnebje je pretežno gorsko z veliko količino padavin, medtem ko je na dnu soške doline čutiti sredozemske vplive tja do Kobarida. Med zgornjim tokom Soče in vzhodnim robom Furlanske nižine se raztezajo južni obronki Julijskih Alp, skoraj polovica površja v tem delu je goratega in petina gričevnatega. Vanj se globoko zajeda Nadiža, ena najtoplejših alpskih rek, ki ima hudourniški značaj in se napaja iz več izvirov. V zgornjem toku se vije proti jugovzhodu do njenega desnega pritoka Legrada pri Logjeh, kjer naredi ovinek proti severovzhodu. Pri Robiču se zopet usmeri proti jugu in teče v soteski med gorama Matajur (1642 m) in Mija/Monte Mia (1237 m) do Špetra Slovenov/S. Pietro al Natisone, kjer vstopa v Furlansko nižino in se po 60 km celotnega to ka izlije v reko Ter/Torre, ta pa v Sočo v zaledju severnega Jadrana. K sredogorju spada tudi Idrijsko hribovje na prehodu iz alpskega v dinarski svet.5 Glavni vodotok Idrijca s pritokoma Bačo in Trebuščico omejuje to pokrajino, ki na jugu meji na Trnovski gozd. Precejšen del površja zavzema uravnan kraški svet, v katerega so reke in potoki vrezali debrske doline in grape ter ga razkosali v posamezne planote, ki se strmo dvigajo nad rečnimi dolinami. Na severnem delu se med Idrijco in Bačo razprostira Šentviška planota (okoli 650 m n. v.), kjer se naj višje povzpne Črvov vrh (974 m), najnižjo točko pa doseže pri 156 m na sotočju Idrijce in Bače. 5 Povzeto po Perko, Orožen Adamič (ur.) 2001, 342–351. 6 Savnik (ur.) 1968, 400 (Daber); Mlinar et al. 2018. 7 Povzeto po Perko, Orožen Adamič (ur.) 2001, 312–323. 3 Povzeto po Perko, Orožen Adamič (ur.) 2001, 72–82. 4 Savnik (ur.) 1968, 77 (Planina pri Cerknem). POKRAJINSKE ZNAČILNOSTI Dna dolin in kotlin so prekrita z nanosi ledeniškega, ledeniško-rečnega, ledeniško-jezerskega in po bočnega nastanka. V Bovški kotlini in Bohinju je prisoten fliš, večinoma pa podlago sestavljajo karbonatne kamnine, na katerih so nastali značilni visokogorski kraški pojavi (konte, kotliči, jame, brezna). V Bohinjsko–Tolminskih gorah se plitvo pojavlja limonitna in piritna železova ruda, ki je bila v preteklosti večinoma izčrpana. Površinske vode so v višjih legah redke ali jih sploh ni. Voda prihaja na dan večinoma v močnih kraških izvirih (Soča, Nadiža, Savica v Bohinju). Na vzhodnem Pokrajinske enote / Landscape units Povprečna n.v. / Average asl Povprečen naklon / Average inclination Julijske Alpe 1107,9 m 25,5o Cerkljansko hribovje 646,5 m 18,7o Idrijsko hribovje 647,8 m 22,8o Banjšice, Kambreško p. 550,3 m 18,5o Sl. 2: Povprečna nadmorska višina (n.v.) in nakloni po krajinskih enot, ki jih zajema poselitveni prostor posoške skupnosti v starejši železni dobi. Sl. 2: Povprečna nadmorska višina (n.v.) in nakloni po krajinskih enot, ki jih zajema poselitveni prostor posoške skupnosti v starejši železni dobi. Fig. 2: Average altitudes (asl) and inclinations of the landscape units in the area of the Early Iron Age Posočje community. Miha MLINAR, Sneža TECCO HVALA 400 delu zbirata vode, pritekajoče iz Julijskih Alp, Bohinjsko jezero v kotanji ledeniško-tektonskega nastanka in Sava Bohinjka, na zahodnem pa Soča, ki teče med Tolminom in Kobaridom v pravem tektonskem jarku. Soča ima več stalnih levih pri tokov, najdaljši je reka Idrijca z Bačo, večja sta še Tolminka s slikovitimi koriti in Koritnica. Relief je v tem alpskem svetu glavni dejavnik za nastanek obdelovalnih površin in prometnic. Glavnino ravnega sveta za obdelovanje predstavljajo terase v rečnih dolinah, za naselitev so marsikje primerne tudi uravnane višje prisojne lege. Podnebje je pretežno gorsko z veliko količino padavin, medtem ko je na dnu soške doline čutiti sredozemske vplive tja do Kobarida. Med zgornjim tokom Soče in vzhodnim robom Furlanske nižine se raztezajo južni obronki Julijskih Alp, skoraj polovica površja v tem delu je goratega in petina gričevnatega. Vanj se globoko zajeda Nadiža, ena najtoplejših alpskih rek, ki ima hudourniški značaj in se napaja iz več izvirov. V zgornjem toku se vije proti jugovzhodu do njenega desnega pritoka Legrada pri Logjeh, kjer naredi ovinek proti severovzhodu. Pri Robiču se zopet usmeri proti jugu in teče v soteski med gorama Matajur (1642 m) in Mija/Monte Mia (1237 m) do Špetra Slovenov/S. POKRAJINSKE ZNAČILNOSTI Na Banjšicah in v Čepovanskem dolu prevladujejo karbonatne kamnine, osrednji del planote na nadmorski višini 700 m prekriva fliš z lapornatim apnencem, konglomeratom, brečo Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 401 in peščenjakom, to je tudi najobsežnejše uravnano območje. V podlagi Soške doline prav tako pre vladujeta fliš in lapor z vložki apnenčastih breč. Soča se med Mostom na Soči in Avčami ter med Plavami in Solkanom komaj prebija skozi ozke soteske; na mestih, kjer se vanjo stekajo izdatnejši pritoki, kot sta Rohot pri Desklah in Avšček pri Avčah, se dolina razširi. Ugodne naravne možnosti za kmetijstvo so okoli Kanalskega Vrha ter Tolmin skega in Kanalskega Loma, kjer je nekaj manjših trajnih izvirov, flišna prst pa omogoča bujno rast trave; z rodovitno zemljo je prekrito tudi dno Grgarske kotlinice. Planota je izdatno namočena in ima celinsko podnebje, medtem ko je v Soški dolini in Grgarski kotlinici čutiti blažilen učinek sredozemskega podnebja. poraščen z mešanim jelovo-bukovim gozdom v sestoju jelke, smreke, hrasta in bukve, človekov vpliv pa je bil zelo šibak. Občutneje se je poveče val od sredine 2. tisočletja pr. n. št., kar nakazuje pojav peloda žit, pašnih indikatorjev (ozkolistni trpotec) in drugih ruderalnih rastlin, ki rastejo na območjih človekove dejavnosti. Spremembe v tem času so zaznavne tudi v gozdnem sestoju – v prevladujočem deležu bukve in znatnejšem upadu jelke, večji je tudi delež trav in leske, ki raste na nepogozdenih območjih. Razmerje med glavnimi drevesnimi vrstami (smreko, jelko in bukvijo) sta sooblikovala klima in človek. Do močnejšega upada bukve je prišlo sredi 1. tisočletja pr. n. št., nekoliko bolj se je uveljavil gaber, pokrajina pa je zaradi človekovih posegov postala dokaj odprta. g p j Proti zahodu se med Soško dolino in mejno reko Idrijo dviga Kambreško pogorje8 z usmerjenostjo slemen SV–JZ. Obsega Livški Kolovrat z najvišjim vrhom na 1243 m, proti jugozahodu se preko sedla nadaljuje v Kanalski Kolovrat z najvišjim vrhom Korado (812 m), ta pa se spušča v gričevnato po krajino Goriških brd, ki že pripada sredozemskemu delu Posočja na obrobju Furlanske nižine. Večina Kambreškega pogorja leži na nadmorskih višinah med 300 in 600 m, njegova pobočja so položnejša zlasti v zgornjih legah ob glavnem slemenu, večje strmine so na spodnjih legah; na nadmorskih višinah 420–460 m so opazne terase. Na zahodu se najnižje spusti v ozko dolino reke Idrije, ki se razširi šele pri Golem Brdu v Goriških brdih. POKRAJINSKE ZNAČILNOSTI Na vzhodu pada k Soči, ki pri Solkanu zapušča hri bovje in nadaljuje pot po ravnini proti Jadranske mu morju. Na območju Kambreškega pogorja se srečujejo sredozemski in alpski vremenski vplivi; kamninska podlaga je podobna kot na Banjšicah, za kmetijsko izrabo je primernih malo površin. Komplementaren pogled na naravno okolje in izkoriščanje naravnih virov ponujajo rastlinski makroostanki, ohranjeni v arheoloških kontekstih, ki prav tako odsevajo okoliško vegetacijo, vendar bolj selektivno. Gre za izbor drevesnih vrst in gr movnic ter večinoma za semena in plodove rastlin, ki jih je uporabljal in gojil človek. Analize oglja iz železnodobne naselbine na Mostu na Soči so pokazale, da so tedanji prebivalci kot stavbni les uporabljali predvsem jelko in hrast, sledijo rdeči bor, bukev in smreka, druge drevesne vrste so bile poleg bukve bolj v uporabi za kurjenje in izdelavo predmetov.10 Iz grobiščnih kontekstov posoške skupnosti pa je med ogljem z grmad, na katerih so sežigali umrle, z največjim deležem zastopana bukev (izjema je tolminsko grobišče, kjer v vzorcih oglja ni bilo niti enega primerka bukve), medtem ko je iglavcev zaradi slabše kurilne vrednosti precej manj.11 Iz tega izhaja, da so tedanji prebivalci z namernim izborom lesa lahko določeno drevesno vrsto v precejšni meri tudi iztrebili, s krčenjem gozda pa spreminjali podobo pokrajine. Te geomorfološke značilnosti in naravne danosti so gotovo pogojevale poselitev in gospodarsko osnovo ter prehodnost ali odročnost tudi v starejši železni dobi, ko je tu živela posoška skupnost oz. svetolucijska kulturna skupina. Nekaj indikacij o tedanji vegetaciji in človekovem vplivu na okolje dajejo nedavno opravljene palinološke raziskave na območju Bohinjskega jezera. To jezero zaradi velikih razsežnosti (318 ha) beleži spremembe ra stlinskega pokrova več deset kilometrov daleč okoli njega, tako v nižini kot na višje ležečih predelih.9 Iz pridobljenega pelodnega diagrama je razvidno, da je bil ta del alpskega sveta v predhodnih dobah 8 Ib. 9 Povzeto po Andrič et al. 2020. 10 Motella De Carlo 2018. 11 Culiberg 2020; M. Culiberg, Poročilo analize oglja iz žganih grobov z Gradca pri Krnu, Ljubljana 2021 (hrani Tolminski muzej Tolmin). 11 Culiberg 2020; M. Culiberg, Poročilo analize oglja iz žganih grobov z Gradca pri Krnu, Ljubljana 2021 (hrani Tolminski muzej Tolmin). Ib. 9 Povzeto po Andrič et al. 2020. 10 Motella De Carlo 2018. 8 Ib. STANJE ARHEOLOŠKIH RAZISKAV Zbiranje in evidentiranje arheoloških najdb in najdišč je v drugi polovici 19. in začetku 20. sto letja – v času avstro-ogrske monarhije – potekalo preko mreže zaupnikov, korespondentov in kon servatorjev, ki jih je imenovala l. 1850 na Dunaju ustanovljena cesarsko-kraljeva centralna komisija Miha MLINAR, Sneža TECCO HVALA 402 za proučevanje in ohranjanje stavbnih spomenikov (Kaiserlich-Königliche Central-Commission zur Erforschung und Erhaltung der Baudenkmale), ta je bila pozneje večkrat reorganizirana.12 Med njimi sta bila odvetnik Paolo Bizzarro iz Gorice/ Gorizia in Enrico Maionica, kustos muzeja v Ogleju/ Aquileia, ki sta nekaj malega izkopavala na naselju in nekropoli iz starejše železne dobe na Mostu na Soči. Med njimi nastopata tudi višji gradbeni inženir Rudolf Mahnič, ki je odkril najdišče Loga pri Bodrežu ter več grobov iz tega časa na Koritnici in na Šentviški Gori, pa tudi Simon Rutar, zgo dovinar, geograf in pisec Zgodovine Tolminskega (1882). Že prej in deloma sočasno z njimi sta na Mostu na Soči iskala starine in jih zbirala v duhu slovenskega narodnega prebujenja lokalna župnika Tomaž Rutar in Alojzij Carli, domačin Anton Vuga pa je l. 1882 na rtu nad sotočjem Idrijce in Soče izkopal 36 grobov.13 za proučevanje in ohranjanje stavbnih spomenikov (Kaiserlich-Königliche Central-Commission zur Erforschung und Erhaltung der Baudenkmale), ta je bila pozneje večkrat reorganizirana.12 Med njimi sta bila odvetnik Paolo Bizzarro iz Gorice/ Gorizia in Enrico Maionica, kustos muzeja v Ogleju/ Aquileia, ki sta nekaj malega izkopavala na naselju in nekropoli iz starejše železne dobe na Mostu na Soči. Med njimi nastopata tudi višji gradbeni inženir Rudolf Mahnič, ki je odkril najdišče Loga pri Bodrežu ter več grobov iz tega časa na Koritnici in na Šentviški Gori, pa tudi Simon Rutar, zgo dovinar, geograf in pisec Zgodovine Tolminskega (1882). Že prej in deloma sočasno z njimi sta na Mostu na Soči iskala starine in jih zbirala v duhu slovenskega narodnega prebujenja lokalna župnika Tomaž Rutar in Alojzij Carli, domačin Anton Vuga pa je l. 1882 na rtu nad sotočjem Idrijce in Soče izkopal 36 grobov.13 tudi na grobišču na Idriji pri Bači. Gradivo iz teh vse do današnjih dni najobsežnejših izkopavanj grobišč je bilo osnova za razreševanje temeljne kronološke in kulturno-historične problematike starejše železne dobe; že l. 1895 je Moritz Hoer nes, prvi profesor za prazgodovinsko arheologijo na dunajski univerzi, v njem prepoznal možnost kronološke delitve na starejšo in mlajšo fazo. STANJE ARHEOLOŠKIH RAZISKAV j j V času med obema vojnama je edina arheološka izkopavanja v Posočju izvedla Bruna Forlati Tama ro, konservatorka spomeniškovarstvene službe v Trstu, ki je na grobišču na Mostu na Soči odkrila štiri grobove.16 V Bohinju pa je v letih 1937–1939 raziskoval Walter Schmid, kustos muzeja Joanne um v Gradcu/Graz in predavatelj arheologije na tamkajšnji univerzi. Izkopaval je na Ajdovskem gradcu in pripadajoči nekropoli v Bitnjah, na Spo dnjem Gradišču pri Lepencah, Dunaju pri Jereki in v Žlanu.17 Do druge svetovne vojne je bilo v Posočju in Bohinju znanih 25 najdišč iz starejše železne dobe (sl. 3). p g Za razvoj prazgodovinske arheologije je bil pomemben zlasti program l. 1878 ustanovljene Prazgodovinske komisije v okviru Kraljeve aka demije za znanost (Prähistorische Kommission der kaiserlichen Akademie der Wissenschaften).14 Najmočnejši pečat pa je v Posočju nedvomno pustilo raziskovanje Carla Marchesettija, direktorja muzeja v Trstu/Trieste, ki je v svojem topografskem delu zabeležil domala vsa znana prazgodovinska naselja na tem območju, znan mu je bil tudi Ajdovski gradec pri Bohinjski Bistrici.15 V letih 1885–1904 je izvedel izkopavanja na več grobiščih v dolinah Nadiže in Soče ter na Šentviški planoti. V Špetru/S. Pietro al Natisone je odkril 41 grobov, v Kobaridu 1100, na Mostu na Soči (z imenom Sveta Lucija/S. Lucia pred l. 1952) pa kar 3960. Še 2480 grobov je na tem grobišču odkril Josef Szombathy, vodja antropološko-prazgodovinske zbirke Prirodoslov no-zgodovinskega muzeja na Dunaju, izkopaval je Nov zamah so arheološka raziskovanja dobila po drugi svetovni vojni z ustanovitvijo muzejev v Tolminu (1951) in Novi Gorici (1954) ter Zavoda za varstvo spomenikov Nova Gorica (1961), kajti skoraj vse dotlej odkrite arheološke najdbe so hranili muzeji zunaj meja nove Jugoslavije. Prvi arheolog v tolminskem muzeju Niko Mozetič je med letoma 1956 in 1958 opravil manjše terenske akcije na Mostu na Soči in v Bovcu. Kmalu zatem je bil tolminski muzej pridružen novogoriškemu (1958), kjer se je l. 1964 zaposlil Drago Svoljšak, ki je postavil temelje in začrtal smeri delovanja tamkajšnjega arheološkega oddelka.18 V tistem času je na nacionalni ravni potekal pomemben projekt arheološke karte kot osnove za nadaljnje preučevanje poselitve v arheoloških dobah na Slovenskem. Gradivo zanjo so iz objavljenih in arhivskih virov ter muzejskih evidenc zbirali do mala vsi slovenski arheologi, sodelovali so tudi študentje arheologije, uredila in za objavo pa ga je pripravila posebna komisija pod okriljem Sekcije za arheologijo pri SAZU. Arheološka najdišča Slo venije so bila objavljena l. 15 Marchesetti 1903, 88–92, vključno s topografsko karto. Nekaterih prazgodovinskih gradišč, ki jih navaja Marchesetti, nismo vključili: npr. Grad (Pavlinčev grad), ker je tam pobrana keramika verjetno bronastodobna; na Kozlovem robu nad Tolminom je bil med izkopavanji l. 2020 sicer najden košček keramike, ki bi lahko bil iz starejše železne dobe, vendar je to preskromen dokaz za naselje iz tega časa (Mlinar 2021a, 31); premalo indicev imamo tudi za najdišče na Deru pri Robiču, ki je bilo v prvi svetovni vojni močno poškodovano. 13 Izčrpneje o zgodovini raziskovanj Gabrovec, Svoljšak 1983, 12–24; Gabrovec 1987, 121–123; id. 1999, 175–177; Svoljšak, Dular 2016, 17–24. j 14 Gabrovec, Svoljšak 1983, 13; izčrpno o komisiji Mader 2018. 16 Forlati Tamaro 1930, 419–428; Mlinar 2020a, sl. 3. 17 Gabrovec 1958–1959; id. 1966b; id. 1974. 18 O razvoju arheološkega oddelka Goriškega muzeja v Novi Gorici gl. Kruh 2012. 19 O zgodovini arheološke karte na Slovenskem in projektu gl. prispevek Petra Petruja in predgovor v publi kaciji Arheološka najdišča Slovenije, 1975 (poslej ANSl). 12 Baš 1955. 12 Baš 1955. 13 Izčrpneje o zgodovini raziskovanj Gabrovec, Svoljšak 1983, 12–24; Gabrovec 1987, 121–123; id. 1999, 175–177; Svoljšak, Dular 2016, 17–24. 14 Gabrovec, Svoljšak 1983, 13; izčrpno o komisiji Mader 2018. 15 Marchesetti 1903, 88–92, vključno s topografsko karto. Nekaterih prazgodovinskih gradišč, ki jih navaja Marchesetti, nismo vključili: npr. Grad (Pavlinčev grad), ker je tam pobrana keramika verjetno bronastodobna; na Kozlovem robu nad Tolminom je bil med izkopavanji l. 2020 sicer najden košček keramike, ki bi lahko bil iz starejše železne dobe, vendar je to preskromen dokaz za naselje iz tega časa (Mlinar 2021a, 31); premalo indicev imamo tudi za najdišče na Deru pri Robiču, ki je bilo v prvi svetovni vojni močno poškodovano. 24 Svoljšak, Dular 2016. 25 Gabrovec, Svoljšak 1983, 7–9; Gabrovec 1999, 175–177. 26 Gabrovec 1964–1965, 24–25. 27 Teržan 2016. 28 Teržan, Trampuž 1973; Svoljšak 1973; Kos 1973. Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 403 Sl. 3: Število znanih najdišč posoške skupnosti iz starejše železne dobe po obdobjih raziskovanj. Fig. 3: Number of known sites of the Early Iron Age Posočje community according to periods of investigation. in najodmevnejša so bila naselbinska izkopavanja Svoljšaka in njegove muzejske ekipe na Mostu na Soči, ki so potekala celo desetletje in se po izjemnem pomenu postavljajo ob bok odkritju tamkajšnje nekropole. Z modernimi metodami in pristopi ter natančnim dokumentiranjem je bilo pridobljenih ogromno dragocenih podatkov o naselbinskem rastru, stavbarstvu, gospodarskih dejavnostih in materialni kulturi v starejši železni dobi Posočja.24 V šestdeseta leta prejšnjega stoletja segajo zametki še drugega pomembnega projekta za preučevanje starejše železne dobe na Sloven skem s formiranjem Vzhodnoalpskega komiteja/ Ostalpenkomitée/Comitato per le Alpi orientali, sestavljenega iz slovenskih, avstrijskih in italijan skih strokovnjakov, ki so organizirali mednarodno razstavo situlske umetnosti (1962). V njegovem programu je bila predvidena objava nekropole in pozneje tudi naselbine na Mostu na Soči.25 Nalogo publiciranja gradiva s slovenskega ozemlja v tujih muzejih z namenom, da bi postalo dostopnejše za znanstveno ovrednotenje, je Stane Gabrovec kot prioriteto postavil že v referatu, ki ga je imel l. 1963 na VI. kongresu Arheološkega društva Jugoslavije v Ljubljani; v njem je tudi opredelil svetolucijsko skupino kot samostojno entiteto v okviru jugovzhodnoalpske halštatske kulture.26 Kot profesorju za prazgodovinsko arheologijo na Filo zofski fakulteti Univerze v Ljubljani mu je s študenti svojega seminarja in z zglednim sodelovanjem z dunajskim prirodoslovno-zgodovinskim muzejem to v dobršni meri uspelo tudi izpeljati.27 Glavne značilnosti in kronološke faze razvoja posameznih halštatskih kulturnih skupin jugovzhodnoalpskega prostora je skupaj s svojimi učenci predstavil l. 1972 na mednarodnem kolokviju v Novem mestu. Njihovi izsledki so bili objavljeni v Arheološkem vestniku 24, 1973 (1975). V njem sta Biba Teržan in Neva Trampuž podali predlog podrobnejše kronologije za svetolucijsko skupino, utemeljen na gradivu z Mosta na Soči v dunajskem muzeju, Drago Svoljšak je na primeru odkritega grobišča v Tolminu predstavil bistvene značilnosti pokopa, Peter Kos pa je objavil in ovrednotil grobove, ki jih je izkopal Mahnič na Koritnici ob Bači.28 Svoj predlog kronoloških faz je l. 1973 podala Fulvia Sl. 3: Število znanih najdišč posoške skupnosti iz starejše železne dobe po obdobjih raziskovanj. Fig. 3: Number of known sites of the Early Iron Age Posočje community according to periods of investigation. l. 20 ANSl 1975 (regije Tolmin, Gorica in Radovljica), 114–127, 164–168. Od teh 27 najdišč je pri sedmih nave deno le, da gre za prazgodovinska najdišča, zato ni nujno, da so iz železne dobe. 20 ANSl 1975 (regije Tolmin, Gorica in Radovljica), 114–127, 164–168. Od teh 27 najdišč je pri sedmih nave deno le, da gre za prazgodovinska najdišča, zato ni nujno, da so iz železne dobe. 21 V digitalni obliki so vse doslej izdane številke te revije dostopne na spletu (https://www.zvkds.si/sl/knjiznica/ varstvo-spomenikov). 22 Gabrovec, Svoljšak 1983; Svoljšak 1990; id. 2005; Svoljšak, Pogačnik 2001–2002. 23 Osmuk 1997; v pripravi je monografska publikacija. STANJE ARHEOLOŠKIH RAZISKAV 1975.19 V tej leksikonski publikaciji (vanjo so vključeni zbrani podatki do Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 21 V digitalni obliki so vse doslej izdane številke te revije dostopne na spletu (https://www.zvkds.si/sl/knjiznica/ varstvo-spomenikov). 22 Gabrovec, Svoljšak 1983; Svoljšak 1990; id. 2005; Svoljšak, Pogačnik 2001–2002. 23 Osmuk 1997; v pripravi je monografska publikacija. 29 Lo Schiavo 1984; Gabrovec 1987, 121–123; id. 1999, 175–177. 30 Preistoria del Caput Adriae 1983; Ruaro Loseri, Montagnari Kokelj (ur.) 1984. 31 Gabrovec, Svoljšak 1983. 32 Teržan, Lo Schiavo, Trampuž-Orel 1984–1985. 33 Montagnari Kokelj (ur.) 1993. 34 Teržan 2019; Brišnik, Kajzer 2019. Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 1965) je iz starejše železne dobe v Posočju in Bohinju zabeleženih 27 najdišč, o katerih so po samezna gesla napisali Stane Gabrovec, Peter in Sonja Petru, Mehtilda Urleb in Franc Truhlar.20 Ob tem projektu so snovali tudi načrte za regionalne arheološke topografije z namenom, da bi s siste matičnimi terenskimi pregledi in rekognosciranjem dopolnili evidence temeljnih arheoloških virov in pridobili podatke o novih najdiščih. Na Goriškem in Tolminskem sta se tej nalogi posvetila Drago Svoljšak (najprej skupaj s Petrom Petrujem) in Nada Osmuk, konservatorka spomeniškovarstve nega zavoda v Novi Gorici, kjer se je zaposlila l. 1973. Opravila sta vrsto arheoloških terenskih pregledov in preverjala podatke z manjšimi son diranji, o vsem pa največkrat poročala v strokovni reviji Varstvo spomenikov.21 Prav tako sta izpeljala številne akcije reševanja arheoloških ostalin, ki so ponekod prerasle v raziskovalna izkopavanja. Naj omenimo samo najodmevnejša, ki so povezana s starejšo železno dobo, kot so odkritje 465 grobov v Tolminu, novih 287 grobov v Kobaridu, sondiranja na naselbini na Sv. Katarini (Kekec) nad Novo Gorico22 ter izkopavanja na Gradiču v Kobaridu, ki so razkrila stavbne ostaline in svetiščni prostor iz železne in rimske dobe.23 A zdaleč najobsežnejša Miha MLINAR, Sneža TECCO HVALA 404 Lo Schiavo na osnovi Marchesettijevih odkritij na Mostu na Soči,29 njena periodizacija pa se ne razhaja bistveno s kronološko shemo, ki je v slovenski arheologiji v uporabi še danes. Posoške najdbe iz starejše železne dobe so bile vključene tudi v razstavo z naslovom Preistoria del Caput Adriae l. 1983 pod okriljem tržaškega muzeja, ob kateri je bil organiziran mednarodni simpozij.30 Tega leta sta Gabrovec in Svoljšak izdala v knjižni seriji Katalogi in monografije Narodnega muzeja v Ljubljani prvi zvezek o Mostu na Soči31 z izčrpno predstavitvijo topografije in zgodovine raziskav ter rekonstruiranim načrtom grobišča, kjer sta kopala Marchesetti in Szombathy. Takoj zatem sta izšla dva zvezka z gradivom Szombathyjevih izkopavanj, za objavo so ju pripravile Biba Ter žan, Fulvia Lo Schiavo in Neva Trampuž-Orel.32 Italijanski kolegi pa so l. 1993 ponatisnili zbrane Marchesettijeve razprave,33 medtem ko večina gradiva njegovih raziskav v tržaškem muzeju še vedno ostaja širši strokovni javnosti nedostopna za nadaljnje analize. naselbini v Grgarju (2005).35 Po l. 2009 je nekaj novih arheoloških lokacij sondiral Center za pre ventivno arheologijo (ZVKDS CPA), in sicer ob vznožju Gradiča v Kobaridu ter na Berjaču nad Podbelo v Breginjskem kotu.36 Večino zaščitnih akcij na tem območju pa po l. Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 2000 prevzema Tolminski muzej, potem ko je vnovič pridobil samostojnost in zaposlil arheologa (Miho Mlinarja). Med pomembnejše terenske raziskave, ki jih je opravil, spadajo odkritja stavbnih ostankov na severnem in zahodnem obrobju železnodobne naselbine na Mostu na Soči (2001, 2004, 2010) ter grobov na najnižji terasi na levem bregu Idrijce, pod njimi pa ostankov naselbinske plasti iz mlajše bronaste dobe (2000–2002 in 2013), nadalje odkritje izjemnega kultnega mesta z zakopom konjev in bojevniških oprav v Kobaridu (2010), kjer priha jajo ob zemeljskih delih na robu v preteklosti že kopanega grobišča na dan tudi vedno novi grobovi (2002, 2021/22). Med nova odkritja na dotlej arheološko povsem neznanih lokacijah pa lahko prištejemo grobove pri Srpenici na Bovškem (2003), v Jerovci na Šentviški planoti (2007) in v Čadrgu visoko nad dolino Tolminke (2014).37 Podatkov o novih najdiščih si ne prizadeva pridobiti zgolj z zaščitnimi akcijami, ki jih narekujejo predvsem gradbeni in drugi zemeljski posegi, temveč tudi od zbirateljev in iskalcev z detektorji kovin, ki so zadnja desetletja zelo dejavni. Iz teh virov izhaja večina novih podatkov in posamičnih najdb na tem območju (sl. 3; 4); verodostojnost teh podatkov pa je različna, saj pogosto niso znani konteksti in tudi sporočene lokacije niso najbolj zanesljive. Z novimi odkritji Tolminski muzej sprotno seznanja zainteresirano občinstvo na razstavah in strokovnih srečanjih ter z objavami. Med novejšimi publikaci jami si posebno mesto zaslužijo tri monografije o Mostu na Soči v železni dobi, ki so plod sodelovanja Goriškega in Tolminskega muzeja z Inštitutom za arheologijo ZRC SAZU ter raznimi strokovnjaki; v dveh so celovito predstavljene ter moderno analizi rane ostaline in najdbe iz železnodobne naselbine, odkrite na desnem bregu Idrijce, tretja pa ponuja Novo prelomnico pomenita reorganizacija spomeniškovarstvene službe in sprememba njene strategije po ustanovitvi samostojne slovenske države. Vloga konservatorjev na območnih enotah zavoda za spomeniško varstvo se je spremenila, težišče njihovega dela se je premaknilo na uprav ne postopke evidentiranja varovanih območij in določanje pogojev za posege vanje ter na nadzor nad izvedbo arheoloških terenskih raziskav. 35 Za Jelenšek pri Godoviču glej prispevek Bratina, Laharnar, Svoljšak v tej publikaciji, za Grgar pa prispe vek Bratina. 35 Za Jelenšek pri Godoviču glej prispevek Bratina, Laharnar, Svoljšak v tej publikaciji, za Grgar pa prispe vek Bratina. 36 Vinazza 2015; T. Fabec, T. Nanut, B. Laharnar, B. Kramberger, T. Leskovar, T. Tolar, K. Kavkler, E. Menart, Poročilo o izvedenih arheoloških raziskavah v Podbeli, 2021 (hrani ZVKDS CPA). 37 Mlinar, Klasinc, Knavs 2008; Mlinar 2009–2010; Mlinar, Gerbec 2011; Laharnar, Mlinar 2013; Laharnar, Mlinar 2019; Mlinar 2020a; id. 2020b. 36 Vinazza 2015; T. Fabec, T. Nanut, B. Laharnar, B. Kramberger, T. Leskovar, T. Tolar, K. Kavkler, E. Menart, Poročilo o izvedenih arheoloških raziskavah v Podbeli, 2021 (hrani ZVKDS CPA). 37 Mlinar, Klasinc, Knavs 2008; Mlinar 2009–2010; Mlinar, Gerbec 2011; Laharnar, Mlinar 2013; Laharnar, Mlinar 2019; Mlinar 2020a; id. 2020b. 38 Svoljšak, Dular 2016; Dular, Tecco Hvala (ur.) 2018; Mlinar 2020a. 39 Svoljšak, Pogačnik 2001–2002. 40 Mlinar, Žbona Trkman 2008; Laharnar, Mlinar 2013; Laharnar 2018b; Mlinar et al. 2018. 41 Mlinar, Gerbec, Laharnar 2014; Gerbec, Vinazza 2018; Gerbec 2021. 42 Horvat 2015–2016; Mlinar, Turk 2016; Horvat 2020. Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja Pri teh gre pretežno za spremljanje gradbenih in infrastrukturnih posegov ter prostorskih načrtov, medtem ko je delež terenskih akcij z raziskovalnimi cilji, ki bi bili usmerjeni v razreševanje konkretnih arheoloških vprašanj, zelo majhen.34 V Posočju je novogoriška enota spomeniškovar stvenega zavoda s konservatorkama Nado Osmuk in Patricijo Bratina zaključila izkopavanja na Gradiču v Kobaridu (1997) in skupaj z Narodnim muzejem Slovenije rešila pred izropanjem grobišče iz starejše železne dobe na Jelenšku pri Godoviču (1993) ter izvedla sondiranja na prazgodovinski Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 405 reliefa ter orodji geografskega informacijskega sistema (GIS) omogoča različne prostorske analize. Na ta način so bila denimo pregledana in ovre dnotena najdišča na Kobariškem.43 V zaznavanju arheološkega potenciala so si mesto zagotovile tudi geofizikalne meritve.44 Z vključevanjem naravoslovnih disciplin, kot so arheobiološke, arheometalurške, radiokarbonske in še vrsta dru gih analiz, postajajo arheološke raziskave čedalje bolj interdisciplinarne. Kljub vsemu napredku pa je čutiti manko ciljnega raziskovanja, s čimer bi izboljšali poznavanje posameznih faz in značilnosti poselitvenega vzorca ter ugotavljanje kontinuitete in prelomnic v poselitvi. 43 Štular 2011a; Laharnar, Štular, Mlinar 2015. 44 Mlinar, Mušič 2020; Tecco Hvala, Mušič 2021. 45 Mlinar 2018 z navedeno starejšo literaturo; Gerbec 2018; Gerbec Vinazza 2018. 46 V katalogu najdišč so podani podatki o geografski legi, stanju raziskav, najdbah in njihovi hrambi ter okvirna datacija in sklic na referenčne objave. Sklici na kataloške številke so uporabljeni v preglednicah in v besedilu. Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja sodoben uvid v strukturo in kronološko sosledje grobov, način pokopavanja in mesto sežiganja na severnem obrobju prostranega grobišča na levem bregu Idrijce.38 Posebej kaže omeniti tudi celovito objavo in podrobno analizo tolminskega grobišča v Katalogih in monografijah Narodnega muzeja Slovenije.39 Od arheoloških topografij pa kaže omeniti sodelovanje Mihe Mlinarja z Beatriče Žbona Trkman, preminulo kustosinjo Goriškega muzeja v Novi Gorici, pri objavi novih arheoloških najdb z Banjške planote in Trnovskega gozda ter njegovo sodelovanje z Boštjanom Laharnarjem iz Naro dnega muzeja Slovenije pri predstavitvi najdišč na Šentviški planoti.40 Pri orisu arheološke podobe Breginjskega kota se jima je pridružila Teja Gerbec, zdaj zaposlena v Goriškem muzeju Nova Gorica, ki je zbrala tudi podatke o najdiščih na Kanalskem Kolovratu in skupaj z Manco Vinazza z Oddelka za arheologijo Filozofske fakultete v Ljubljani še za zahodni del Banjške planote.41 V posoških in bohinjskih hribih pa poteka sistematično evi dentiranje in vrednotenje visokogorskih najdišč v okviru programa Inštituta za arheologijo ZRC SAZU, ki ga izvajata Jana Horvat skupaj z Lucijo Lavrenčič in Matija Turk v sodelovanju s Tolmin skim muzejem ter zunanjimi arheološkimi (Marija Ogrin, ArheoAlpe) in nearheološkimi sodelavci (Janez Bizjak, Miran Bremšak (†), Gorazd in Jani Kutin, Pavel Jamnik), ti so kot odlični poznavalci lokalnega okolja in z veliko zavzetostjo odkrili večino novih arheoloških lokacij iz različnih dob v visokogorju.42 Vse te akcije so prispevale k temu, da se je število znanih najdišč posoške skupnosti iz starejše železne dobe povzpelo na 85, pri čemer je med njimi nekaj večjih kompleksov, ki vključujejo naselje s pripadajočim grobiščem in/ali drugimi vrstami najdb v neposredni bližini (sl. 3). Po do sedaj zbranih podatkih je med najdišči največ po samičnih naključnih najdb, medtem ko so naselja in grobišča zastopana z enakim deležem (sl. 4). NASELJA Od potencialnih naselij, omenjenih v virih,45 smo v katalog vključili le tista, za katera obstajajo bolj ali manj zanesljivi materialni dokazi o njihovi obljudenosti v starejši železni dobi (sl. 4; 5).46 Ta kih je 24, vendar na večini še ni bilo arheoloških raziskav, temveč kažejo na njihovo poseljenost poleg okopov in teras predvsem najdbe, pobrane 46 V katalogu najdišč so podani podatki o geografski legi, stanju raziskav, najdbah in njihovi hrambi ter okvirna datacija in sklic na referenčne objave. Sklici na kataloške številke so uporabljeni v preglednicah in v besedilu. Sl. 4: Deleži posameznih vrst najdišč posoške skupnosti iz starejše železne dobe, znanih do l. 2022 (skupaj n = 85). Fig. 4: Shares of the types of sites in the Early Iron Age Posočje community as known to 2022 (total n = 85). V novejšem času se je pri topografskih vre dnotenjih uveljavila uporaba podatkov zračnega laserskega snemanja površja (LIDAR), ki skupaj z arheološkimi evidencami in digitalnim modelom Sl. 4: Deleži posameznih vrst najdišč posoške skupnosti iz starejše železne dobe, znanih do l. 2022 (skupaj n = 85). Fig. 4: Shares of the types of sites in the Early Iron Age Posočje community as known to 2022 (total n = 85). Miha MLINAR, Sneža TECCO HVALA 406 dobe v ozki dolini med Mostom na Soči in Gorico niso bila odkrita, temveč ležijo na planotah. Na Banjški planoti jih zasledimo v bližini trajnih iz virov in območij z rodovitno prstjo; na severnem delu sta iz tega časa Grad pri Levpi in domnevno V Glavi pri Kanalskem Lomu (št. 50, 48), na zaho dnem robu planote leži Gradišče pri Desklah (št. 51), na jugozahodnem delu pa sta znani naselbini v Grgarski kotlinici (št. 52) in na Sv. Katarini nad Novo Gorico (št. 53). Nekaj naselij je bilo tudi na Šentviški planoti (št. 57, 62), na njenem južnem delu, ki se dviga nad Idrijco, medtem ko v odročnejših predelih Idrijskega hribovja za zdaj niso znana. Vzhodno mejo teritorija te skupnosti morda nakazuje naselje Štalca nad Železniki (št. 70) nad dolino Selške Sore, katerega pripadnost se predvideva predvsem na osnovi naselbinske keramike, ki je podobna tisti z Mosta na Soči. Najbolj vzhodno je naselje Žirk pri Žireh (št. 71), na površju ali pridobljene z detektorjem kovin. Arheološka sondiranja ali izkopavanja so bila opravljena le na devetih. Sl. 5: Velikost, tip in nadmorska višina zanesljivih in domnevnih (?) naselij posoške skupnosti iz starejše železne dobe po posameznih pokrajinskih enotah. p p p j Fig. 5: Size, type and altitude of positively identified and presumed (?) settlements of the Early Iron Age Posočje com munity according to landscape units. Lega in velikost V alpskem visokogorju so naselja iz tega časa umeščena na vzpetine pri dnu dolin glavnih vo dotokov Soče in Nadiže ter Save Bohinjke, ki ne pomenijo zgolj stalnega vira za oskrbo z vodo, temveč so tudi glavni orientacijski označevalci v težko prehodni pokrajini. Te poseljene vzpetine (sl. 5) se pnejo na obrobju širših plodnih ravnic. Poleg stalnih naselij sta znani tudi dve sezonski postojanki, ena na območju Trente – V plazeh (št. 29), druga na Dolgi planji na Voglu (št. 38), obe ležita nad 1500 m nadmorske višine. V nasprotju z zgornjim Posočjem pa naselja iz starejše železne Kat. št. / Cat. No. Naselja / Settlements n. v. (m) / asl (m) Utrjeno / Fortification Velikost (ha) / Size (ha) Pokrajinske enote / Landscape units 33a Jereka – Dunaj 655 - 2 Julijske Alpe 36a Boh. Bistrica – Ajdovski gradec 580 + 1,5 " 35a Lepence – Sp. Gradišče (?) 554 - ? " 26a Bovec – Ravelnik 519 + 3 " 22a Podbela – Sv. Helena 363 + 0,8 " 21 Robič – Sv. Volar 317 + 0,7 " 18a Kobarid – Gradič 297 + 4 " 3 Volče – Sv. Lenart (?) 212 - ? " 1a Most na Soči 173 + 13 " 48 Kanalski Lom – V Glavi (?) 580 + 0,6 Banjšice 50 Levpa – Grad 574 - 0,9 " 52 Grgar – Grašišče 351 + 1,7 " 53 Nova Gorica – Sv. Katarina 327 + 1 " 51 Deskle – Gradišče (?) 246 - 0,5 " 43 Golo Brdo – sv. Marija na Jezeru 189 + 1 Kambreško pogorje 62 Šentviška Gora – Berlotov rob (?) 730 + 0,2 Šentviška planota 57 Pečine – Vrh gradu 588 + 0,5 " 72a Godovič – Jelenšek 817 + 0,7 Cerkljansko hribovje 69 Novaki – Mali Njivč (?) 758 - 0,5 " 71 Žiri – Žirk (?) 663 + 1 " 70 Železniki – Štalca 639 + 9 " 65 Police – Pri cierki (?) 531 - ? " 68 Cerkno – Gradišče 450 + 0,5 " 66 Reka – Grad (?) 346 - ? " l l k d k l h d h ( ) l k k l d b Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 407 Sl. 6: Lidarski posnetek naselja domnevno iz starejše železne dobe na Ravelniku pri Bovcu (št. 26a) z okolico. Fig. 47 Najdišč v Vipavski dolini (gl. npr. Bratina 2009–2010) in na južnih obronkih Trnovskega gozda v tem članku ne obravnavava, ker jih zaradi skromne raziskanosti ni mogoče nedvomno opredeliti v starejšo železno dobo in jih pripisati posoški skupnosti. Lega in velikost 6: LiDAR-derived image of a presumably Early Iron Age settlement on Ravelnik near Bovec (No. 26a) and the surrounding area. (Vir / Source: © ARSO) Sl. 6: Lidarski posnetek naselja domnevno iz starejše železne dobe na Ravelniku pri Bovcu (št. 26a) z okolico. Fig. 6: LiDAR-derived image of a presumably Early Iron Age settlement on Ravelnik near Bovec (No. 26a) and the surrounding area. (Vir / Source: © ARSO) (Vir / Source: © ARSO) ob naravni komunikaciji z dolino Nadiže (Koba rid). Daleč pred vsemi prednjači Most na Soči, ki je s pribl. 13 ha površine do štirikrat večje od bovškega Ravelnika (3 ha) (sl. 6) ali kobariškega Gradiča (4 ha); med največja se pogojno uvršča tudi mejno naselje Štalca nad Železniki (9 ha) (sl. 5: št. 1a, 18a, 26a, 70). Srednje velikih naselij, ki merijo od 1 do 2 ha, je šest in so razmeščena ob Savi Bohinjki (št. 33a, 36a) na severni meji in v Grgarski kotlinici (št. 52) na jugozahodnem koncu Čepovanskega dola, ki predstavlja naravno komu nikacijo prek Banjške planote v dolino Idrijce. V ta velikostni razred spadajo še Sv. Katarina nad Novo Gorico na stičišču Soške in Vipavske doline (št. 53), Golo Brdo ob reki Idriji na zahodni meji (št. 43) ter domnevno naselje Žirk pri Žireh ob Poljanski Sori (št. 71) na vzhodni meji. Lega in velikost omenjenih naselij nakazujeta pomembno vlogo, ki so jo ti naravni koridorji igrali pri izbiri njihove lokacije. Naselja v njihovem zaledju so manjša od enega hektarja, najmanjša so na Šentviški planoti (št. 57, 62). V petih primerih pa njihove površine ni mogoče oceniti, ker v reliefu ni opaziti umetnih preoblikovanj. na jugovzhodu pa Jelenšek pri Godoviču (št. 72a), oba ležita na ozkih grebenih tako kot druga naselja v Cerkljanskem hribovju (št. 66, 68, 69). O poselitvi Kambreškega pogorja zahodno od Soče v tem času za zdaj nimamo trdnih dokazov. Naselje Golo Brdo (št. 43), ki nemara pripada posoški skupnosti, leži v okljuku mejne reke Idrije na prehodu v Furlansko nižino.47 Ker je bila samobitnost svetolucijske kul turne skupine prepoznana prvenstveno po njenih posebnostih v načinu pokopa in grobnih pridatkih, lahko pripadnost mejnih naselij, ob katerih grobi šča še niso bila odkrita, obravnavamo le pogojno oziroma z vprašajem. Stanje raziskav in objav v obravnavo vključenih naselij za zdaj dopušča le primerjave po njihovi legi in velikosti ter fortifikacijah (sl. 5). 50 Svoljšak 2005, 660, sl. 3. 51 Osmuk 1977; ead. 1993; Bratina 2001; ead. 2009a. 52 Glej prispevek Bratina v tej publikaciji. 53 Tecco Hvala, Mušič 2021; E. Leghissa, Arheološke raziskave na najdišču Most na Soči – prazgodovinsko obzidje. Končno strokovno poročilo o raziskavi 21-0230, marec 2022, Ljubljana (hrani arhiv Iza ZRC SAZU). Fortifikacije Večina naselij je bila že naravno dobro zavaro vana z bolj ali manj strmimi pobočji ali globokimi rečnimi strugami in grapami. Sledi dodatnega umetnega utrjevanja na lažje dostopnih predelih so opazne pri dveh tretjinah (sl. 5). Delno so bila utrjena vsa velika naselja (sl. 6), tudi največje med njimi na Mostu na Soči, kot je pokazalo sondiranje l. 2021 (sl. 7). Doslej je namreč zanj veljala domneva, da dodatnih obrambnih zidov ni potrebovalo ob naravni zaščiti, ki sta mu jo nudili reki Soča in Idrijca, in ob varovanju, ki so ga zagotavljale utrjene strateške točke ob vstopih v porečje Soče (št. 26a, 21, 53).48 V Bohinju je bil utrjen le Ajdovski gradec (št. 36a), vendar naj bi bilo sklenjeno obzidje rimskodobno. Devet jih ni imelo posebej grajene zaščite, iz česar sklepamo, da gre v teh primerih za drug tip ali rang naselja. Na naselju pri cerkvi sv. Marije na Jezeru nad Golim Brdom (št. 43) so bile prav tako odkrite kulturne plasti in suhozidne konstrukcije iz več dob, zanesljivo je bilo utrjeno v rimski dobi.51 Izsledki sondiranja na grgarskem Grašišču (št. 52) pa so v tej publikaciji posebej predstavljeni.52 Na Mostu na Soči (št. 1a) poteka na vzhodnem robu naselja peščen greben v smeri S–J z najvišjega vrha proti rečnemu koritu Idrijce. Na dveh odsekih v razmiku 60 m so bile na njem l. 2021 opravljene geofizikalne meritve in izkopana manjša sonda, ki so razkrile kamnito obzidje (sl. 7; 8B).53 Na območju izkopa je bilo obzidje sicer poškodovano z dvema okroglima vkopoma (verjetno iz prve svetovne vojne) (sl. 7A: a,b), kljub temu se je dokaj dobro ohranila vzhodna fronta (sl. 7B). Vkopana je bila v peščen greben brez vsakršnih kulturnih ostankov (sl. 7C: c), grajena je bila iz ploščatih kamnov, mestoma postavljenih v stolpiče. Ob njej je potekala še ena linija zloženih kamnov, morda za dodatno utrditev. Zahodna fronta, ki je gledala proti notranjosti naselja, je bila razrušena. Vmesni prostor so zapolnjevali manjši zaobljeni kamni. Razpon med vzhodno in zahodno fronto obzidja znaša okoli tri metre, podobno kot na Sv. Katarini nad Novo Gorico. Način gradnje je enak, kot so bili grajeni drenažni zidovi pri železnodobnih hišah v naselbini in obzidja v notranjsko-kraški regiji. 48 Svoljšak 1984a; id. 1986; Gabrovec 1999, 182–183; Mlinar 2018. 49 Svoljšak 1990; id. 2005. Lega in velikost Največja so bila naselja v zgornji Soški dolini ob sotočju Soče z Idrijco (Most na Soči) in s Koritnico (Bovec) ter Miha MLINAR, Sneža TECCO HVALA 408 sta pozneje izrabila rimskodobni in zgodnjesre dnjeveški kastel.50 Fortifikacije Na vzhodni strani grebena, na območju izven nasel bine, pa je bila odkrita črnikasta zemljena plast z veliko drobci oglja in živalskih kosti; ležala je na konglomeratu, ki je bil ponekod zaradi intenziv nejše ali pogoste prisotnosti ognja že poapnel (sl. 7D; 7E). Ta plast se je deloma raztezala čez enovit peščeni greben, v njej odkrite najdbe pa kažejo na ostanke metalurške dejavnosti (kosi žlindre, drobci bronaste taline, del ostenja livarske posode, polizdelek bronaste večglave igle, košček kalupa iz peščenjaka). Na vrhu sta ruševine obzidja na zahodni strani in plast žganine na vzhodni pre krivali humusna plast z razpršenimi recentnimi in rimskodobni najdbami ter travnata ruša. Žganinsko plast na vzhodnem delu izven obzidja lahko na g p g p g j Arheološka sondiranja, ki so bila opravljena na nekaterih okopih, so pokazala, da se način gradnje utrdbenih struktur ni razlikoval od tistih na notranjsko-kraških kaštelirjih. Na Sv. Katarini nad Novo Gorico je bilo ugotovljenih več faz utrjevanja (št. 53).49 Prvo sondiranje na skrajnem severnem koncu nasipa je razkrilo, da je bilo na skalnati naravni podlagi zgrajeno do 4 m debelo obzidje z močno zunanjo in notranjo fronto, ki sta bili preprosto zloženi iz velikih ploščatih kamnov, vmesni prostor pa zapolnjen s kamnitim materialom. Nanj je bil pozneje v enaki širini postavljen zid iz lomljencev, vezanih z malto. V plasteh, ki sodijo k prvemu obzidju, grajenem v suhozidni tehniki, so bile odkrite črepinje lončenine s primerjavami na utrjenih naseljih v Vipavski dolini in na kra ških kaštelirjih. V črnikasti plasti s sledmi ognja na zunanji strani obzidja je bilo nekaj železnih, bronastih in koščenih predmetov pa tudi glinast omet z odtisi protja ter prostoročno izdelana lončenina. Najdbe iz vrhnjih plasti ter z malto vezana zid in obrambni stolp so iz rimske dobe in zgodnjega srednjega veka. S poznejšim sondira njem na južni strani so bile jasno ugotovljene tri faze utrjevanja. Prvo in drugo obzidje je ločevala tanka zemljena plast, ki bi kazala na opustelost naselja v času njenega nastanka; obe obzidji sta bili enako grajeni in približno enake debeline (3,3 in 2,9 m). Te prazgodovinske obrambne strukture Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 409 Sl. 7: Most na Soči (št. 1a), arheološka sonda leta 2021. Fortifikacije A kumulativni tloris, na zahodnem delu je ruševina obzidja z recentnima vkopoma (a, b), v sredini je peščen greben brez kulturnih ostankov, na vzhodnem delu pa kotanja s sledmi metalurške dejavnosti; B ostanki obzidja z ohranjeno vzhodno fronto in vmesnim zidom ter razrušeno zahodno fronto, pogled z juga; C južni profil z vkopom v peščen greben (c) za vzhodno fronto obzidja, vmesni zid in ostanki zahodne fronte, pogled s severa; D, E kotanja na vzhodnem delu sonde, zapolnjena z žganino in ostanki metalurške dejavnosti, pogled s severa. Fig. 7: Most na Soči (No. 1a), trial trenching in 2021. A cumulative plan, the west part shows the ruins of the stonework rampart with two recent cuts (a, b), in the centre is a sandy ridge without cultural remains, the east part has a depression with traces of metallurgic activities; B remains of the stonework rampart with the surviving east face, core and collapsed west face, view from the south; C south section with a cut into the sandy ridge (c) behind the east face of the stonework rampart, core and the remains of the west face, view from the north; D, E depression in the eastern part of the trench, filled with burnt debris and the remains of metallurgic activities, view from the north. Sl. 7: Most na Soči (št. 1a), arheološka sonda leta 2021. A kumulativni tloris, na zahodnem delu je ruševina obzidja z recentnima vkopoma (a, b), v sredini je peščen greben brez kulturnih ostankov, na vzhodnem delu pa kotanja s sledmi metalurške dejavnosti; B ostanki obzidja z ohranjeno vzhodno fronto in vmesnim zidom ter razrušeno zahodno fronto, pogled z juga; C južni profil z vkopom v peščen greben (c) za vzhodno fronto obzidja, vmesni zid in ostanki zahodne fronte, pogled s severa; D, E kotanja na vzhodnem delu sonde, zapolnjena z žganino in ostanki metalurške dejavnosti, pogled s severa Sl. 7: Most na Soči (št. 1a), arheološka sonda leta 2021. p g Fig. 7: Most na Soči (No. 1a), trial trenching in 2021. A cumulative plan, the west part shows the ruins of the stonework rampart with two recent cuts (a, b), in the centre is a sandy ridge without cultural remains, the east part has a depression with traces of metallurgic activities; B remains of the stonework rampart with the surviving east face, core and collapsed west face, view from the south; C south section with a cut into the sandy ridge (c) behind the east face of the stonework rampart, core and the remains of the west face, view from the north; D, E depression in the eastern part of the trench, filled with burnt debris and the remains of metallurgic activities, view from the north. Fortifikacije Izmerjen je bil 60 m severno od arheološke sonde l. 2021 in kaže na različno strukturo sedimentov pod površjem do globine pribl. 4 m (po Tecco Hvala, Mušič 2021, sl. 4). Sl. 8: Most na Soči (št. 1a). A Radiokarbonska datacija vzorcev oglja in živalskih kosti iz žganinske plasti v vzhodnem delu arheološke sonde leta 2021 (VZ 6 – oglje) in iz plasti nad njo (VZ 8 – kost) ter iz ruševine obzidja (VZ 9 – kost). (Poznań Radiocarbon Laboratory, Poljska). B Georadarski profil prečno čez greben, potekajoč v smeri S–J po pobočju. Izmerjen je bil 60 m severno od arheološke sonde l. 2021 in kaže na različno strukturo sedimentov pod površjem do globine pribl. 4 m (po Tecco Hvala, Mušič 2021, sl. 4). Fig. 8: Most na Soči (No. 1a). A radiocarbon dating of the charcoal and animal bone samples taken in 2021 from the layer of burnt debris in the eastern part of the trial trench (VZ 6 – charcoal), from the layer above it (VZ 8 – bone) and from the rampart ruins (VZ 9 – bone). (Poznań Radiocarbon Laboratory, Poland). B GPR (ground-penetrating radar) profile across the ridge running N–S along the slope. It was measured 60 m north of the 2021 trial trench and shows a diverse structure of sediments to the depth of roughly 4 m (from Tecco Hvala, Mušič 2021, Fig. 4). Fig. 8: Most na Soči (No. 1a). A radiocarbon dating of the charcoal and animal bone samples taken in 2021 from the layer of burnt debris in the eastern part of the trial trench (VZ 6 – charcoal), from the layer above it (VZ 8 – bone) and from the rampart ruins (VZ 9 – bone). (Poznań Radiocarbon Laboratory, Poland). B GPR (ground-penetrating radar) profile across the ridge running N–S along the slope. It was measured 60 m north of the 2021 trial trench and shows a diverse structure of sediments to the depth of roughly 4 m (from Tecco Hvala, Mušič 2021, Fig. 4). 56 Svoljšak, Dular 2016; Dular, Tecco Hvala (ur.) 2018; Tecco Hvala 2020. 57 Pozneje so bili iz halštatske dobe odkriti ostanki še 4 stavb (Mlinar, Klasinc, Knavs 2008), iz rimske dobe pa 5 stavb. 54 Vzorec oglja (VZ 7) iz ruševine obzidja pri kalibraciji 2 σ kaže večji odstotek verjetnosti (68,6 %) za razpon od 1811 do 1918 cal AD, kar bi pritrjevalo domnevi, da je bilo poškodovano v prvi svetovni vojni. 55 Prim. Štular 2011a; Mlinar 2018. Fortifikacije A kumulativni tloris, na zahodnem delu je ruševina obzidja z recentnima vkopoma (a, b), v sredini je peščen greben brez kulturnih ostankov, na vzhodnem delu pa kotanja s sledmi metalurške dejavnosti; B ostanki obzidja z ohranjeno vzhodno fronto in vmesnim zidom ter razrušeno zahodno fronto, pogled z juga; C južni profil z vkopom v peščen greben (c) za vzhodno fronto obzidja, vmesni zid in ostanki zahodne fronte, pogled s severa; D, E kotanja na vzhodnem delu sonde, zapolnjena z žganino in ostanki metalurške dejavnosti, pogled s severa. p g Fig. 7: Most na Soči (No. 1a), trial trenching in 2021. A cumulative plan, the west part shows the ruins of the stonework rampart with two recent cuts (a, b), in the centre is a sandy ridge without cultural remains, the east part has a depression with traces of metallurgic activities; B remains of the stonework rampart with the surviving east face, core and collapsed west face, view from the south; C south section with a cut into the sandy ridge (c) behind the east face of the stonework rampart, core and the remains of the west face, view from the north; D, E depression in the eastern part of the trench, filled with burnt debris and the remains of metallurgic activities, view from the north. 410 Miha MLINAR, Sneža TECCO HVALA Sl. 8: Most na Soči (št. 1a). A Radiokarbonska datacija vzorcev oglja in živalskih kosti iz žganinske plasti v vzhodnem delu arheološke sonde leta 2021 (VZ 6 – oglje) in iz plasti nad njo (VZ 8 – kost) ter iz ruševine obzidja (VZ 9 – kost). (Poznań Radiocarbon Laboratory, Poljska). B Georadarski profil prečno čez greben, potekajoč v smeri S–J po pobočju. Izmerjen je bil 60 m severno od arheološke sonde l. 2021 in kaže na različno strukturo sedimentov pod površjem do globine pribl. 4 m (po Tecco Hvala, Mušič 2021, sl. 4). Fig. 8: Most na Soči (No. 1a). A radiocarbon dating of the charcoal and animal bone samples taken in 2021 from the Sl. 8: Most na Soči (št. 1a). A Radiokarbonska datacija vzorcev oglja in živalskih kosti iz žganinske plasti v vzhodnem delu arheološke sonde leta 2021 (VZ 6 – oglje) in iz plasti nad njo (VZ 8 – kost) ter iz ruševine obzidja (VZ 9 – kost). (Poznań Radiocarbon Laboratory, Poljska). B Georadarski profil prečno čez greben, potekajoč v smeri S–J po pobočju. Značilnosti stavbarstva in notranja ureditev naselij osnovi delčkov loka kačaste fibule in kalotastih dvodelnih obeskov pa tudi po lončenih črepinjah z vodoravnimi gladkimi rebri datiramo v stopnjo Sv. Lucija IIa oz. v 6. st. pr. n. št. Takšni kronolo ški interpretaciji pritrjujejo tudi radiokarbonske analize vzorcev oglja in kosti (sl. 8A).54 Moderna izkopavanja Draga Svoljšaka in njegove ekipe na Mostu na Soči ter celovita objava nasel binskih ostankov in najdb z desnega brega Idrijce so najizčrpnejši in pravzaprav edini razpoložljivi vir podatkov o posoškem stavbarstvu v starejši železni dobi, urbanistični zasnovi, dejavnostih in bivalni kulturi.56 Na raziskanem območju, velikem 4 ha, na vzhodnem delu današnje vasi so odkrili ostanke 36 stavb iz starejše in 3 iz mlajše železne dobe ter 10 iz rimske dobe.57 Na sledi hiše iz mlajše bronaste dobe so naleteli na zahodnem delu bližje Lidarski posnetki nekaterih utrjenih naselij oziroma gradišč, kot so Ravelnik pri Bovcu (sl. 6), Gradič v Kobaridu, Sv. Volar pri Robiču, Sv. Helena pri Podbeli, Vrh gradu pri Pečinah, Gra dišče v Cerknem in Žirk pri Žireh (št. 26a, 18a, 21, 22a, 57, 68, 71), razkrivajo različne obrambne sisteme. Ker pa še niso arheološko raziskani, ostaja odprto vprašanje, ali so vse obrambne strukture iz halštatske dobe.55 Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 411 dejavnost, v nekaterih drugih na metalurško. Glavne značilnosti keramičnega zbira iz naselbine so sestava lončarske gline z obilnim dodajanjem peska, tipične oblike loncev, pitosov, kelihov in situl ter okras (vodoravna gladka rebra, rdeče-črno barvanje, metličenje) pa tudi številčnost glinastih svitkov.63 Sodeč po ostankih žlindre in bronaste taline, livarskih pripomočkih ter najdenih ingotih in železnem kvadru se je v naselbini odvijala tudi sekundarna predelava kovin in izdelava predmetov.64 sotočju,58 skromna naselbinska plast iz tega časa je bila pozneje odkrita tudi onkraj Idrijce blizu njenega izliva v Sočo.59 Stavbe iz starejše železne dobe so bile grajene po ustaljenih stavbarskih pravilih, značilnih za vzhodnoalpski krog, a izkazujejo tudi svoje lokalne posebnosti. S treh strani so bile vkopane v prisojno pobočje, glavna pročelja z vhodom so večinoma gledala na jug. Stene gradbene jame so bile obložene z drenažnimi zidovi, kar je posebnost posoškega stavbarstva oziroma posoške železnodobne hiše, kot ju je označil Drago Svoljšak.60 Kamniti temelji stavb so bili malce odmaknjeni od njih in posta vljeni na poravnana tla. Na njih je počivala lesena konstrukcija, sestavljena iz temeljnega praga, soh, ročic, poveznika ali oklepa ter opaža iz klanih desk. Značilnosti stavbarstva in notranja ureditev naselij Za gradnjo je bil največkrat izbran trd in odporen hrastov les.61 Bolje ohranjeni tlorisi stavb kažejo, da so bile velike od 6 do 28,5 m2. Bile so eno-, dvo- ali troprostorne, kar nakazujejo vmesne vrste temeljnih kamnov, ki so služili kot podlaga predel nim stenam.62 Tla v notranjosti so bila največkrat iz phane ilovice, v enem primeru so bile po tleh položene kamnite plošče, v treh pa lesene podnice v enem izmed prostorov. Odstopajo enoprostorne stavbe, v katere se je vstopalo z zahoda in so bile z vhodne strani odprte, hodna površina je bila le poravnana naravna osnova, streha pa je bila ver jetno enokapna in podprta z lesenimi stojkami; druge stavbe so imele dvokapne strehe. Razlike med stavbami so opazne tudi v njihovi notranji opremi, h kateri spadajo med drugim glinaste stenske plošče, okrašene z bogatimi geometrijski mi motivi, ki sodijo med znamenitosti tukajšnje naselbine. Nekatere stavbe so bile opremljene z ognjišči, izdelanimi iz drobnih oblic in manjših kamnov ter premazanimi z glino; v eni je bila odkrita tudi kalotasta peč iz lapornatih plošč. K notranji opremi so sodile še jame. Manjše jame okrogle oblike so bile v večprostornih stavbah običajno umeščene v manjši prostor in so verjetno služile za shrambo, tako kot glinasti okrogli silosi. Večje pravokotne jame so bile odkrite večinoma v enoprostornih stavbah, kjer so zavzemale večidel funkcionalnega prostora in so služile delovnim procesom, kar kažejo ohranjeni ostanki v njih. V enem primeru je mogoče sklepati na lončarsko p p Razlike v kakovosti gradnje, notranji opremi in inventarju nakazujejo ločnico med stanovanjskimi stavbami in delavnicami.65 Dve stavbi na vzho dnem delu sta nemara služili kot javni prostor, namenjen skupnim potrebam, glede na izjemno količino živalskih kosti (tudi najbolj mesnatih delov živali) in posodja v primerjavi z drugimi;66 v severovzhodnem predelu pa je mogoče sklepati na kultni prostor po spektru in časovnem razponu najdb, ki izrazito odstopajo od drugih odkritih območij.67 Elemente prostorskega načrtovanja in organizacije naselja iz starejše železne dobe na Mostu na Soči je mogoče videti v ločevanju stavb po namembnosti in v skupni infrastrukturi, kot sta s peskom nasuta pot, odkrita vzdolž niza delavnic na zahodnem delu izkopišča, in jarek za odvodnjavanje, ki je prečil pobočje, ter v enaki usmerjenosti stavb SZ–JV in njihovi obnovi ali novi pozidavi znotraj prvotnih stavbišč. Po omembah v virih naj bi bile podobne stavbe in jame odkrite v naseljih na Bohinjskem, kjer je med obema vojnama izkopaval W. 58 Svoljšak 1988–1989. 59 Mlinar 2020a, 36–39, t. 15A. 60 Svoljšak 2018. 61 Motella De Carlo 2018. 62 Dular, Tecco Hvala 2018, 14–78. 66 Prim. Toškan, Bartosiewicz 2018, tab. 4–7; Grahek 2018a, sl. 2. ; , ; 65 Kriteriji opredelitve posameznih funkcij so ekspli citno navedeni v razpravi Dular, Tecco Hvala 2018, 73–78, pri čemer so bile upoštevane kvalitativne in kvantitativne razlike v zvrsteh najdb, živalskih in rastlinskih ostankih, v kakovosti gradnje, velikosti in zaprtosti stavb, njihovi notranji opremi pa tudi večfaznost gradnje stavb na isti lokaciji, kar je vse podrobneje razčlenjeno v razpravah v monografiji Dular, Tecco Hvala (ur.) 2018. Ob tem kaže opozoriti na razhajanje v interpretaciji namembnosti stavb, ki jo podaja Teržanova v uvodniku in kratkem ori su svetolucijske halštatske kulturne skupine v tej številki Arheološkega vestnika. Njena interpretacija temelji na izbiri zgolj ene zvrsti najdb (skromnih ostankov žlindre in talin), pripadnost teh k stavbam pa je večkrat vprašljiva. 67 Dular, Tecco Hvala 2018, 79–85; Laharnar 2018a, 224–234. 64 Laharnar 2018a; Šmit, Laharnar 2018; Lamut 2018. 63 Grahek 2018a. 64 Laharnar 2018a; Šmit, Laharnar 2018; Lamut 2018. 65 Kriteriji opredelitve posameznih funkcij so ekspli citno navedeni v razpravi Dular, Tecco Hvala 2018, 73–78, pri čemer so bile upoštevane kvalitativne in kvantitativne razlike v zvrsteh najdb, živalskih in rastlinskih ostankih, v kakovosti gradnje, velikosti in zaprtosti stavb, njihovi notranji opremi pa tudi večfaznost gradnje stavb na isti lokaciji, kar je vse podrobneje razčlenjeno v razpravah v monografiji Dular, Tecco Hvala (ur.) 2018. Ob tem kaže opozoriti na razhajanje v interpretaciji namembnosti stavb, ki jo podaja Teržanova v uvodniku in kratkem ori su svetolucijske halštatske kulturne skupine v tej številki Arheološkega vestnika. Njena interpretacija temelji na izbiri zgolj ene zvrsti najdb (skromnih ostankov žlindre in talin), pripadnost teh k stavbam pa je večkrat vprašljiva. 66 Prim. Toškan, Bartosiewicz 2018, tab. 4–7; Grahek 2018a, sl. 2. 67 Dular, Tecco Hvala 2018, 79–85; Laharnar 2018a, 224–234. 63 Grahek 2018a. 62 Dular, Tecco Hvala 2018, 14–78. Značilnosti stavbarstva in notranja ureditev naselij Schmid za graški muzej Joanneum, a rezultati niso celovito objavljeni. Na Ajdovskem gradcu pri Bohinjski Miha MLINAR, Sneža TECCO HVALA 412 Bistrici (št. 36a) je odkril devet v skalno osnovo vkopanih stavb; nekatere so imele kamnite temelje in ognjišča, narejena iz kamnov ali poglobljena v tla; ob stavbah je bilo tudi po več ovalnih jam, ki so služile metalurški dejavnosti. S posoškimi stav bami primerljivo suhozidno gradnjo objektov in sledi metalurških dejavnosti iz halštatske dobe so na tem najdišču pokazala tudi sondiranja Gorenj skega muzeja l. 2003.68 Podobne stavbne ostanke in ognjišče iz zloženih kamnov ter pet topilniških jam je Schmid izkopal na Dunaju pri Jereki (št. 33a).69 Primerljive ostaline so bile ugotovljene prav tako na Štalci nad Železniki (št. 70).70 delu pomola in na najnižji terasi na levem bregu Idrijce blizu sotočja s Sočo, so datirane v mlajšo bronasto dobo in kažejo drugačen tip gradnje s stojkami in stenami iz prepletenega protja z glinastim premazom.75 Iz tega izhaja, da naselje verjetno ni bilo neprekinjeno poseljeno niti z enako intenzivnostjo in da je doseglo največji obseg v mlajšem halštatskem obdobju. g j j V mlajši ali pozni bronasti dobi so bile poleg Mosta na Soči poseljene še vzpetine Sv. Volar nad Robičem na Kobariškem (št. 21), grajski grič Zuc cola na desnem bregu Nadiže severno od Čedada/ Cividale del Friuli,76 na Banjški planoti Grad pri Levpi, Grašišče pri Grgarju in Sv. Katarina ter Gradišče pri Desklah na njenih zahodnih obronkih (št. 50–53), prav tako Golo Brdo ob reki Idriji (št. 43), na Šentviški planoti pa Vrh gradu pri Pečinah (št. 65) poleg še nekaterih drugih domnevnih prazgodovinskih naselij, kjer iz starejše železne dobe sicer ni prepričljivih materialnih dokazov.77 To pomeni, da je bila pokrajina, v kateri so zrasla železnodobna naselja, deloma že kultivirana. Skoraj na vseh obravnavanih naseljih kažejo kronološko oprijemljivejše najdbe obljudenost v mlajšem halštatskem obdobju (št. 1a, 18a, 21, 22a, 26a (t. 2: 5), 33a, 36a, 50, 57, 62, 65, 66, 68, 69, 70, 71, 72a), tudi na Sv. Katarini nad Novo Gorico (št. 53), kot bi lahko domnevali po lončenini, ki spominja po sestavi in nekaterih oblikah, predvsem pa po okrasu vodoravnih gladkih plastičnih reber in glavničenju na naselbinsko keramiko z Mosta na Soči.78 Starejšehalštatskodobna faza, ki je zanesljivo izpričana na nekaterih grobiščih (sl. 9), je v nase ljih precej slabše prepoznavna. 75 Svoljšak 1988–1989; Mlinar 2020a, 36–39, t. 15A. 76 Med arheološkimi izkopavanji srednjeveškega gradu Zuccola l. 1989 so odkrili odlomke posod iz časa med koncem srednje bronaste dobe (Bronzo recente) in začet kom halštatske dobe; hrani jih MAC (Tomadin, Visintini, Colussa 1989). 77 Npr. na gradišču Pavlinčev grad (gl. op. 15). 78 Prim. Svoljšak 1990, t. 2–3 in Grahek 2018a, 249–306, sl. 28. 74 Poselitev v starejšem halštatskem obdobju posre dno dokazujejo grobovi iz stopnje Sv. Lucija I na levem bregu Idrijce (prim. Marchesetti 1886; id. 1893; Teržan, Lo Schiavo, Trampuž-Orel 1984–1985). Značilnosti stavbarstva in notranja ureditev naselij Večina jih je bila poseljenih še v poznejših dobah – v latenski in/ali rimski dobi, pozni antiki ter zgodnjem srednjem veku. Vendar kontinuiteta iz starejše v mlajšo že lezno dobo v naselbinskih kontekstih do zdaj še ni bila potrjena, saj z redkimi izjemami prevladujejo najdbe iz poznega latenskega obdobja, kar kaže na upad v srednjelatenskem obdobju. 71 Na nekaterih za poselitev primernih lokacijah so bile naključno odkrite posamične najdbe, kot npr. na Vrsnem – Strničelo (Strenčl), v Trnovem ob Soči – Trno všček, Debenju – sv. Jakob, na Goljevici – sv. Volbenk, v Tolminskem Lomu – Kal, Dolenji Trebuši – Sovodenj (sl. 11: št. 14, 20, 44, 45, 47, 56). 73 Mlinar, Klasinc, Knavs 2008, 197. 72 Dular 2018. 70 Grahek 2018b, 267–268. 78 Prim. Svoljšak 1990, t. 2–3 in Grahek 2018a, 249–306, sl. 28. 81 Naselju na Ravelniku pripadajoče grobišče bi lahko bilo na bližnji lokaciji s toponimom Gomilce (Na Gomilcah) v Mali vasi na jugovzhodnem območju Bovca. Na širšem območju ob vznožju Rombona se omenjajo še druge grobne najdbe (“človeške kosti in en lonec”, “žara in nekaj brona”, “lončene posode”), in sicer na prostoru Kasarn, v Bislih ob poti proti Plužni ter na Ograjnicah v predelu današnje Kaninske vasi (Svoljšak 2002, 272, 276), kar bi kazalo na obstoj še druge naselbine poleg Ravelnika. Kronološka slika Za časovno opredelitev nastanka in trajanja naselja ter ugotavljanje kontinuitete in vrzeli v poselitvi bi bila potrebna raziskovalna izkopa vanja domala na vseh obravnavanih naseljih in tudi na potencialnih lokacijah, ki so primerne za poselitev,71 predvsem pa celovite objave rezultatov dosedanjih akcij. Na najbolje in najobsežneje raziskanem Mostu na Soči (št. 1a) sodi izkopan vzhodni predel naselja v celoti v mlajše halštatsko obdobje (6.–4. st. pr. n. št.), obljuden je bil tudi pozneje – v skromnem obsegu v poznem latenskem obdobju, konec 2. in 1. st. pr. n. št. (3 hiše),72 v večji meri pa v rimski dobi (15 hiš). V mlajše halštatsko obdobje (6. in 5. st. pr. n. št.) so datirani tudi ostanki štirih stavb, ki so bile delno raziskane l. 2001 na ledini Maregova guna na skrajnem zahodnem robu na selbinskega areala blizu sotočja Idrijce in Soče.73 Lokacija naselja iz predhodnega, tj. starejšega halštatskega obdobja še ni bila ugotovljena.74 Sledi najstarejše poselitve, ki so bile odkrite na zahodnem Jasnejšo kronološko sliko ponujajo grobišča, vendar so bila le v sedmih primerih odkrita v neposredni okolici znanih naselij, in sicer na Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 413 Mostu na Soči, v Kobaridu in Podbeli v zgornjem Posočju (št. 1b, 18d, 22b), na Bohinjskem v Jereki, na Lepencah in v Bohinjski Bistrici (št. 33b, 35b, 36b) ter v Godoviču (št. 72b). rob, na katerem pa sočasna poselitev doslej ni bila zadostno potrjena oziroma grobišču pripadajoče naselje še ni bilo odkrito.80 V Bovcu odkriti grob Na Raduljah (št. 26b) je ležal na terasi ob potoku, toda zagotovo ni pripadal naselju na Ravelniku, saj je od njega oddaljen 3 km zračne črte.81 Po dobno lego v bližini vodotokov imajo še druga znana grobišča ali grobovi v zgornji dolini Soče (št. 16, 25), ob Idrijci (št. 58), na sotočju Bače in Koritnice (št. 40) ter v Bohinju (št. 32–37). Ob vznožju vzpetin na obrečni ravnici sta ležali tudi nekropoli v Špetru/S. Pietro al Natisone in Der nazzaccu (št. 41 in 42). Med izjeme spada Berjač nad Podbelo (št. 22b) v okljuku Nadiže, kjer so bili nedavno odkriti grobovi na terasastem platoju na vzpetini blizu naselja pri sv. Heleni (Na Lupu), od katerega jih ločuje sedlo, a je po drugi strani lega primerljiva z grobišči na sotočjih, saj se nedaleč stran pod naselbino izliva v Nadižo potok Bela. Grobišče Jelenšek pri Godoviču (št. 72b) leži na sedlu blizu naselja. 79 O nekaterih grobiščih/grobovih, ki so bila v preteklosti nestrokovno izkopana, ni na voljo nobenih podatkov, da bi jih lahko zanesljivo opredelili v starejšo železno dobo, npr. grobišče Pod Cerkvijo pri vasi Sedlo (Osmuk 1985, 297) in 36 žarnih grobov, ki jih je izkopal Anton Vuga na Mostu na Soči, na rtu ob sotočju Idrijce in Soče (Marchesetti 1886, 97). Poleg tega omenjajo razni viri še druge potencialne lokacije grobišč, npr. najdbo štirih loncev s pepelom, kostmi in ogljem v Doblarju, odkritje bronastih loncev s pepelom v Modreju (ANSl 1975, 117, 124); na Libušnjah naj bi bila najdena pri pokopališki cerkvi sv. Lovrenca “dva prstena lonca s pepelom”, v Smasteh naj bi bilo “prastaro groblje” (Knific et al. 2021, 20, 22), na Policah pa “lonci s pepelom” (Flego 2005); žare naj bi našli tudi na Krnicah/ Šancah nad Poljubinjem/Žabčami (Mlinar, Turk 2016, 28–29), na Pečinah pa “ilirske” žare (Mlinar et al. 2018, 26). A ti podatki niso arheološko preverjeni, zato njihova datacija ni jasna. Lega in velikost Tako kot naselja so tudi največja grobišča znana v zgornji Soški in v Nadiški dolini (sl. 9), čeprav se o njihovi velikosti lahko samo ugiba, saj so bila izkopana v zelo različnem obsegu. V največji meri so bila izkopana grobišča na Mostu na Soči, v Kobaridu in Tolminu ter v Dernazzaccu pri Gaglianu in Špetru/S. Pietro al Natisone. Grobišče iz starejše železne dobe na Mostu na Soči (št. 1b) se je razprostiralo na terasastem pobočju na drugem bregu Idrijce nasproti naselju, v Kobaridu je ležalo na rečnih terasah Soče ob vznožju naselja na Gradiču (št. 18d), v Tolminu (št. 5) je bilo umeščeno na rečni terasi nad sotočjem Soče in Tolminke pri vznožju osamelca Kozlov GROBIŠČA Čeprav je število znanih grobišč in naselij iz halštatske dobe enako, pa v dveh tretjinah pri merov vzajemne povezave med njimi ni. Številčna razmerja po pokrajinskih enotah so sledeča: na alpskem območju je znanih skoraj dvakrat toliko grobišč kot naselij (prim. sl. 5 in 9), enako velja za Idrijsko hribovje s Šentviško planoto, medtem ko na Banjški planoti in Kanalskem Kolovratu ob šestih zabeleženih naseljih ni bilo odkrito še nobeno grobišče, na Cerkljanskem pa le eno.79 Kronološka slika Precej višje v gorskem svetu sta bili izkopani manjši skupini grobov pri Krnu (št. 13) in v Čadrgu (št. 8) ter posamičen grob v Rutu (št. 39). Njihova lega se navezuje na poti med Posočjem in Bohinjem oziroma na bližino nahajališč železove rude. Naselbinske sledi v njihovi okolici niso bile zaznane, zato je mogoče domnevati, da so pripadali neutrjenim naseljem. Na Šentviški planoti je grobišče Jerovca (št. 61) morda pripadalo domnevnemu naselju na vzpe tini Dobje, na kateri so vidni nasipi in terase, a še ni arheološko preverjeno. Grobovi na Lipcah/ Prevali (št. 63) bi lahko sodili k majhnemu naselju na Berlotovem robu pri Šentviški Gori, medtem ko v bližini grobišča Na Dobcu pri Dabru (št. 64) naselbinskih sledov ni opaziti. 80 Prazgodovinsko naselje na Kozlovem robu omenja Marchesetti (1903, 90). Glej op. 15. Glavne značilnosti grobnega rituala Plan žgani pokop v preprosti jami z raztrese nimi ostanki sežganih kosti in grmade, obeležen Miha MLINAR, Sneža TECCO HVALA 414 Kat. št. / Cat. No. Grobišča / Cemeteries n. v. (m) / asl (m) Št. grobov / No. of graves Sv.Lucija / Faze / Phases Pokrajinske enote / Landscape units 13 Krn – Gradec 923 5+ IIb/c Julijske Alpe 39 Rut – V trojah 741 1 IIc " 8 Čadrg – Laze I 735 4 IIc–III " 33b Jereka – Na sedlu 635 3 Ic–IIa " 37 Žlan – Groblje 571 2 Ic " 32 Brod 512 1 IIa " 36b Boh. Bistrica – Osnovna šola 508 1 IIa " 34 Bitnje – Krašica 505 25 Ic–IIc " 35b Lepence – Na Kremnu 505 4+ IIa–c " 26b Bovec – Na Raduljah 450 1 ? " 25a,b Srpenica – Ograjenca, Lanišča 372 4 IIa–III " 22b Podbela – Berjač 377 x IIc " 40 Koritnica – Lajišče 276 52 IIa–c " 16 Ladra – Na Goricah 237 2 Ia " 18d Kobarid – V Logu 225 1413 Ia–IIc " 5 Tolmin – Pod gradom 212 465 Ia–c " 1b Most na Soči 191 6847 Ia–IIc " 41 Špeter/S. Pietro al Natisone 162 116 Ia–IIa " 42 Gagliano – Dernazzacco 123 287 IIa–c " 61 Gorski vrh – Jerovca 790 22 IIa–c Šentviška planota 64 Daber – Na Dobcu 656 19 IIa–c " 63 Šentviška Gora – Lipce-Prevala 605 9 IIb/c? " 58 Idrija pri Bači – Na Robu 172 16 IIb/c Idrijsko hribovje 72b Godovič – Jelenšek 795 37 IIb/c Cerkljansko Sl. 9: Število odkritih grobov na posameznih grobiščih posoške skupnosti iz starejše železne dobe ter njihov časovni razpon in lega po posameznih pokrajinskih enotah. Fig. 9: Number of graves unearthed in individual cemeteries of the Early Iron Age Posočje community and their chro nological span and location according to landscape units. sintetičen pregled strukture in načina pokopa.84 Kot navaja, so bili kostni ostanki redko močno prežgani, nekateri so bili le komajda. Žganinske lise in prežgana zemlja z obilico oglja, drobci lončenih črepinj in brona pričajo, da je sežiganje umrlih v njihovih opravah potekalo na posebnih mestih v nekropoli ali izven nje. Kosti so po sežigu pobrali in prenesli v grobno jamo skupaj z ostanki lesnega oglja grmade. 84 Marchesetti 1886; id. 1893, 133–141; glej še Bergonzi et al. 1981; Boiardi 1983. 82 Svoljšak 1973; Teržan, Trampuž 1973, 418–419; Boiardi 1983, 164–166; Gabrovec 1987, 138–141. 83 Glej Teržan v tej publikaciji; za Kobarid pa prispe vek Ane Kruh. Sl. 9: Število odkritih grobov na posameznih grobiščih posoške skupnosti iz starejše železne dobe ter njihov časovni razpon in lega po posameznih pokrajinskih enotah. Fig. 9: Number of graves unearthed in individual cemeteries of the Early Iron Age Posočje community and their chro nological span and location according to landscape units. Glavne značilnosti grobnega rituala Grobne jame so bile različno velike in globoke, v njih pa so bili običajno odloženi sežgani ostanki le ene osebe, izjemoma dveh (v 5 grobovih). Jame so pokrivale ena ali več plošč in kamni iz različnih kamnin (apnenca, skrilavcev, konglomerata). Samo tre tjina pokopov je odstopala od ustaljenih uzanc. s kamnito pokrivno ploščo, ter odsotnost orožja med grobnimi pridatki so poglavitne značilnosti, po katerih je bila prepoznana samoniklost in samostojnost svetolucijske skupine v okviru hal štatske kulture.82 Največji potencial za raznovrstne in večplastne analize zagotovo skrivata največji grobišči z naj daljšim časovnim razponom – na Mostu na Soči in v Kobaridu (sl. 9),83 a še ni bil docela izkoriščen, saj gradivo ni v celoti objavljeno. Marchesetti je poleg opisa 2950 grobov in manjšega izbora najdb, ki jih je na Mostu na Soči izkopal do l. 1892, podal Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 415 Mednje sodi pet skeletnih grobov, v katerih so bili odkriti deli človeških okostij, v dveh prime rih pa konjski skeleti. Pokopov v žare je slaba desetina (9,5 %). Samo 13,2 % grobov ni bilo pokritih s kamnito ploščo ali kamni, ponekod je ena plošča pokrivala dva groba, v okoli 30 primerih (1 %) pa je skupino grobov prekrivala groblja kamenja, največja med njimi (10 × 5 m) je prekrivala 41 grobov (25 odraslih oseb in 16 otrok). V 58 grobovih (2 %) so bile jame obdane s kamni ali so imele stene obložene z lomljenci v obliki skrinj, v 21 med njimi so bile žare. V nekropoli se pojavljajo tudi vrste kamnov, ki so bili zloženi z drugačnim namenom (morda za označevanje meje), saj pod njimi ni bilo grobov. Na podlagi ocene biološke starosti pokopanih oseb je Marchesetti poskušal ugotoviti demografsko sliko tedanjega prebivalstva. Med njimi je zabeležil 26 % otrok, v njihovih grobovih večinoma naj ne bi bilo pridatkov. Med grobovi odraslih je bilo takih brez pridatkov manj kot desetina, v 14 % jim je bila pridana samo lončenina. Kosi orožja ali orodja predstavljajo 2,3-odstotni delež najdb, a so bili večkrat najdeni ob grobu ali na njem, nekateri so iz latenske dobe. prav tako kamnit pokopališki zidec.87 Le trije od skupno 87 grobov na Pucarjevem robu in Repel cu so bili žarni, v dveh sta bili pokopani odrasli ženski z dragocenimi pridatki, v tretji pa odrasla oseba, katere spola zaradi preskromnih kostnih ostankov ni mogoče opredeliti. 87 Mlinar 2020a. 85 Gr. Sz 592 in 2304: Teržan, Lo Schiavo, Trampuž -Orel 1984–1985, 120, 363, t. 51; 52A; 242B. 86 Te ocene temeljijo na evidencah iz lastne podatkovne zbirke S. Tecco Hvala, ki je bila ustvarjena na podlagi podat kov v objavi Teržan, Lo Schiavo, Trampuž-Orel 1984–1985. 86 Te ocene temeljijo na evidencah iz lastne podatkovne zbirke S. Tecco Hvala, ki je bila ustvarjena na podlagi podat kov v objavi Teržan, Lo Schiavo, Trampuž-Orel 1984–1985. 85 Gr. Sz 592 in 2304: Teržan, Lo Schiavo, Trampuž -Orel 1984–1985, 120, 363, t. 51; 52A; 242B. Glavne značilnosti grobnega rituala Količina sežganih kosti v grobovih je bila zelo različna, od 1 do 523 g. V povezavi z dvema žganima grobovoma so bile odkrite tudi nežgane konjske kosti in zobje. Orožje in orodje je bilo najdeno v grobovih iz latenske dobe, razen enega noža, pridanega v žarni grob (antropološko opredeljen kot ženski) iz poznega halštatskega obdobja. Na območju Repelca so bili deli orožja in orodja najdeni tudi na območju sežiganja in v premešani plasti, ki je prekrivala grobove na tem delu grobišča. A Sl. 10: Most na Soči (št. 1b). Grobovi 2, 3 in 6 v prerezu, odkriti leta 2001 na Pucarjevem robu (A), ter sežigališče (ustrina) na Repelcu (B), odkrito l. 2002 na najnižji terasi v bližini sotočja Soče in Idrijce (po Mlinar 2020a, sl. 5 in 40). Fig. 10: Most na Soči (No. 1b), Graves 2, 3 and 6 in section, investigated in 2001 at Pucarjev rob (A), and the ustrinum at Repelc (B), found in 2002 on the lowest terrace near the confluence of the Soča and Idrijca (from Mlinar 2020a, Fig. 5 and 40). A B A Szombathyjeva izkopavanja na tem grobišču orisujejo podobno sliko. Od 2478 grobov je manj kot desetina žarnih (7,5 %), pridatkov ni imela dobra četrtina (27 %), lončenina kot edini pri datek nastopa v 16 %. Primerljiv je delež grobov (1,4 %), ki so bili obdani s kamni ali z lomljenci, zloženimi v obliki skrinje, v 11 od njih so bile žare. Na dveh mestih se omenjata ustrini. Dva grobova sta skeletna, v enem od njiju je bil pokopan konj z opremo.85 Orožje ali orodje je zabeleženo v 65 grobovih (2,6 %) ali njihovi neposredni bližini, največkrat gre za nože.86 B B B Primerljive sestavne elemente grobišča in gro bov ter način pokopa, kakršni so bili ugotovljeni s starejšimi raziskavami, so razkrila najnovejša izkopavanja na severnem obrobju tega prostranega grobišča, le da so bili na enem delu (na Repelcu) poleg grobov iz mlajšega halštatskega obdobja tudi poznejši pokopi – iz latenske in rimske dobe ter zgodnjesrednjeveška jama. Poleg žganih pokopov je bil odkrit skeletni grob iz rimske dobe. Odkrito je bilo tudi mesto, kjer so umrle sežigali (sl. 10), Sl. 10: Most na Soči (št. 1b). Grobovi 2, 3 in 6 v prerezu, odkriti leta 2001 na Pucarjevem robu (A), ter sežigališče (ustrina) na Repelcu (B), odkrito l. 2002 na najnižji terasi v bližini sotočja Soče in Idrijce (po Mlinar 2020a, sl. Sl. 10: Most na Soči (št. 1b). Grobovi 2, 3 in 6 v prerezu, odkriti leta 2001 na Pucarjevem robu (A), ter sežigališče (ustrina) na Repelcu (B), odkrito l. 2002 na najnižji terasi v bližini sotočja Soče in Idrijce (po Mlinar 2020a, sl. 5 in 40). Fig. 10: Most na Soči (No. 1b), Graves 2, 3 and 6 in section, investigated in 2001 at Pucarjev rob (A), and the ustrinum at Repelc (B), found in 2002 on the lowest terrace near the confluence of the Soča and Idrijca (from Mlinar 2020a, Fig. 5 and 40). Glavne značilnosti grobnega rituala 5 in 40). Fig. 10: Most na Soči (No. 1b), Graves 2, 3 and 6 in section, investigated in 2001 at Pucarjev rob (A), and the ustrinum at Repelc (B), found in 2002 on the lowest terrace near the confluence of the Soča and Idrijca (from Mlinar 2020a, Fig. 5 and 40). Sl. 10: Most na Soči (št. 1b). Grobovi 2, 3 in 6 v prerezu, odkriti leta 2001 na Pucarjevem robu (A), ter sežigališče (ustrina) na Repelcu (B), odkrito l. 2002 na najnižji terasi v bližini sotočja Soče in Idrijce (po Mlinar 2020a, sl. 5 in 40). Fig. 10: Most na Soči (No. 1b), Graves 2, 3 and 6 in section, investigated in 2001 at Pucarjev rob (A), and the ustrinum at Repelc (B), found in 2002 on the lowest terrace near the confluence of the Soča and Idrijca (from Mlinar 2020a, Fig. 5 and 40). Miha MLINAR, Sneža TECCO HVALA 416 pritrjuje groba primerjava s pribl. 5 km oddaljenim tolminskim grobiščem iz starejšega halštatskega obdobja (št. 5), kjer je bil odkrit le en žarni pokop in noben v kamniti skrinji. Poleg tega je čutiti raz like v grobnih pridatkih: v tolminski nekropoli ni imelo pridatkov pribl. 10 % grobov, medtem ko je takih na Mostu na Soči dvakrat več (pribl. 23 %), iz česar bi bilo mogoče sklepati na porast grobov brez pridatkov v mlajšem halštatskem obdobju. Tak trend nakazujeta tudi grobišči Koritnica ob Bači in Jerovca na Šentviški planoti (št. 40, 61), ki sta mlajšega nastanka, delež grobov brez pridatkov je tam večji (pribl. 18 do 22 %) kot na tolminskim grobišču. Na drugi strani pa na Mostu na Soči sodi v ta čas večina najbogatejših grobov, v njih so med grobnimi pridatki tudi importi, kar bi kazalo na večje družbeno razlikovanje.90 Uvoženi predmeti in surovine oddaljenega izvora so bili odkriti tudi v tamkajšnjem naselju, ki je takrat doseglo največje razsežnosti, saj na raziskanem območju ni bilo najdb iz predhodnega obdobja.91 V tolminski nekropoli (št. 5) način pokopa ni bistveno drugačen, le da tam med 465 grobovi ni nobenega v obliki kamnite skrinje in samo en je bil žarni, v njem je bil po antropološki opredelitvi spola in pridani igli pokopan moški. Večji je delež grobov, v katerih je bila pridana samo keramika (30 %), manjši pa brez pridatkov (10 %). Glavne značilnosti grobnega rituala Primer ljiva je zastopanost orožja in orodja med pridatki (2,2.%), največ je nožev, ki se pojavljajo tako skupaj z značilnimi moškimi kot ženskimi pridatki, od bronastih suličnih osti je bila ena najdena v grobu, druga pa v zasipu med grobovi, tako kot britev. Enake značilnosti in podobno strukturo poko pov imajo tudi manjša grobišča v Špetru/S. Pietro al Natisone in Dernazzaccu v Nadiški dolini (št. 41, 42), na Koritnici ob Bači (št. 40), Jerovci na Šentviški planoti (št. 61) in Bitnjah v Bohinju (št. 34). V njih se posamično pojavljajo žarni grobovi, na Koritnici in Bitnjah pa izjemoma tudi skeletni. V grobovih v Čadrgu na Tolminskem (št. 8) in Krnu na Kobariškem (št. 13) ter na Jelenšku pri Godoviču (št. 72b) je orožje zastopano v znatno večji meri, delež grobov z orožjem je večji tudi na Koritnici (11 %). Na prehodu v mlajše halštatsko obdobje je zaznan pojav novih grobov pri Srpenici na Bovškem (št. 25a), na Koritnici ob Bači in Šentviški planoti (št. 40, 61, 64) (sl. 9). V ta čas segajo začetki grobišč v Bohinju (št. 32–37);92 v poslednjih fazah starejše železne dobe (Sv. Lucija IIb–IIc) pa zasledimo grobove tudi na odročnejših in višje ležečih kra jih (št. 8, 13, 22b, 39 (t. 2: 11), 58, 63, 72b), kar kaže na ekspanzijo poselitve v stranske doline in hribovite predele. 88 V Špetru so iz mlajšega halštatskega obdobja znane certoška fibula ter železni tulasta in uhata sekira (Pettarin 2006a, kat. št. 174, 393, 588; Pettarin 2006b). 94 V neposredni bližini Jerovce je bil pri izvirčku Pri B'ču sicer najden delček železne žičnate fibule srednjelatenske sheme, kar pa še ne dokazuje, da so se tudi pokopi nada ljevali v latensko dobo (Laharnar, Mlinar 2013, sl. 11: 3). 90 Teržan, Trampuž 1973, 429; Bergonzi et al. 1981, 214–215, 226–228, 246–248; Boiardi 1983, 165–166; ead. 1984; glej še Teržan v tej publikaciji. 91 Dular, Tecco Hvala 2018, 109–130; Dular 2018. 92 Na konec starejšega halštatskega obdobja morda sodita grob 2 z Jereke in grob 5 z Bitenj (Gabrovec 1974, 300; t. 4: 5,6; 11: 1–7). 93 Guštin 1991, sl. 1; Božič 1999a, 213, sl. 1; glej še Mlinar, Gerbec 2011, 23; Mlinar 2020a, 93–97; id. 2020b. 94 V neposredni bližini Jerovce je bil pri izvirčku Pri B'ču sicer najden delček železne žičnate fibule srednjelatenske sheme, kar pa še ne dokazuje, da so se tudi pokopi nada ljevali v latensko dobo (Laharnar, Mlinar 2013, sl. 11: 3). 95 Guštin 1991, 91–93, sl. 7 in 21. 93 Guštin 1991, sl. 1; Božič 1999a, 213, sl. 1; glej še Mlinar, Gerbec 2011, 23; Mlinar 2020a, 93–97; id. 2020b. 92 Na konec starejšega halštatskega obdobja morda sodita grob 2 z Jereke in grob 5 z Bitenj (Gabrovec 1974, 300; t. 4: 5,6; 11: 1–7). , , , ; ) 89 Bergonzi et al. 1981, 187–188, tab. 23; 24; 26. 95 Guštin 1991, 91–93, sl. 7 in 21. 90 Teržan, Trampuž 1973, 429; Bergonzi et al. 1981, 214–215, 226–228, 246–248; Boiardi 1983, 165–166; ead. 1984; glej še Teržan v tej publikaciji. 90 Teržan, Trampuž 1973, 429; Bergonzi et al. 1981, 214–215, 226–228, 246–248; Boiardi 1983, 165–166; ead. 1984; glej še Teržan v tej publikaciji. g j j p j 91 Dular, Tecco Hvala 2018, 109–130; Dular 2018. 88 V Špetru so iz mlajšega halštatskega obdobja znane certoška fibula ter železni tulasta in uhata sekira (Pettarin 2006a, kat. št. 174, 393, 588; Pettarin 2006b). 89 Bergonzi et al. 1981, 187–188, tab. 23; 24; 26. Kronološka slika Najstarejši grobovi iz železne dobe (Sv. Lucija I) so zgoščeni v zgornji Soški in Nadiški dolini – v Tolminu, na Mostu na Soči, v Kobaridu, Ladrah (t. 1: 1–6) in Špetru/S. Pietro al Natisone (sl. 9), kar bi pomenilo, da je bilo na tem območju prvotno jedro poselitve. Iz predhodnega obdobja grobov na teritoriju te skupnosti ne poznamo. Domala na vseh grobiščih v zgornji Soški in Nadiški dolini ter na Cerkljanskem so zabeleženi pokopi ali najdbe iz latenske dobe,93 razen v Tolminu (št. 5), Jerovci (št. 61)94 in na Jelenšku (št. 72b). Na grobišču na Idriji pri Bači (št. 58) so grobovi iz latenske dobe v prevladi in ležijo v glavnem na jugovzhodnem delu, grobovi iz halštatske dobe pa na severozahodnem delu.95 Na Mostu na Soči in v Kobaridu (št. 1b, 18d) se je pokopavanje neprekinjeno nadaljevalo v mlajše halštatsko obdobje (Sv. Lucija II), medtem ko je v Tolminu usahnilo (št. 5). V Špetru/S. Pietro al Natisone (št. 41) je iz mlajšega obdobja znanih le nekaj najdb,88 na grobišču v Dernazzaccu (št. 42) pa so takrat šele začeli pokopavati in nadaljevali še v latensko dobo. V zgornji Soški dolini je v tem času doživljal vzpon in vrhunec Most na Soči, kjer je iz 6. in 5. st. pr. n. št. (Sv. Lucija IIa in IIb) po ocenah največ grobov; opazne so tudi strukturne spremembe, kajti večina pokopov v žare in kamnite skrinje sodi v mlajše halštatsko obdobje.89 Temu 417 Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja Če primerjamo strukturo pokopov v tolminski nekropoli kot predstavnici zgodnje faze razvoja posoške skupnosti (Sv. Lucija I) in grobove iz pozne faze (Sv. Lucija IIc), lahko opazimo največje razlike v zastopanosti orožja. V tolminski nekro poli sta bili od orožja najdeni le dve (bronasti) sulični osti,96 medtem ko je denimo na Jelenšku pri Godoviču grobov z orožjem že skoraj polovica,97 med halštatskodobnimi grobovi na Idriji pri Bači je takih 37,5 %,98 v Čadrgu na Tolminskem so orožje imeli trije od štirih tam odkritih grobov, eden od njih je latenski.99 Porast orožja v grobovih bi tako lahko imeli za znak nemirnih časov, ki so zaznamovali konec halštatske dobe in prehod v latensko dobo.100 Največ posamičnih najdb je zabeleženih v ne posredni okolici in v zaledju Kobarida in Tolmina (sl. 11). Pojavljajo se celo nad 1000 m n. v., na visokogorskih pašnikih in ob poteh na planine (št. 96 Svoljšak, Pogačnik 2001–2002. Bronasta sulična ost je bila najdena tudi na grobišču Ladra (št. 16; t. 1: 6). 97 Glej prispevek Bratina, Laharnar, Svoljšak v tej publikaciji. 98 Guštin 1991, t. 23: 6,7,11,15; 24: 15,16; 25: 1,7,8; 26: 1,2; 27: 11,12. 99 Mlinar 2020b. 100 Teržan, Trampuž 1973, 437. Kronološka slika 6, 7, 10, 11, 12, 28; t. 1: 7,9; 2: 2,4,10; 3: 6), gre pa za dele noše (fibule, igla, prstan) in orožje/ orodje (sekira). Glede na naravno okolje bi jih morda lahko povezovali s transhumanco ali z nabiranjem rude pa tudi s potmi na bohinjsko stran. Sezonsko postojanko v bohinjskih gorah na 1680 m n. v. na Dolgi Planji na Voglu (št. 38) smo že omenili; tam je bila poleg ostankov stavbe iz zgodnjega srednjega veka odkrita žganinska plast, v njej pa železova ruda, živalske kosti in drobci keramike iz starejše železne dobe, v ruši v neposredni bližini je bila najdena še certoška fibula XI. vrste iz 5./4. st. pr. n. št.101 Podobno je najdišče V plazeh v Trenti (št. 29) na 1542 m n. v., kjer so bili odkriti suhozidni temelji stavbe in z žganino zapolnjena kotanja, v kateri so bili drobci prazgodovinske keramike in košček tra kaste fibule iz 6. st. pr. n. št. (t. 2: 6).102 Nekje na Kobilniku na Tolminskem (št. 7) je bil pobran surovec iz bakrove zlitine, vendar ne skupaj s certoško fibulo s tega hriba (t. 2: 2),103 podobno kot ni bil na območju Fiščeva pri Ajbi ob Soči (št. 46), kjer naj bi ležal pribl. 50–100 m stran od certoške fibule (t. 2: 7).104 Iz starejše železne dobe bi lahko bili tudi bronasti ingoti in surovci, najdeni okoli cerkvice sv. Jakoba nad Debenjem (št. 44) v Kambreškem pogorju.105 101 Ogrin 2020, 63, sl. 3; za tipokronološko opredelitev glej Teržan 1976. 106 Železna sekira je bila najdena pri separaciji peska pri Volčah, pesek pa je izviral iz rečnega proda Soče med Idrskim in Tolminom (št. 4; t. 3: 8). 101 Ogrin 2020, 63, sl. 3; za tipokronološko opredelitev glej Teržan 1976. 102 Za podatke in risbo fibule se zahvaljujeva Jani Horvat (Inštitut za arheologijo ZRC SAZU). Za tipokro nološko opredelitev glej npr. Tecco Hvala 2012, 243–244, op. 1029 s sklici na primerke z grobišča na Mostu na Soči. 103 Mlinar, Turk 2016, 23–24, kat. št. 22 in 24. 104 Gerbec 2021, 137–138. 105 Nanut 2018, sl. 4; t. 2–4. 106 Železna sekira je bila najdena pri separaciji peska pri Volčah, pesek pa je izviral iz rečnega proda Soče med Idrskim in Tolminom (št. 4; t. 3: 8). 102 Za podatke in risbo fibule se zahvaljujeva Jani Horvat (Inštitut za arheologijo ZRC SAZU). Za tipokro nološko opredelitev glej npr. Tecco Hvala 2012, 243–244, 105 Nanut 2018, sl. 4; t. 2–4. Posamične najdbe Poselitveno sliko dopolnjujejo naključno odkri te najdbe iz zadnjih treh desetletij, ki prispevajo k znatno večji številki arheoloških najdišč na obravnavanem območju (sl. 3; 4; 11). A lokacije niso vselej zanesljive, ker so bile te najdbe veči noma pridobljene z detektorjem kovin, pri čemer okoliščin odkritja največkrat niso dokumentirali, zato v primerih, ko niso arheološko preverjene, njihov kontekst ni najbolj jasen – ali so bili po leg kovinskih predmetov še kaki drugi kulturni ostanki (keramika, oglje, kosti, surovci, žlindra). S tem pa je njihova izpovednost okrnjena, saj je težko opredeliti njihov značaj in pomen (ali gre za grobne, naselbinske ali votivne najdbe ali pa dejansko za izgubljene predmete). Pri preverjanju nekaterih lokacij v sodelovanju z arheologi so bili ponekod ugotovljeni grobovi (sl. 9: št. 8, 13, 16 (t. 1: 1–6), 22b, 25a,b, 39 (t. 2: 11), 72b). Za nekatere bi lahko domnevali, da so naselbinske najdbe, ker so bile odkrite na vzpetinah z okopi ali s terasiranimi pobočji (sl. 5: št. 21, 22a, 26a (t. 2: 5), 66, 68, 69, 71). Drugačen značaj bi lahko pripisali najdbam iz prodišč reke Soče pri Kobaridu in z neznane natančne lokacije med Idrskim in Tolminom (št. 4, 18g,h,i). Gre za železne sulične osti in sekiro (t. 3: 1–4,8), 106 medtem ko je bila na vzhodnem obrežju Bohinjskega jezera pri Stari Fužini (št. 30) najdena certoška fibula (t. 2: 3). Ker je med najdišči posoške skupnosti in vodnimi viri opazna precejšnja soodvisnost, bi nemara lahko domnevali, da so vodam pripisovali čudodelno Miha MLINAR, Sneža TECCO HVALA 418 Kat. št. / Cat. No. Posamične najdbe / Stray finds n. v. (m) / asl (m) Vrste / Types Sv. Lucija / Faze / Phases Tabla: slika / Plate: Figure Julijske Alpe 11 Tolminske Ravne – Zeleni vrh 1775 igla /pin Ib T. / Pl. 1: 7 28 Trenta – Pod Razorci 1687 fibula IIb T. / Pl. 2: 4 10 Tolminske ravne – Na kalu 1475 sekira / axe IIc T. / Pl. 3: 5 6 Zatolmin – Zavrh 965 prstan / finger ring IIa/b T. / Pl. 1: 9 12 Selišče – Mrzli vrh 867 fibula IIb/c T. / Pl. 2: 10 14 Vrsno – Strničelo 795 fibula IIc T. / Pl. 2: 8 7 Zadlaz-Čadrg – Kobilnik 651 fibula IIb T. / Pl. 2: 2 2 Bača pri Modreju – Senica 600 sekira / axe IIc T. / Pl. p p j Fig. 11: Type and findspot of the stray finds recovered in the area of the Early Iron Age Posočje community according to chronological phases and landscape units. Sl. 11: Vrsta in lega posamičnih najdb na območju posoške skupnosti po kronoloških fazah starejše železne dobe in po posameznih pokrajinskih enotah. Posamične najdbe Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 419 moč in ob njih izvajali očiščevalno ali daritveno obredje.107 Iz te predpostavke bi izhajal nasledek, da so najdbe iz neposredne bližine rečnih strug in jezera morda povezane z votivnimi daritvami in čaščenjem. Seveda pa so bili vodni viri prven stveno povezani z oskrbo v vsakdanjem življenju. Poleg tega predstavljajo rečne doline, kjer niso stisnjene med strma pobočja, tudi prometne kori dorje, skozi katere je najlažji prehod preko gorate pokrajine. Zato bi lahko v naključnih najdbah videli predvsem indikatorje poti in izkoriščanja naravnih virov oziroma običajnih človekovih opravil v zaledju naselij. v Nadiški dolini.111 Antropomorfni obeski za po soško skupnost niso ravno običajni, pogostejši so v obliki košarice, taki so znani iz grobov mlajšega halštatskega obdobja in tudi iz naselbine na Mostu na Soči.112 Tu predstavljeni posamični primerki so bili najdeni na Kobariškem (št. 15, 18g, 23; t. 1: 12,13,18). Bronasta zapestnica s presegajočima koncema, okrašena s snopi vrezov, iz Loga pod Mangartom (št. 27; t. 1: 11) predstavlja redkost na tem območju,113 primerjamo jo lahko z najdbami na Notranjskem in z zapestnicami, ki so značilne za dolenjsko nošo s konca 7. do 5. st. pr. n. št.114 V posoški noši ni običajna niti bronasta samostrelna fibula z naprej gledajočo konjsko glavico (t. 2: 1), najdena na Jajnkovcu ob levem bregu Soče pri Kobaridu (št. 19); enak primerek je bil odkrit na kobariškem grobišču, podoben z ovnovo glavico na zaključku noge pa na Mostu na Soči.115 Tovrstne fibule so znane iz dolenjskega kulturnega prostora v 5. st. pr. n. št., kjer jih zasledimo v moških opravah.116 V povezavi s posoškimi primerki bi kazalo omeniti podobni fibuli s sosednjih območij – v Podgori ob Poljanski Sori in na Gurini na avstrijskem Koro škem.117 Ostale naključno najdene fibule (sl. 11) pripadajo certoškim fibulam vrste VI (št. 12; t. 2: 10), X (št. 46, 14, 55, 20; t. 2: 7,8,12,13), XI (št. 60; t. 2: 9) in XIII (št. 7, 28, 30; t. 2: 2–4), ki so jih – tako kot orožje – nosili moški v zadnjih fazah starejše železne dobe, X. vrsto lahko še dlje.118 V zadnjo fazo se uvršča še živalska fibula zgodnjelatenske sheme (št. Posamične najdbe 3: 7 9 Zatolmin – Javorca 589 jagoda / bead sekira / axe IIb/c T. / Pl. 1: 16; 3: 11 17 Koseč 575 jagoda / bead IIb/c T. / Pl. 1: 15 23 Homec – Na Mlakah 558 obesek / pendant IIb T. / Pl. 1: 12 24 Sedlo – Pod cerkvijo 547 obesek / pendant prstan / finger ring IIb T. / Pl. 1: 10,14 30 Stara Fužina – Veliki Vegl 527 fibula IIb T. / Pl. 2: 3 27 Log pod Mangartom 393 zapestnica / bracelet IIa T. / Pl. 1: 11 20 Trnovo ob Soči – Trnovšček 288 fibula IIc T. / Pl. 2: 13 18h Kobarid – V mevcah 280 sulična ost / spearhead Ha ali LT T. / Pl. 3: 4 19 Magozd – Jajnkovec 269 fibula IIb T. / Pl. 2: 1 18g Kobarid – Za gradom 229 obesek / pendant sulična ost / spearhead IIb T. / Pl. 1: 13; 3: 3 15 Smast 212 obesek / pendant IIb T. / Pl. 1: 18 18i Kobarid – Mlinsko, reka Soča 194 2 sulični osti / spearheads Ha ali LT T. / Pl. 3: 1,2 4 Idrsko-Tolmin, reka Soča 162 sekira / axe IIc T. / Pl. 3: 8 Banjšice 54 Trnovo – Kamni breg 916 sekira / axe Ha ali LT T. / Pl. 3: 9 47 Tolminski Lom – Kal 680 igla / pin fibula Ib T. / Pl. 1: 8 Kambreško pogorje 45 Goljevica – sv. Volbenk 373 sekira / axe IIc T. / Pl. 3: 6 46 Ajba – Fiščevo 150 fibula IIc T. / Pl. 2: 7 Idrijsko & Cerkljansko hribovje 55 Gor. Trebuša – Obenčel 880 fibula IIc T. / Pl. 2: 12 59 Ponikve – Kračice 640 jagoda / bead IIb/c T. / Pl. 1: 17 67 Dol. Ravne – Gastabil 616 fibula IIc 60 Kneža – Grebljica 271 fibula IIb/c T. / Pl. 2: 9 56 Dol. Trebuša – Sovodenj 186 sekira / axe Ha ali LT T. / Pl. 3: 10 Sl. 11: Vrsta in lega posamičnih najdb na območju posoške skupnosti po kronoloških fazah starejše železne dobe in po posameznih pokrajinskih enotah. Fig. 11: Type and findspot of the stray finds recovered in the area of the Early Iron Age Posočje community according to chronological phases and landscape units. p 115 Marchesetti 1903, t. 18: 5; glej še Mlinar 2020a, 70, t. 42: 1. 113 Podobni fragmenti so znani z Repelca na Mostu na Soči (Mlinar 2020a, 75, t. 29: 4,5) in z grobišč ob Nadiži (Pettarin 2006a, t. 20: 298,317; 21: 328,330; Pettarin 2006b). 117 Gabrovec 1966a, 31, 34, karta 3 in seznam najdišč z referenčno literaturo. O povezavah s koroško skupino oz. skupino Frög/Breg glej prispevek Gleirscher v tej publikaciji. 111 Za Krn glej prispevek Laharnar, Mlinar (t. 4: 1) v tej publikaciji, za Dernazzacco pa Pettarin 2006a, t. 26: 451; Pettarin 2006b. 114 Za Notranjsko glej Guštin 1979 (t. 22: 8–22; 66: 24–26,28,29), za Dolenjsko npr. Tecco Hvala 2012, sl. 109: 6 (različica IIb), 294, 298, op. 1270–1273. 112 Laharnar 2018a, 213; Mlinar 2020a, 29; glej še Pavlin 2014. 108 Teržan, Trampuž 1973, 421, pril. 1; Teržan 2002, 89 (igle vrste III. in V. po Pogačnikovi). Prim. še Most na Soči: Teržan, Lo Schiavo, Trampuž-Orel 1984–1985, t. 14G: 1; 37G; 43C: 1; 75E: 1; 95B: 1; 101B: 1; 102B: 1; 112E: 3; 119A: 1; 126R: 2. 107 Podobno kot drugod v jugovzhodnih Alpah so bili tudi v reki Soči oziroma njenih prodiščih odkriti kosi orožja iz pozne bronaste dobe, ki jih lahko prav tako razlagamo kot votivne najdbe (prim. Gerbec, Mlinar 2011). g g g j j j 118 Teržan 1976; prim. npr. še Teržan, Lo Schiavo, Trampuž-Orel 1984–1985, t. 7B; 27F; 29D; 39B; 47D; 50B; 124A; Mlinar 2020b, sl. 5 in 8; Guštin 1991, t. 23: gr. 30; 25: gr. 37; 26: gr. 40. 116 Tecco Hvala 2012, 262–263, sl. 99: 5,6. 109 Božič 2011, 248; Laharnar 2018a, 213–214; Mlinar 2020a, 84; glej še Laharnar, Mlinar (t. 6 in 7) v tej publikaciji. 110 Nanut 2021, 78–81. Posamične najdbe 67), ki je verjetno iz delek lokalne delavnice, saj so podobni primerki z redkimi izjemami omejeni na prostor posoške Med posamičnimi najdbami so zastopani deli noše (fibule, igli, zapestnici, prstani, obeski, ste klene jagode) ter kosi orožja (sekire in sulične osti). Najstarejši med njimi sta bronasti večglavi igli (t. 1: 7,8), kakršne so v tolminskem grobišču značilna prvina moških oprav v stopnji Sv. Lucija Ib,108 ena je bila najdena pod Zelenim vrhom visoko nad Tolminskimi Ravnami (št. 11), druga v Tolminskem Lomu na Banjšicah (št. 47) na ob močju, kjer naj bi bila že prej najdena nekakšna bronasta fibula, vendar je izgubljena. Vse druge najdbe so iz mlajšega halštatskega obdobja (Sv. Lucija II), nekatere bi lahko bile tudi iz latenske dobe. Ženskemu nakitu gre pri pisati rumene steklene jagode z modro-belimi plastovitimi očesci (sl. 11: št. 9, 17; t. 1: 15–17), ki se pojavljajo v stopnjah Sv. Lucija IIb in IIc, sicer pa so bile v 5. in 4. st. pr. n. št. široko raz prostranjene.109 Dva od prstanov (št. 6, 24; t. 1: 9,10) sta okrašena z vtisnjenimi krožci s piko in snopi prečnih vrezov, tovrsten okras je pogost na nakitu posoške skupnosti, izdelanem verjetno v lokalnih delavnicah v stopnjah Sv. Lucija IIb in IIc.110 Enako okrašen je obesek iz Sedla (št. 24; t. 1: 14), ki morda v stilizirani obliki predstavlja žensko figuro, po obliki in okrasu pa je še najbolj podoben obeskoma iz Krna na Kobariškem in Dernazzacca Miha MLINAR, Sneža TECCO HVALA 420 skupnosti, kjer so jih izdelovali najbrž po vzoru sočasnih južnoalpskih zgodnjelatenskih fibul.119 bil zakopan nekje na pobočju Gradiča pri Kobaridu (št. 18c), le da je za poldrugo stoletje mlajši od njiju (4. st. pr. n. št.).128 Na podlagi poročil (najdbe niso v celoti objavljene) je bilo v veliki bronasti posodi situlaste oblike deponirano železno orožje (ena sekira z dvostranskimi plavutmi, ena uhata in 4 tulaste sekire ter 8 suličnih osti, poleg tega še nekakšno koničasto orodje ali sulično kopito, brusni kamen in železna zapestnica). Podobno lego na pobočju hriba kot v Kobaridu je imel depo iz Rubij/Rubbia, ki ju družijo tudi tulaste sekire, medtem ko ga z depojem Porpetto povezuje način deponiranja v posodo ali zaboj ter sekira z obojestranskimi plavutmi, teh je bilo v depoju iz Porpetta kar 23.129 Po teh indicih je videti, da so se z dejavnostmi v hribovitem zaledju ukvarjali predvsem moški. 121 Teržan, Trampuž 1973, 430–434, sl. 4: karta 3, pril. 1; glej še Kos 1973, t. 5: 1; 11: 1; Teržan, Lo Schiavo, Trampuž-Orel 1984–1985, t. 7B: 2; Laharnar, Mlinar 2019; Mlinar 2020b, sl. 5. 126 Glej prispevek Vitri, Corazza (sl. 2; 18; 20) v tej publikaciji. Depoji Podoben depo orožja, kot sta znana iz Rubij/ Rubbia ob spodnjem toku Soče blizu izteka reke Vipave ter iz Porpetta ob Tilmentu/Tagliamento, je 123 Sekira iz Javorce, ki ima obojestranske plavuti in sedlast prehod v rezilo, je primerljiva z najdbami na Tri dentinskem, kjer jih uvrščajo med starejšeželeznodobne oblike (prim. Marzatico 1997, kat. št. 1986). 122 Npr. Guštin 1991, t. 6: 2; 10: 11; 14: 4 itd.; prim. še Turk, Svetličič 2018 ter prispevek Laharnar, Mlinar v tej publikaciji (t. 13: 5; 14). Posamične najdbe Med posamičnimi najdbami so zastopane še uhate sekire (št. 2, 4, 10, 45; t. 3: 5–8);120 primerjave imajo v grobnih kontekstih na Mostu na Soči, Srpenici in v Čadrgu, kjer se pojavljajo skupaj s certoško fibulo X. vrste, na Koritnici pa s prej omenjeno živalsko fibulo zgodnjelatenske sheme.121 V rabi so bile še v poznem latenskem obdobju, vendar so nekoliko drugačne – s kladivastim čelom.122 Težje je časovno opredeliti posamične najdbe sekir z dvostranskimi plavutmi (št. 9, 54, 56; t. 3: 9–11),123 ki jih denimo na Idriji pri Bači (št. 58) zasledimo v grobovih iz pozne halštatske in latenske dobe.124 V njih na stopajo tako skupaj z orožjem kot z orodjem, zato tudi njihova funkcija ni povsem enoznačna, ali so služile kot orožje in/ali kot orodje. Mnogo prej jih zasledimo v bojevniških grobovih na Dolenjskem (že na samem začetku železne dobe),125 v osrednje furlanski skupnosti se pojavljajo v grobovih skupaj s kačastimi fibulami s konca 7. in začetka 6. st. pr. n. št.126 Na Notranjskem (Tržišče) in v Furlaniji (Porpetto) so znane tudi v depojih iz sredine in druge polovice 6. st. pr. n. št.127 Po sestavi je povsem drugačna depojska najdba z Gastabila pri Dolenjih Ravneh na Cerkljanskem, odkrita z detektorjem kovin (št. 67). V manjši kotanji ob naravnem skalnem grebenu so skupaj ležali razlomljeni koščki paličastih in ploščatih bronastih ingotov ter bronastih uhatih sekir z veliko vsebnostjo svinca. Slednje so se na prelomu iz 2. v 1. tisočletje z izvornega območja na Apeninskem polotoku razširile v prostor zahodne in osrednje Slovenije, kjer se pojavljajo vse do 6. st. pr. n. št.130 Podobne najdbe so bile naključno odkrite na Kanalskem Kolovratu pri cerkvici sv. Jakoba nad Debenjem, ležale so na kotanjastem delu zahodne terase tik pod vrhom hriba (št. 44).131 125 Tecco Hvala 2012, 111–114, sl. 46: 1. 130 Nanut 2016; ead. 2018. Podobni odlomki bronastih uhatih sekir, pogač, paličastega ingota in ploščatih ingotov so bili najdeni tudi v naselbini pri cerkvi sv. Helene (Na Lupu) nad Podbelo, vendar ni podatka o tem, ali so ležali skupaj, da bi jih lahko opredelili kot depo (prim. Mlinar, Gerbec, Laharnar 2014, 30–31). 119 Nanut 2021, 73–83, prim. sl. 1 in 6 (karti) ter sl. 7. 120 Uhata sekira s planine Kal pod Tolminskim Mi govcem je morebiti služila kot orodje, saj je bila najdena na območju, bogatem z železovo rudo (prim. Mlinar, Turk 2016, 25). 127 Guštin, Božič 2021, 482–485, sl. 2: D. 128 Ib., 491–492. 129 Ib., 483–484, 490–492, sl. 2. 130 Nanut 2016; ead. 2018. Podobni odlomki bronastih uhatih sekir, pogač, paličastega ingota in ploščatih ingotov so bili najdeni tudi v naselbini pri cerkvi sv. Helene (Na Lupu) nad Podbelo, vendar ni podatka o tem, ali so ležali skupaj, da bi jih lahko opredelili kot depo (prim. Mlinar, Gerbec, Laharnar 2014, 30–31). 131 Nanut 2018. 124 Npr. Guštin 1991, t. 2: 3; 15: 5; 26: 2. 131 Nanut 2018. 119 Nanut 2021, 73–83, prim. sl. 1 in 6 (karti) ter sl. 7. 120 Uhata sekira s planine Kal pod Tolminskim Mi govcem je morebiti služila kot orodje, saj je bila najdena na območju, bogatem z železovo rudo (prim. Mlinar, Turk 2016, 25). 121 Teržan, Trampuž 1973, 430–434, sl. 4: karta 3, pril. 1; glej še Kos 1973, t. 5: 1; 11: 1; Teržan, Lo Schiavo, Trampuž-Orel 1984–1985, t. 7B: 2; Laharnar, Mlinar 2019; Mlinar 2020b, sl. 5. 122 Npr. Guštin 1991, t. 6: 2; 10: 11; 14: 4 itd.; prim. še Turk, Svetličič 2018 ter prispevek Laharnar, Mlinar v tej publikaciji (t. 13: 5; 14). 123 Sekira iz Javorce, ki ima obojestranske plavuti in sedlast prehod v rezilo, je primerljiva z najdbami na Tri dentinskem, kjer jih uvrščajo med starejšeželeznodobne oblike (prim. Marzatico 1997, kat. št. 1986). 124 Npr. Guštin 1991, t. 2: 3; 15: 5; 26: 2. 125 Tecco Hvala 2012, 111–114, sl. 46: 1. 126 Glej prispevek Vitri, Corazza (sl. 2; 18; 20) v tej publikaciji. 127 Guštin, Božič 2021, 482–485, sl. 2: D. 128 Ib., 491–492. 129 Ib., 483–484, 490–492, sl. 2. Kultno-daritvena mesta Po primarnih virih so pri cestnih delih na le vem bregu Soče nekje pri zaselku Loga severno od Bodreža konec 19. stoletja naleteli na globoko skalno razpoko, zapolnjeno z žganino, v njej pa na lončene črepinje, kose bronastih posod in nakita ter železno orožje in orodje (št. 49). Najdbe kažejo velik časovni razpon, od mlajšega halštatskega obdobja (Sv. Lucija II), kamor sodijo nekateri tipi fibul, obeski, prstani, zapestnica, del ročaja in ataše situle, morda tudi železna sekira z obojestranskimi plavutmi, do poznolatenskega Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 421 košček bronastega srpa (11/10. st.),135 med najz govornejšimi pa sta najdbi bronastega sceptra in daritvena pravokotna ploščica iz srebra z napisom v venetski pisavi. obdobja, sodeč po značilnem železnem orodju.132 Glede na omembo v poročilu, da v okolici tega mesta ni bilo sledov žganine niti najdb, najbrž ne gre za grobišče. Naravne okoliščine bi govorile bolj v prid domnevi o depoju, kar omenja že primarni vir. Ker pa žganina v depojih ni običajna, bi lahko šlo za kultno mesto. Podobna bronasta daritvena ploščica je bila najdena pri raziskavah utrjene poznoantične nasel bine na Tonovcovem gradu pri Kobaridu (št. 18f), kjer so bili poleg treh cerkva, vodnega zbiralnika in bivalnih stavb iz pozne antike odkriti skromni sledovi iz prazgodovine. V ruševinah ene od po znoantičnih stavb so bili poleg daritvene ploščice najdeni tudi razlomljeni deli mladohalštatske in latenske noše in obeski ter košček negovske če lade, koničnik nožnice in branik meča.136 Še več daritvenih ploščic in podobnih najdb širokega časovnega razpona je bilo odkritih na zahodnem robu naselbine na Gradiču v Kobaridu (št. 18b),137 in sicer na južnem delu raziskane terase, kjer je bilo v rimski dobi svetišče nadregionalnega pomena; približno 20 m severneje pa so bili ugotovljeni ostanki mladohalštatske stavbe s tipičnim hišnim inventarjem, z drenažnima zidovoma, temeljnimi kamni in z lomljenci tlakovanimi tlemi. Posebno mesto s povsem drugačnim značajem leži ob juž nem vznožju naselbine na Gradiču (št. 18e), kjer so naleteli na zakop konjskih okostij in zgodnjela tenskih bojnih oprav iz časa na prelomu 4. v 3. st. pr. n. št. Konjski skeleti šestih ali sedmih osebkov so bili brez lobanj z izjemo enega (ta lobanja je bila zdrobljena), od dveh osebkov pa je bila najdena samo polovica okostja. 135 Božič 1999b; Turk, Božič, Istenič 2009, 57–59; Božič 2011, 256–258; Laharnar, Mlinar 2014; Laharnar, Turk 2017, 166–169, 168, sl. 192; Mlinar et al. 2018, 24–26, kat. št. 32–36, 38, 40, 45, 52–54, 60, 62–65, 69, 70, 72, 74, 84–86, 92–97, 100–111. 136 Božič 2011, 239–260, sl. 6.2. 137 Osmuk 1997; ead. 1998; Božič 2011, 262, sl. 6.17: 1,8. Monografija je v pripravi, Nadi Osmuk in soavtoricam se najlepše zahvaljujeva za vpogled v gradivo. 138 Toškan 2011. 132 Mittheilungen der k.k. Central-Commission (MZK) 24, 1898, 111 in MZK 27, 1901, 77; Guštin 1991, 11–12, t. 38–40. 133 Božič 2011, 265; Laharnar, Mlinar 2014; Laharnar, Turk 2017, 166–169, sl. 164; 188–190; 191; 193; 195; 196; Mlinar et al. 2018, 17–19, sl. 11, kat. št. 28–31, 37, 42–44, 46–48, 50, 51, 55–59, 61, 66–68, 71, 73, 75–77, 79–83. 134 Laharnar, Mlinar 2014; Laharnar, Turk 2017, 166–168; glej še Turk, Božič, Istenič 2009, 57–59. 132 Mittheilungen der k.k. Central-Commission (MZK) 24, 1898, 111 in MZK 27, 1901, 77; Guštin 1991, 11–12, t. 38–40. 133 Božič 2011, 265; Laharnar, Mlinar 2014; Laharnar, Turk 2017, 166–169, sl. 164; 188–190; 191; 193; 195; 196; Mlinar et al. 2018, 17–19, sl. 11, kat. št. 28–31, 37, 42–44, 46–48, 50, 51, 55–59, 61, 66–68, 71, 73, 75–77, 79–83. 134 Laharnar, Mlinar 2014; Laharnar, Turk 2017, 166–168; glej še Turk, Božič, Istenič 2009, 57–59. 135 Božič 1999b; Turk, Božič, Istenič 2009, 57–59; Božič 2011, 256–258; Laharnar, Mlinar 2014; Laharnar, Turk 2017, 166–169, 168, sl. 192; Mlinar et al. 2018, 24–26, kat. št. 32–36, 38, 40, 45, 52–54, 60, 62–65, 69, 70, 72, 74, 84–86, 92–97, 100–111. Kultno-daritvena mesta Kosti so ležale bolj ali manj v anatomski legi in so se deloma prekrivale; pripadale so jezdnim konjem “zahodnega” nizko raslega tipa, starih med 7 in 13 let. Poleg tega so bile odkrite kosti in rogovje še drugih živali (koz, ovc, goveda, prašiča in gamsa).138 Najdena je bila tudi človeška nadlahtnica, vendar njena lega ne kaže nedvomne povezanosti oziroma sočasnosti z zakopom konj. Med najdbami je nekaj delov konjske opreme (dvoje železnih brzd in polovi ca tretje, 4 manjši in 3 večji bronasti gumbi za jermenje), med železnimi predmeti je 8 suličnih osti, 5 mečev v nožnicah, več kosov dvodelnih Posebno daritveno mesto med skalnimi stenami, škrapljami in brezni se predvideva na Berlotovem robu na Šentviški planoti (št. 62); tam je bilo z nestrokovnimi detektorskimi posegi v zadnjih desetletjih nabranih kar okoli 120 predmetov, že sredi 19. stoletja pa naj bi bil najden bronast kipec boginje Izide, a se ni ohranil.133 Tudi te najdbe kažejo velik časovni razpon – od poznega halštatskega obdobja do začetka rimske dobe – med njimi so podobni obeski, fibule, železno orodje in sekire ter deli bronastih posod kot v Bodrežu. Najdišče je precej uničeno, vendar so opazne umetne poravnave in terase, ki bi lahko bile naselbinske. Po približni oceni površine bi šlo za zelo majhno naselje (0,2 ha), medtem ko sta bogastvo in izjemnost najdb neobičajna za naselbinski kontekst nasploh, zato je prepričljivejša razlaga, da gre za kultno-daritveno mesto dolgega trajanja. Temu pritrjujejo med drugim obeski, ki bi jim lahko pripisali apotropejski značaj, kot je antropo-ornitomorfni obesek iz 5./4. st. pr. n. št. s primerjavami v ikonografskih upodobitvah gospodarice živali, ali pa srebrna okrogla ploščica s konca 2. ali iz 1. st. pr. n. št., ki naj bi prikazo vala lunine mene (podobne so bile najdene tudi v Bodrežu), ter ustje bronaste situle z napisom v venetski pisavi in navsezadnje neohranjeni kipec boginje.134 Na Šentviški planoti je znano še eno tako najdišče – Vrh gradu pri Pečinah (št. 57), ki je od Berlotovega roba oddaljeno dobre 3 km proti zahodu. Najdišče leži na robu planote, kjer se ta prevesi v pobočja, ki strmo padajo proti Idrijci in v grapo hudourniškega potoka. Na ozkem skalnem grebenu so vidne umetne terase s sledovi stavb in okopi manjšega naselja (0,5 ha). Spekter tu odkritih najdb prav tako kaže na kultno-daritveno mesto dolgega trajanja, od 6.–1. st. pr. n. 143 Svoljšak, Dular 2016, 71–74, sl. 57–60; t. 26; 27 (hiša 6/faza 2); Dular, Tecco Hvala, 2018, 79–85; Laharnar 2018a, 224–234, sl. 7–9; Motella De Carlo 2018, 379; Grömer et al. 2018; Toškan, Bartosiewicz 2018, 491, tab. 11. 144 Svoljšak, Dular 2016, 67–70, sl. 50–56; Dular, Tecco Hvala 2018, 78, sl. 74; Svoljšak 2018, sl. 11. 145 Mlinar 2020a, 58–60, 94–96, sl. 40–42; t. 36–41. Kultno-daritvena mesta št., starejši je Miha MLINAR, Sneža TECCO HVALA 422 imel posvečenih prostorov. Eden je bil prepoznan pod zidovi rimskega stavbnega kompleksa v se verovzhodnem delu naselbine. Čeprav so rimski zidovi v dobršni meri uničili starejše ostaline, se je ob njih ohranil slab meter širok pas žganine, ki je segala okoli 75 cm globoko do naravne osno ve. Na severozahodni in severovzhodni strani je bila obdana z linijama kamnov, ki sta nakazovali tloris štirioglate oblike (6 × 4 m). V žganini ni bila zaznana večplastnost, v njej pa je bilo kljub zelo majhni prostornini odkritih prek 200 najdb, ki odstopajo od inventarja v drugih stavbah. Ve činoma so bile razkosane in izpostavljene ognju, največ je delov bronastega nakita, obeskov, drobnih steklenih jagod, nekaj obdelanih in neobdelanih rdečih koral, koščkov bronastega posodja in izjemno malo lončenine, v severnem kotu pa so ležali trije noriški srebrniki. Poleg lesnega oglja je bilo v tej plasti tudi zoglenelo zrnje, raztreseno ali sprijeto v kepe (te so dajale vtis pogače), plodovi lešnikov in orehov, ohranil se je še košček platna ter živalske kosti, od katerih so zastopani samo deli lobanj in stopal.143 V bližini je stala stavba iz mlajšega halštatskega obdobja, tlakovana s kamnitimi plo ščami v glavnem prostoru, v stranskem pa je bil najden atiški skyphos, kakršni so se uporabljali pri religioznih obredjih.144 verig za pripenjanje meča ter 7 polovic dvodelnih ščitnih grb, ampak nobene dvojice, ki bi sestavlja la komplet, v enem primeru sta bili dve različni polovici položeni skupaj. Ščiti niso bili odloženi celi, temveč zgolj njihovi kovinski deli. Orožje ni bilo obredno poškodovano, prav tako ne železna kosa ali srp in nož. Od nakita so bile najdene ena bronasta fibula in 4 različne zapestnice (bronasta narebrena, vozlasta in votla z reliefnim okrasom ter ena železna). Najdene so bile tudi lončene črepinje, okrašena železna ploščica, kosi železove žlindre in bronaste taline. Pokop je prekrivala in zamejevala kamnita obloga. Najdbe imajo primerjave v sre dnjeevropskem keltskem prostoru, domnevati pa je mogoče, da je šlo za enkratno ritualno dejanje vojaškega značaja.139 V Nadiških dolinah se kultno mesto s keltskim orožjem iz srednjelatenskega obdobja domneva na hribu Barda/Monte Barda v Špetru/S. Pietro al Natisone (št. 41), prav tako na gradišču na vzpetini Madonna delle Grazie z grobiščem Dernazzacco ob njenem vznožju (št. 42) ter pri cerkvi sv. 139 Mlinar, Gerbec 2011. 140 Rupel 2004, 67–69. 141 Glej Turk, Božič, Istenič 2009, 57–59; glej še pris pevek L. Repanška v tej publikaciji. 142 Josipovič, Gaspari, Miškec 2012. 140 Rupel 2004, 67–69. Kultno-daritvena mesta Matije v Hostnah pri Garmaku/ Costne – Grimacco, kjer sta bila najdena bronast kipec in keltski srebrnik.140 Na Cerkljanskem je bilo predrimsko kultno mesto najbrž na Gradišču v Cerknem (št. 68), sodeč po zabeležki o najdbi kipca (ta ni ohranjen) in naključno odkritih bro nastih obeskih iz mlajšega halštatskega obdobja ter železnim srpom, kopačo in sulično ostjo iz poznolatenskega obdobja poleg najdb iz rimske dobe. V Posočju na kultna mesta opozarjajo tudi ohranjeni napisi v venetski pisavi.141 Podobna jama z žganino, obdana s kamni, je bila odkrita tudi na najnižji terasi blizu sotočja Idrijce in Soče na severnem robu grobišča na Mostu na Soči (sl. 10B). Zavzemala je pribl. 12 m2 površine in je bila debela okoli 30 cm. Poleg drobcev sežganih človeških kosti so bile v njej razpršene tudi nežgane živalske kosti in obilica najdb (deli bronastega nakita, železnega orožja, steklene jagode in črepinje posod) iz mlajšega halštatskega, poznolatenskega in zgodnjerimskega obdobja.145 V Bohinju je bilo nedavno odkrito posebno dari tveno mesto dolgega trajanja na vzhodnem obrežju jezera (št. 31), kjer so izkopavanja v neposredni bližini cerkve sv. Janeza Krstnika na območju med tremi vodami (jezerskim izlivom, Savo Bohinjko in Mostnico) razkrila s suhim zidom obdan prostor (pribl. 3 × 4 m), v njegovi notranjosti pa 50 cm globoko jamo, zapolnjeno z žganino. V njej so bile najdbe iz različnih dob, iz mlajšega halštatskega obdobja sta dva obeska, drugi deli noše, keramika in novci so iz latenske in rimske dobe, kar kaže, da je bilo to mesto v uporabi od 5. st. pr. n. št. do konca 4. st. n. št.142 Za interpretacijo takih prostorov so okoli ščine odkritja tako kot struktura najdb izjemno pomembne, saj pripomorejo k razumevanju, za kakšna obredja bi lahko šlo, denimo za čaščenje naravnih sil, plodnosti in virov življenja v poseb nem naravnem ambientu (npr. Berlotov rob) ali za Ob vsem tem bi bilo zelo nenavadno, če Most na Soči z največjim naseljem in grobiščem ne bi Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 423 POSELITVENI VZOREC IN KOMUNIKACIJE čaščenje prednikov, kar bi bilo možno domnevati po daritvenih prostorih v grobiščih (npr. Most na Soči – Repelc), ali božanskih zavetnic/zavetnikov lokalnih skupnosti v primeru takih mest v naselbini (npr. Kobarid – Gradič, Most na Soči – naselbina), ali pa za marcialne kulte na bojnih poljih. A vse to so bolj ali manj hipoteze, ker zgodovinskih pisnih virov, ki bi to potrdili, ni. Domnevamo pa lahko, da je bilo življenje prazgodovinskih skupnosti, ko se še ni oblikovala znanstveno-historična misel, prežeto z miti, ljudskimi verovanji in običaji. Po zbranih podatkih ter na podlagi dosedanjih raziskav in objav lahko skiciramo sledečo poselitve no sliko posoške skupnosti v starejši železni dobi. Najstarejše jedro stalne poselitve se je izoblikovalo ob naravnih poteh, ki vodijo iz Furlanske nižine oz. iz zaledja severnega Jadrana v alpski visoko gorski svet. Na vprašanje, kakšna so bila prvotna naselja, še ni mogoče odgovoriti, ker so podatki Sl. 12: Najdišča posoške skupnosti oz. svetolucijske kulturne skupine iz starejše železne dobe po zvrsteh. Fig. 12: Sites of the Early Iron Age Posočje community/Sv. Lucija cultural group according to types. Sl. 12: Najdišča posoške skupnosti oz. svetolucijske kulturne skupine iz starejše železne dobe po zvrsteh Fig. 12: Sites of the Early Iron Age Posočje community/Sv. Lucija cultural group according to types. 424 Miha MLINAR, Sneža TECCO HVALA Sl. 13: Hipotetična mreža komunikacij v starejši železni dobi na območju posoške skupnosti oz. svetolucijske kulturne skupine. Fig. 13: Hypothetical network of Early Iron Age com munications in the area of the Posočje community/Sveta Lucija cultural group. preskromni. Iz mlajše in pozne bronaste dobe se domnevajo utrjena naselja kaštelirskega tipa na Banjšicah, na vzpetinah ob robovih Nadiških do lin ter ob vstopu v dolino mejne reke Idrije.146 Iz tega časa so na Mostu na Soči ohranjeni ostanki s stojkami grajene stavbe na desnem bregu Idrijce ter skromna naselbinska plast na njenem levem bregu.147 Človekovo prisotnost v visokogorju v tistem času nakazujejo tudi redke depojske in po samične najdbe ter nekaj skromnih sledi občasnih postojank.148 O stalni poselitvi na začetku železne dobe pričajo predvsem grobišča (sl. 9 in 12). Grobovi v Tolminu in Špetru/S. p 151 Do l. 1892 je bilo izkopanih vsaj 1343 grobov s fibulami mladohalštatskih oblik, ko so jih namesto igel nosili tudi moški. 150 Upoštevaje opredelitev vodilnih tipov za posame zne kronološke faze po Teržan, Trampuž 1973 je grobov s starohalštatskimi fibulami, iglami in ovratnicami, ki sta jih do l. 1892 izkopala Marchesetti in Szombathy, vsaj 1150. Glej Marchesetti 1886, 114–125; id. 1893, 141–169; za Szombathyjeva izkopavanja pa Teržan, Lo Schiavo, Trampuž-Orel 1984–1985. 148 Teržan (ur.) 1995–1996 (posamične najdbe: kat. št. 61, 66, 146, 216, 221, 234 in Addendum 1–3; depoji: kat. št. 11, 17, 18 in 30); za vodne najdbe glej Gerbec, Mlinar 2011; za visokogorska najdišča pa Horvat 2020, sl. 1, tab. 1. 149 Glej tu poglavje o grobiščih. 146 Svoljšak 1988–1989, sl. 7; Tomadin, Visintini, Colussa 1989; Mlinar, Gerbec, Laharnar 2014, 24 (karta), 27–29; Gerbec 2018; Gerbec, Vinazza 2018; Gerbec 2021. 147 Svoljšak 1988–1989, sl. 3–5, pril. 1; 2; t. 5: 3–6,8,10; 6: 1,4–10,12,13; 7: 1,4–6; 8: 1–5,8; Mlinar 2020a, 37–39, sl. 23–27; t. 15A. 152 Glej op. 63, 64, 89–91 ter še Gabrovec 1987, 141–150. 153 Trasa poti ob zahodnih pobočjih Gradiča in Tonov covega grada v Kobaridu je zaobšla tesen doline Soče nad Kobaridom (prim. Štular 2011b, sl. 1.41), pri Trnovem se je 152 Glej op. 63, 64, 89–91 ter še Gabrovec 1987, 141–150. 147 Svoljšak 1988–1989, sl. 3–5, pril. 1; 2; t. 5: 3–6,8,10; 6: 1,4–10,12,13; 7: 1,4–6; 8: 1–5,8; Mlinar 2020a, 37–39, sl. 23–27; t. 15A. 146 Svoljšak 1988–1989, sl. 7; Tomadin, Visintini, Colussa 1989; Mlinar, Gerbec, Laharnar 2014, 24 (karta), 27–29; Gerbec 2018; Gerbec, Vinazza 2018; Gerbec 2021. 147 Svoljšak 1988–1989, sl. 3–5, pril. 1; 2; t. 5: 3–6,8,10; 6: 1,4–10,12,13; 7: 1,4–6; 8: 1–5,8; Mlinar 2020a, 37–39, sl. 23–27; t. 15A. 148 Teržan (ur.) 1995–1996 (posamične najdbe: kat. št. 61, 66, 146, 216, 221, 234 in Addendum 1–3; depoji: kat. št. 11, 17, 18 in 30); za vodne najdbe glej Gerbec, Mlinar 2011; za visokogorska najdišča pa Horvat 2020, sl. 1, tab. 1. 149 Glej tu poglavje o grobiščih. 150 Upoštevaje opredelitev vodilnih tipov za posame zne kronološke faze po Teržan, Trampuž 1973 je grobov s starohalštatskimi fibulami, iglami in ovratnicami, ki sta jih do l. 1892 izkopala Marchesetti in Szombathy, vsaj 1150. Glej Marchesetti 1886, 114–125; id. 1893, 141–169; za Szombathyjeva izkopavanja pa Teržan, Lo Schiavo, Trampuž-Orel 1984–1985. 151 Do l. 1892 je bilo izkopanih vsaj 1343 grobov s fibulami mladohalštatskih oblik, ko so jih namesto igel nosili tudi moški POSELITVENI VZOREC IN KOMUNIKACIJE Pietro al Natisone iz starejšega hal štatskega obdobja kažejo na manjše lokalne sku pnosti, če upoštevamo število grobov in približno stopetdeset- do dvestoletni razpon pokopavanja; grobni pridatki pa ne izkazujejo večjih družbenih razlik.149 Koliko je bilo prebivalstva v starejšem halštatskem obdobju in kakšna je bila njegova družbena struktura na sosednjem Mostu na Soči in v Kobaridu, še ni mogoče oceniti. Glede na po vršno oceno števila grobov, v katerih so zastopane fibule, ovratnice in igle, značilne za stopnjo Sv. Lucija I,150 se zdi se, da je bilo na Mostu na Soči več prebivalcev kot v Tolminu, hkrati pa manj kot v mlajšem halštatskem obdobju,151 ko so se tudi v načinu pokopa in grobnih pridatkih pojavile večje razlike, ki kažejo na družbeno razslojevanje. Most na Soči se je takrat razvil v glavno regional no središče z zgodnjeurbanimi značilnostmi in s proizvodno dejavnostjo kovinskih in lončarskih izdelkov ter blagovno menjavo, ki sta presegla Sl. 13: Hipotetična mreža komunikacij v starejši železni dobi na območju posoške skupnosti oz. svetolucijske kulturne skupine. Fig. 13: Hypothetical network of Early Iron Age com munications in the area of the Posočje community/Sveta Lucija cultural group. okvire hišnega gospodarstva in samozadostno sti.152 Rastoče potrebe po surovinah in naravnih resursih so bržkone botrovale intenzivnejšemu izkoriščanju hribovitega zaledja vse do Bohinja, ob tem so zrasla nova manjša naselja, verjetno tudi zaselki in sezonske postojanke (sl. 12). Domnevati je mogoče, da je prišlo v 6. in 5. st. pr. n. št. do močnejše integracije te skupnosti in utrjevanja njene kulturne identitete, hkrati pa so občutnejši tudi stiki z drugimi kulturnimi regijami. Trgovske in tovorne poti so potekale delno ob rekah, na mestih večjih kanjonskih zožitev pa so se preusmerile na planote (sl. 13). Glavna komunikacija, ki je povezovala ključni najdišči posoške skupnosti oziroma svetolucijske kulture skupine – Most na Soči in Kobarid, je tekla ob levem bregu Soče, kar nakazujejo železnodobna najdišča Tolmin, Volarje in Ladra. Pri Kobaridu se je razcepila na dva kraka – severni je prečkal Sočo in nadaljeval pot po desnem bregu proti bovški kotlini ter čez prelaz Predel/Predil (1156 m) na trbiško območje in avstrijsko Koroško, zahodni krak pa mimo gradišča na Sv. Volarju nad Robičem in ob Nadiži proti Furlaniji.153 Glavna povezava 425 Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja proti jugu, proti Goriški in Jadranskemu morju, je od Mosta na Soči verjetno šla po zahodnem robu Banjške planote do utrjene naselbine na Sv. POSELITVENI VZOREC IN KOMUNIKACIJE 156 Poti med Posočjem in Bohinjem so verjetno vodile tudi čez nekatere druge prelaze ali vrhove Bohinjsko-Tol minskih gora, npr. Škrbinsko sedlo, Dobrenjska Planja in Tolminski Migovec (Mlinar, Turk 2016, 9–14). g 157 Komunikacijo s Selško dolino nakazuje najdišče Štalca pri Železnikih (prim. Grahek 2018b; Mlinar 2018). V 157 Komunikacijo s Selško dolino nakazuje najdišče Štalca pri Železnikih (prim. Grahek 2018b; Mlinar 2018). V 155 To komunikacijo nakazujejo železnodobna najdišča na Šentviški planoti (prim. Mlinar et al. 2018) in odsotnost najdišč v težko prehodni soteski med Dolenjo Trebušo in Reko pri Cerknem. zopet približala Soči, med Srpenico in Bovcem je potekala bolj ali manj po trasi današnje ceste; težje je rekonstruirati njen nadaljnji potek mimo Kluž in Loga pod Mangartom do prelaza na Predelu. POSELITVENI VZOREC IN KOMUNIKACIJE Katarini nad Novo Gorico na stičišču z Vipavsko dolino.154 Pot proti vzhodu, Panoniji in Balkanu, je vodila po dolini Idrijce do Slapa ob Idrijci, kjer se je povzpela na Šentviško planoto in se šele pri Reki pri Cerknem zopet spustila v dolino Idrijce ter nadaljevala ob Zali naprej do Godoviča.155 Druga trasa proti vzhodu je potekala ob spodnjem toku reke Bače in se pri Koritnici vzpela do Ruta ter nadaljevala čez prelaz Vrh Bače v Bohinj.156 Pri Koritnici se je odcepil krak poti, ki je prečil reko Bačo in se usmeril prek Cerkljanskega hribovja in Poljanske doline proti vzhodu. Potek trase med Koritnico in Petrovim Brdom ter mimo Soriške planine naprej do Bohinja z najdbami doslej še ni potrjen, a se zdi zelo verjeten.157 Vzponu in razcvetu v 6. in 5. st. pr. n. št. so sledili pretresi in spremembe v 4. st. pr. n. št., ki so privedli do zatona središča na Mostu na Soči in dezintegracije skupnosti. Občuten je upad prebi valstva, ki se je umaknilo v stranske doline in bolj hribovite predele. V grobnih in daritvenih obredjih je v znatnejši meri prisotno orožje, v katerem so zastopani tudi elementi srednjeevropske keltske oborožitve in namensko deformiranje. Poveličevanje vojaškega statusa bi lahko videli tudi v bronastem kipcu z negovsko čelado z Idrije ob Bači, ki je na območju posoške skupnosti najstarejša mala polna plastika v realistični in individualizirani podobi človeka.158 Po drugi strani se pojavijo prve pismenke na daritvenih posodah v venetski pisavi, kot pričata bronasti situli z Gradu pri Reki in Berlotovega roba.159 Za jasnejšo sliko procesov v železni dobi na ob močju posoške skupnosti in za iskanje odgovorov na številna odprta vprašanja pa bodo potrebna nadaljnja ciljno usmerjena arheološka raziskovanja in temeljne objave raziskanih najdišč. zopet približala Soči, med Srpenico in Bovcem je potekala bolj ali manj po trasi današnje ceste; težje je rekonstruirati njen nadaljnji potek mimo Kluž in Loga pod Mangartom do prelaza na Predelu. prid tej komunikaciji morebiti govorijo žrmlje iz naselbine na Mostu na Soči, ki so iz kremenovega konglomerata, najbližja ležišča te kamnine pa so v okolici Cerknega ter v Poljanski in Selški dolini (Dular, Tecco Hvala 2018, 94). 158 Gabrovec 1987, 144–145; Guštin 1991, 51–52; t. 22: 1 (grob 25 iz stopnje IIc). 159 Turk, Božič, Istenič 2009, 57–59; Laharnar, Turk 2017, 139–141, sl. 158–160, 164; glej še Repanšek 2020 in njegov prispevek v tej publikaciji. 154 Mlinar, Žbona Trkman 2008, sl. 1. Iza ZRC SAZU = Inštitut za arheologijo Znanstvenoraz iskovalnega centra Slovenske akademije znanosti in umetnosti ZVKDS OE NG = Zavod za kulturno dediščino Slovenije, Območna enota Nova Gorica * Register kulturne dediščine [gisportal.gov.si] Arheološki kataster Slovenije[http: //arkas.zrc-sazu.si/] * Atlas okolja [http://gis.arso.gov.si/atlasokolja/profile. aspx?id=Atlas_Okolja_AXL@Arso] prid tej komunikaciji morebiti govorijo žrmlje iz naselbine na Mostu na Soči, ki so iz kremenovega konglomerata, najbližja ležišča te kamnine pa so v okolici Cerknega ter v Poljanski in Selški dolini (Dular, Tecco Hvala 2018, 94). 158 Gabrovec 1987, 144–145; Guštin 1991, 51–52; t. prid tej komunikaciji morebiti govorijo žrmlje iz naselbine na Mostu na Soči, ki so iz kremenovega konglomerata, najbližja ležišča te kamnine pa so v okolici Cerknega ter v Poljanski in Selški dolini (Dular, Tecco Hvala 2018, 94). 158 Gabrovec 1987, 144–145; Guštin 1991, 51–52; t. 22: 1 (grob 25 iz stopnje IIc). 159 Turk, Božič, Istenič 2009, 57–59; Laharnar, Turk 2017, 139–141, sl. 158–160, 164; glej še Repanšek 2020 in njegov prispevek v tej publikaciji. KATALOG NAJDIŠČ V katalog so vključena le najdišča, ki so podprta z bolj ali manj zanesljivimi materialni dokazi. Upoštevani so tudi podatki iz Registra kulturne dediščine (RKD),* ki ga vodi Ministrstvo za kulturo, ter iz Arheološkega katastra Slovenije (ARKAS), ki je spletna podatkovna zbirka arheoloških naj dišč, vzpostavljena na Inštitutu za arheologijo ZRC SAZU. Koordinate so podane v državnem (Gauss -Kruegerjevem) koordinatnem sistemu po digitalnem viru Atlas okolja,* ki je javni pregledovalnik prostorskih podatkov Agencije Republike Slovenije za okolje. Med objavami so navedena samo glavna dela, ki podajajo primarne vire oz. referenčno literaturo in temeljne podatke o okoliščinah odkritja ter slikovno gradivo. Pojasnila kratic: CMATS = Civico Museo di Antichità J.J. Winckelmann, Trieste/Trst GMNG = Goriški muzej, Nova Gorica GMKR = Gorenjski muzej, Kranj MAC = Museo Archeologico Nazionale di Cividale del Friuli NHMW = Naturhistorisches Museum, Wien NMS = Narodni muzej Slovenije TMT = Tolminski muzej, Tolmin Iza ZRC SAZU = Inštitut za arheologijo Znanstvenoraz iskovalnega centra Slovenske akademije znanosti in umetnosti ZVKDS OE NG = Zavod za kulturno dediščino Slovenije, Območna enota Nova Gorica * Register kulturne dediščine [gisportal.gov.si] Arheološki kataster Slovenije[http: //arkas.zrc-sazu.si/] * Atlas okolja [http://gis.arso.gov.si/atlasokolja/profile. aspx?id=Atlas_Okolja_AXL@Arso] Pojasnila kratic: CMATS = Civico Museo di Antichità J.J. Winckelmann, Trieste/Trst GMNG = Goriški muzej, Nova Gorica GMKR = Gorenjski muzej, Kranj MAC = Museo Archeologico Nazionale di Cividale del Friuli NHMW = Naturhistorisches Museum, Wien NMS = Narodni muzej Slovenije TMT = Tolminski muzej, Tolmin Iza ZRC SAZU = Inštitut za arheologijo Znanstvenoraz iskovalnega centra Slovenske akademije znanosti in umetnosti ZVKDS OE NG = Zavod za kulturno dediščino Slovenije, Območna enota Nova Gorica * Register kulturne dediščine [gisportal.gov.si] Arheološki kataster Slovenije[http: //arkas.zrc-sazu.si/] * Atlas okolja [http://gis.arso.gov.si/atlasokolja/profile. aspx?id=Atlas_Okolja_AXL@Arso] 4 Soča – med Idrskim in Tolminom, posamična najdba (votivna?) Pojasnila kratic: 426 Miha MLINAR, Sneža TECCO HVALA 1 2 a. Most na Soči (desni breg Idrijce), delno utrjeno naselje Lega: pomol, ki se razteza v smeri V–Z od zahodnega vznožja hriba Senica do sotočja Idrijce in Soče, je oblikovan v tri vzpetine (kuke) s strmim padcem na severni strani proti ravnici ob Soči, južno pobočje pomola pa se v terasah spušča v rečno korito Idrijce. Na dostopnejši vzhodni strani poteka 164 m dolg greben/okop v smeri S–J, zahodno od njega so bili na prisojni legi pomola odkriti naselbinski ostanki. Ocenjena površina nasel binskega areala je pribl. 13 ha. Bača pri Modreju – Senica, posamična najdba Lega: na jugovzhodnem pobočju Senice nad vasjo Bača pri Modreju je bila naključno odkrita železna sekira. Lega: na jugovzhodnem pobočju Senice nad vasjo Bača pri Modreju je bila naključno odkrita železna sekira. Raziskave: odkritje z detektorjem kovin okoli l. 2010, najditelj Primož Kos s Prapetna. Najdba: železna uhata sekira (t. 3: 7). Datacija: mlajše halštatsko obdobje (Sv. Lucija IIc). Objava: Datacija: mlajše halštatsko obdobje (Sv. Lucija IIc) Datacija: mlajše halštatsko obdobje (Sv. Lucija IIc). Objava: - Koordinate: 403295, 112791, n. v. 173 m. Soča – med Idrskim in Tolminom, posamična najdba (votivna?) b. Most na Soči – grobišče I (levi breg Idrijce), plano grobišče Lega: na separaciji pri Volčah so delavci CPG Nova Gorica pri ločevanju peska naleteli na železno sekiro, ki izvira verjetno iz prodišča reke Soče nekje na območju med Idrskim in Tolminom. Lega: na vzpenjajočih se terasah na levem bregu Idrijce JV od njenega izliva v Sočo je bilo odkrito eponimno grobišče svetolucijske kulturne skupine. Koordinate: 403259, 112557, n. v. 191 m. Koordinate (sekundarnega najdišča): 400346, 117218, n. v. 162 m. Raziskave: na najvišji in najširši terasi je l. 1880/81 P. Bizzarro odkril okoli 70 grobov, 52 grobov je izkopal E. Maionica med l. 1881 in 1891; z obsežnimi izkopavanji C. Marchesettija v l. 1885–1899 in 1902 je bilo odkritih okoli 3960 grobov, z izkopavanji J. Szombathyja v l. 1886–1887 in 1890 pa 2450 grobov. Na malce nižji terasi je B. Forlati Tamaro l. 1927 izkopala 4 grobov, še 3 pa domačin. V neposredni okolici območij, prekopanih v 19. stoletju, je bilo ob manjših zaščitnih akcijah GMNG in TMT odkritih 29 grobov. Na spodnji terasi (Repelc in Pucarjev rob) tik nad Idrijco pa je bilo ob arheološkem nadzoru in z izkopavanji TMT v l. 2000–2002 in 2013 odkritih 87 grobov iz halštatske, latenske in rimske dobe ter sežigališče ali žgalno daritveno mesto (sl. 10). Raziskave: naključno odkritje okoli l. 2000. Najdba: železna uhata sekira, odlomljena pri ušesu (t. 3: 8). Hrani: TMT (darovalec Štefan Konec, Poljubinj). Najdba: železna uhata sekira, odlomljena pri ušesu (t. 3: 8). Š Hrani: TMT (darovalec Štefan Konec, Poljubinj). Datacija: mlajše halštatsko obdobje (Sv. Lucija IIc). Objava: Gerbec, Mlinar 2013–2014, 406, kat. št. 2. 5 Tolmin – Pod gradom, plano grobišče Tolmin Pod gradom, plano grobišče Lega: na izteku strmega vzhodnega pobočja kopastega osamelca Kozlov rob, ki se pne nad ravnico v sotočju Tolminke in Soče, je bilo ob gradbeni delih odkrito grobišče. g Koordinate: 402275, 116750, n. v. 212 m. Najdbe: doslej odkritih grobov na levem bregu Idrijce je okoli 6847, večinoma žganih iz halštatske dobe ter maloštevilni iz latenske in rimske dobe. Raziskave: l. 1965 je bilo ob gradnji hiše na Ilirski ulici 11 poškodovanih ali uničenih 9 grobov, sledila so arheo loška izkopavanja GMNG na širšem območju v l. 1965 in 1966 ter 1968–1970. Hrani: CMATS, NHMW, NMS, TMT. Hrani: GMNG in TMT. Hrani: GMNG in TMT. Najdbe: iz domnevno starejše železne dobe je nekaj frag. prostoročno izdelanih in rjavo-rdeče žganih loncev. Hrani: TMT. Najdbe: iz domnevno starejše železne dobe je nekaj frag. prostoročno izdelanih in rjavo-rdeče žganih loncev Datacija: mlajša bronasta doba, mlajše halštatsko obdobje (Sv. Lucija IIa–IIc), pozno latensko obdobje (LT D), rimska doba. Datacija: starejša železna doba, zgodnji srednji vek. Objava: Mlinar 2019 12 21 kat št 4 7 Objava: Gabrovec, Svoljšak 1983, 34–35, sl. 18; Mlinar, Klasinc, Knavs 2008; Svoljšak, Dular 2016; Dular, Tecco Hvala (ur.) 2018; Mlinar 2021b; Tecco Hvala, Mušič 2021. Datacija: starejša železna doba, zgodnji srednji vek. Objava: Mlinar 2019, 12–21, kat. št. 4–7. 3 Raziskave: v l. 1971–1984 so potekala zaščitna izkopavanja GMNG, v l. 2001, 2004, 2010, 2015 in 2016 pa arheo loško dokumentiranje TMT ob zemeljskih posegih. L. 2021 je Iza ZRC SAZU izkopal testno sondo na okopu z namenom ugotavljanja utrdbenih struktur (sl. 7; 8). Volče – sv. Lenart, neutrjeno naselje (?) Lega: na ravnici ob desnem bregu Soče med pritoki Ka mnica, Gunjač in Hotevlje je bila pri obnovi cerkve sv. Lenarta v Volčah med drugim odkrita prazgodovinska naselbinska keramika. Potencialno območje naselbine se domneva na dominantni terasi s toponimom Zajčca ob vzhodnem vznožju hriba Veliki Špik. Najdbe: iz železne dobe so bili doslej odkriti ostanki 39 hiš, kultno mesto, pot in drenažni jarek, s sondo na vzhodnem grebenu je bilo ugotovljeno obzidje. Poleg tega so bili odkriti ostanki ene stavbe iz pozne bronaste dobe in 15 hiš iz rimske dobe. Koordinate: 400475, 115520, n. v. 212 m. Raziskave: l. 2016 je TMT opravil arheološke raziskave v okviru popotresne obnove cerkve sv. Duha v Volčah. Hrani: GMNG in TMT. Hrani: CMATS, NHMW, NMS, TMT. Hrani: CMATS, NHMW, NMS, TMT. Datacija: starejše in mlajše halštatsko obdobje (Sv. Lucija Ia–IIc), srednje in pozno latensko obdobje (LT C1–LT D), rimska doba. Najdbe: 465 žganih grobov. Najdbe: 465 žganih grobov. j g Hrani: GMNG. Objava: Marchesetti 1886; Marchesetti 1893; Gabrovec, Svoljšak 1983; Teržan, Lo Schiavo, Trampuž-Orel 1984–1985; Mlinar 2020a, sl. 3. Datacija: starejše halštatsko obdobje (Sv. Lucija Ia–Ic). Objava: Svoljšak, Pogačnik 2001–2002. Datacija: starejše halštatsko obdobje (Sv. Lucija Ia–Ic). Obj S ljš k P č ik 2001 2002 Objava: Svoljšak, Pogačnik 2001–2002. Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 427 Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja Raziskave: ob cerkvici je bila l. 2009/10 naključno najdena steklena jagoda, sekiro pa so hranili na bližnji domačiji. Raziskave: ob cerkvici je bila l. 2009/10 naključno najdena steklena jagoda, sekiro pa so hranili na bližnji domačiji. 6 Zatolmin – Planina Zavrh (Vodil vrh), posamična najdba Najdbi: rumena steklena jagoda z modro-belimi plastovi timi očesci, železna sekira z obojestranskimi plavutmi (t. 1: 16; 3: 11). Lega: na gorskem grebenu, ki poteka v smeri SZ–JV med rekama Sočo in Tolminko, je bil na vzhodnem pobočju pod sedlom med vrhovoma Grmuč in Vodil najden bronast prstan. p Koordinate: 401100, 119500, n. v. 965 m. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIc). Objava: Mlinar 2006a, 241–242; Gerbec, Mlinar 2013–2014, 413; Mlinar, Turk 2016, 16, kat. št. 25 in 26. Raziskave: odkritje z detektorjem kovin okoli l. 2000, najditelj Marko Komac iz Bovca. Najdba: nesklenjen bronast prstan, okrašen z vtisnjenimi krožci s piko in snopi prečnih vrezov (t. 1: 9). Čadrg – Laze I, plano grobišče Čadrg – Laze I, plano grobišče Najdba: bronasta večglava igla s trombastim zaključkom (t. 1: 7). Lega: na položnejšem delu pobočja, ki se spušča proti vodotokoma Tolminke na zahodu in vzhodu, je bilo pri zemeljskih delih odkrito manjše grobišče. j j g Koordinate: 402620, 121570, n. v. 735 m. Datacija: pozna bronasta doba, igla sodi v starejše halštat sko obdobje (Sv. Lucija Ib), rimska doba, pozna antika. Raziskave: po naključnem odkritju lončenih črepinj in oglja je bilo v sodelovanju z odkriteljem Janijem Kutinom l. 2014 opravljeno arheološko sondiranje ZVKDS OE NG in TMT. Objava: Horvat 2015–2016, 306; Mlinar, Turk 2016, 24, kat. št. 21. Zatolmin – Javorca, posamični najdbi Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb/c). Objava: - ato Javo ca, posa č ajdb Lega: posamični najdbi iz železne dobe domnevno izvirata s položnejšega dela pobočja med pritokoma v zgornjem delu Tolminke, kjer stoji spominska cerkvica sv. Duha v Javorci. p j Lega: posamični najdbi iz železne dobe domnevno izvirata s položnejšega dela pobočja med pritokoma v zgornjem delu Tolminke, kjer stoji spominska cerkvica sv. Duha v Javorci. Zadlaz-Čadrg – Kobilnik, posamični najdbi Najdba: železna uhata sekira z ohranjenim delom lesenega toporišča (t. 3: 5). Lega: v sedlu pod grebenom, ki poteka v severozahodni smeri z vrha Kobilnika med vodotokoma Tolminko in Zadlaščico, je v bližini litoželeznega križa Marko Komac iz Bovca našel v začetku tega stoletja del bronaste fibule; nekje na tem območju je bil že v 60. letih 20. stoletja najden surovec iz bakrove zlitine. Hrani: TMT. Hrani: TMT. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIc). Datacija: mlajše halštatsko obdobje (Sv. Lucija IIc). Objava: Mlinar, Turk 2016, 16, kat. št. 27. Selišče – pobočje Mrzlega vrha, posamična najdba Selišče pobočje Mrzlega vrha, posamična najdba Lega: na strmem zahodnem pobočju Mrzlega vrha je Aleš Hvalič s Selišč v profilu poti na planino Lapač našel bronasto fibulo. Koordinate: pribl. 398196, 120339, n.v. 867 m. Raziskave: naključno odkritje okoli l. 2010. Najdba: bronasta certoška fibula VIb (t. 2: 10). Hrani: TMT. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb/c). Objava: - Selišče pobočje Mrzlega vrha, posamična najdba Lega: na strmem zahodnem pobočju Mrzlega vrha je Aleš Hvalič s Selišč v profilu poti na planino Lapač našel bronasto fibulo. Koordinate: pribl. 398196, 120339, n.v. 867 m. Raziskave: naključno odkritje okoli l. 2010. Najdba: bronasta certoška fibula VIb (t. 2: 10). Hrani: TMT. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb/c). Objava: Lega: na strmem zahodnem pobočju Mrzlega vrha je Aleš Hvalič s Selišč v profilu poti na planino Lapač našel bronasto fibulo. Datacija: grobovi sodijo v mlajše halštatsko obdobje (Sv. Lucija IIc), jama pa v srednje latensko obdobje (LT C1). Objava: Mlinar, Turk 2016, 20–22, kat št. 28–45; Mlinar 2020b. Datacija: grobovi sodijo v mlajše halštatsko obdobje (Sv. Lucija IIc), jama pa v srednje latensko obdobje (LT C1). Koordinate: pribl. 398196, 120339, n.v. 867 m. Objava: Mlinar, Turk 2016, 20–22, kat št. 28–45; Mlinar 2020b. Raziskave: naključno odkritje okoli l. 2010. Najdba: bronasta certoška fibula VIb (t. 2: 10). Hrani: TMT. Zatolmin – Javorca, posamični najdbi 10 Tolminske Ravne – Planina Na kalu, posamična najdba Lega: v kotanji na sedlu pod grebenom, ki poteka v jugo zahodni smeri od vrha Tolminskega Migovca, je bila najdena železna sekira. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIa/b). Objava: Gerbec, Mlinar 2013–2014, 414; Mlinar, Turk 2016, 16, kat. št. 24. Koordinate: 404415, 122760, n. v. 1475 m. Raziskave: naključno odkritje l. 2000. Zadlaz-Čadrg – Kobilnik, posamični najdbi 11 Koordinate: 403650, 118930, n. v. 651 m. Tolminske Ravne – Pod Zelenim vrhom, posamična najdba Raziskave: naključno odkritje l. 1964 (surovec) in z detek torjem kovin v začetku 21. stoletja (fibula). Lega: v globoki konti med Tolminskim Migovcem, Tol minskim Kukom in Zelenim vrhom je bilo odkrito arheološko zanimivo območje z opaženimi tremi rahlimi grbinami – ostanki stavb. Najdba: frag. bronaste certoške fibule XIII. vrste (t. 2: 2), surovec iz bakrove zlitine z visokim deležem svinca. Koordinate: 405034, 124200, n. v. 1775 m. Hrani: TMT in Maksi Cuder, Tolmin. Hrani: TMT in Maksi Cuder, Tolmin. Raziskave: odkritju l. 2010 je sledil arheološki pregled območja l. 2014 z manjšimi sondami in detektorjem kovin v sodelovanju z odkritelji in Iza ZRC SAZU. Poleg ugotovljenih ostankov treh stavb, ki so bili datirani na osnovi oglja v 11.–10 st. pr. n. št., v 1.–2. st. n. št. ter v 5.–6. st. n. št., je bila v njihovi bližini najdena bronasta igla iz starejše železne dobe, vendar ne v arheološki plasti. Datacija: surovec po sestavi morda sodi v pozno bronasto dobo (Ha B), fibula pa v mlajše halštatsko obdobje (Sv. Lucija IIb). Objava: Mlinar, Turk 2016, 23–24, kat. št. 22 in 24. 12 Najdbe: 3 žgani grobovi s pridatki in jama z železnim orožjem (verjetno grob). Selišče – pobočje Mrzlega vrha, posamična najdba Selišče – pobočje Mrzlega vrha, posamična najdba 14 Hrani: najditelj Simon Rutar, Koseč. Vrsno – Strničelo (Strenčl), posamična najdba Hrani: najditelj Simon Rutar, Koseč. Hrani: najditelj Simon Rutar, Koseč. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb/c). Vrsno Strničelo (Strenčl), posamična najdba Lega: na vrhu razglednega hriba Strničelo (Strenčl) nad Vrsnim, ki se pne nad levim bregom Soče med prito koma potoka Volarja, je bil naključno najden bronast gumb fibule. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb/c Objava: Knific et al. 2021, 16. 13 Krn – Gradec, plano grobišče, kultno mesto? Lega: na gričku pri vasi Krn, ki je na vzhodni strani zamejen s prepadnim pobočjem in potokom, je ob poti proti Koordinate: 401595, 122115, n. v. 589 m. Lega: na gričku pri vasi Krn, ki je na vzhodni strani zamejen s prepadnim pobočjem in potokom, je ob poti proti 428 Miha MLINAR, Sneža TECCO HVALA zemljo sprali in presejali ter našli še nekaj drobc keramike in sežganih kosti, kar kaže na grobove. Najdbe: 2 žgana grobova s pridatki (t. 1: 1–6). Hrani: TMT zemljo sprali in presejali ter našli še nekaj drobcev keramike in sežganih kosti, kar kaže na grobove. zemljo sprali in presejali ter našli še nekaj drobcev keramike in sežganih kosti, kar kaže na grobove. planini Pretovč zbiratelj starin Jože Golja iz Trebenč izkopal večje število kovinskih predmetov. Koordinate: 398180, 122420, n. v. 923 m. Najdbe: 2 žgana grobova s pridatki (t. 1: 1–6). Hrani: TMT. Raziskave: odkritje z detektorjem kovin okoli l. 2010, najditelj J. Golja. L. 2020 je TMT preverjal lokacijo z geofizikalnimi meritvami v sodelovanju s podjetjem Gearh in opravil arheološko sondiranje s Skupino STIK. Datacija: pozna bronasta doba/začetek halštatske dobe (Sv. Lucija Ia). Datacija: pozna bronasta doba/začetek halštatske dobe (Sv. Lucija Ia). Objava: Knific et al. 2021, 24, kat. št. 8. Najdbe: s sondiranjem je bilo odkritih 5 žganih grobov, v zbirki Golje pa je bilo železno orožje in orodje, deli oprav ter kovinskih posod, ki verjetno izvirajo iz uni čenih grobov in morda kultnega mesta. 18 g Koordinate: pribl. 396578, 121356, n. v. 795 m. a. Kobarid – Gradič, delno utrjeno naselje Lega: vzpetina Gradič se dviga nad obsežno ravnico ob desnem bregu Soče na prehodu v dolino Nadiže in je v virih poimenovana tudi po cerkvici sv. Antona na njej. Prva svetovna vojna in povojna gradnja kostnice ter drugi moderni posegi so arheološke ostaline precej poškodovali. Kamniti nasipi so se ohranili v odsekih na zahodni, severni in vzhodni strani, v notranjosti in na jugozahodnem pobočju so vidne terase. Ocenjena površina naselbinskega areala je pribl. 4 ha. Raziskave: odkritje z detektorjem kovin v 90. letih 20. stoletja, najditelj Igor Hrast iz Robiča. Najdba: bronast gumb certoške fibule Xc (t. 2: 8). Hrani: TMT. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIc). Objava: Knific et al. 2021, 20. Objava: Knific et al. 2021, 20. Smast, posamična najdba Koordinate: 391190, 123510, n. v. 297 m. Lega: na levem bregu Soče med pritokoma Ročica in Rakovca je bil na terasi z ledinskim imenom Žaršče (Zaršče) med vasema Smast in Libušnje naključno odkrit bronast obesek. Raziskave: na območju naselbine še ni bilo sistematičnih arheoloških raziskav razen topografskih ogledov v 80. in 90. letih prejšnjega stoletja, ki jih je izvajal ZVKDS OE NG. L. 2010 je bila v sodelovanju NMS, TMT in Iza ZRC SAZU opravljena mikrotopografija najdišča z namenom obdelave in arheološkega ovrednotenja lidar skih podatkov. Istega leta je bil opravljen tudi arheološki nazor pri infrastrukturnih delih na Gregorčičevi ulici ob jugozahodnem vznožju griča, pri tem je bila odkrita jama z naselbinsko lončenino in živalskimi kostmi iz železne dobe. Na severnem obrobju Gradiča pa je bil z detektorjem kovin na lokaciji s toponimom Skrinjca odkrit novčni depo keltskega srebrnega drobiža ter rimskih republikanskih srebrnih in bronastih novcev iz začetka druge polovice 2. st. pr. n. št. Koordinate: pribl. 393438, 121423, n. v. 212 m. Raziskave: odkritje z detektorjem kovin l. 2005, najditelj Primož Kos s Prapetna. Najdba: bronast košaričast obesek (t. 1: 18). Najdba: bronast košaričast obesek (t. 1: 18). Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb). Objava: Knific et al. 2021, 22. Objava: Knific et al. 2021, 22. 17 Koseč, posamična najdba Lega: v travnati kotanji ob griču Gorica med potokoma Ročica in Brusnik je bil zahodno od hiše Koseč 3a na ključno najden del steklene jagode. Po pripovedovanju je bilo takih jagod na tem prostoru odkritih več, a so izgubljene. Hrani: NMS in TMT. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb/c), pozno latensko obdobje (LT D1). Objava: Laharnar, Turk 2017, 170, sl. 197; Istenič 2018, 284–289; Knific et al. 2021; glej prispevek Laharnar, Mlinar v tej publikaciji. g j Koordinate: pribl. 394033, 123876, n. v. 575 m. Raziskave: naključno odkritje okoli l. 2010. Najdba: delček rumene steklene jagode z dvojnimi modro -belimi očesci (t. 1: 15). Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja Objava: Gabrovec 1976, 44–64, sl. 1–3; t. 1–11; Svoljšak 1981, 211–212; Mlinar 2005, 299–312; glej še prispevek A. Kruh v tej publikaciji. Objava: Gabrovec 1976, 44–64, sl. 1–3; t. 1–11; Svoljšak 1981, 211–212; Mlinar 2005, 299–312; glej še prispevek A. Kruh v tej publikaciji. b. Kobarid – Gradič, kultno mesto Lega: na zahodnem pobočju Gradiča je bilo na največji terasi, utrjeni s podpornimi zidovi, odkrito kultno mesto. Območje in arheološke kontekste so močno poškodovali nepooblaščeni posegi z detektorji kovin. Koordinate: 391014 123696 n v 283 m Lega: na zahodnem pobočju Gradiča je bilo na največji terasi, utrjeni s podpornimi zidovi, odkrito kultno mesto. Območje in arheološke kontekste so močno poškodovali nepooblaščeni posegi z detektorji kovin. K di t 391014 123696 283 e. Kobarid – Bizjakova hiša, kultno mesto Lega: ob jugozahodnem vznožju vzpetine Gradič je bilo ugotovljeno kultno mesto na Gregorčičevi ulici 19. Koordinate: 391014, 123696, n. v. 283 m. Raziskave: l. 1982 so bile na jugozahodnem delu terase s poskusnimi sondiranji ZVKDS OE NG odkrite v premešanih plasteh najdbe iz več dob. Izkopavanja l. 1993, 1994, 1996 in 1997 so razkrila drenažna zidova in temeljni vogalni kamen, v stavbni ruševini je bilo precej živalskih kosti in hišne lončenine iz mlajšega halštatskega obdobja, na razširjenem južnem delu terase pa ruševino stavbe s kamnitimi temelji, grajenimi z malto. V njej in njeni bližini je bilo kar nekaj predmetov iz halštatske in latenske dobe ter veliko rimskodobne opeke, lončenega in steklenega posodja grško-italske provenience ter razni votivni predmeti (koroplastika, ploščice, obeski, nakit novci). Koordinate: 390943, 123550, n. v. 240 m. Raziskave: ob gradbenih delih l. 2010 je TMT izvajal ar heološki nadzor, ki je prerasel v izkopavanja. Odkrit je bil kultni zakop s konjskimi skeleti in kostmi drugih živali ter predmeti iz mlajše železne dobe. Najdbe: skupaj s konjskimi kostmi so bili najdeni deli konjske opreme (železne žvale, bronasti razdelilni gumbi), železnih mečev in nožnic ter pasne verige, sulične osti, ščitne grbe, bronaste zapestnice in ena železna, pa tudi železen srp in nož ter na lončarskem vretenu izdelan lonec. Hrani: TMT Hrani: TMT Hrani: TMT Najdbe: iz halštatske dobe so bili odkriti deli nakita. Datacija: zgodnje latensko obdobje (LT B2). Datacija: zgodnje latensko obdobje (LT B2). Objava: Mlinar Gerbec 2011 Hrani: TMT, začasno ZVKDS OE NG. Objava: Mlinar, Gerbec 2011. Objava: Mlinar, Gerbec 2011. Datacija: mlajše halštatsko obdobje (Sv. Ladra – Na Goricah, plano grobišče Lega: ob jugozahodnem vznožju Ladrskega vrha, ki se dviga nad levim bregom Soče in njenim pritokom Ročico, je bilo na travnati rečni terasi Na Goricah (Gorice) na ključno odkrito grobišče ob novozgrajeni poti. Najdbe: na površju vzpetine so pogoste keramične najdbe iz rimske dobe; prazgodovinska lončenina je bila zabe ležena ob notranjem robu vzhodnega nasipa ter v jami, odkriti ob jugozahodnem vznožju. Raziskave: pri korenini strojno izruvanega drevesa je domačin v začetku l. 2021 našel bronasto sulično ost, zatem so na tej lokaciji Primož Kos, Jani Kutin in Nejc Maver aprila 2021 našli v zemlji izruvane korenine še delce bronaste zapestnice in fibule ter lončene črepinje in keramično predilno vretence pa tudi drobce sežganih človeških kosti. Avgusta istega leta je TMT z najditelji opravil pregled lokacije, ob tem so iz korenin pobrano Raziskave: pri korenini strojno izruvanega drevesa je domačin v začetku l. 2021 našel bronasto sulično ost, zatem so na tej lokaciji Primož Kos, Jani Kutin in Nejc Maver aprila 2021 našli v zemlji izruvane korenine še delce bronaste zapestnice in fibule ter lončene črepinje in keramično predilno vretence pa tudi drobce sežganih človeških kosti. Avgusta istega leta je TMT z najditelji opravil pregled lokacije, ob tem so iz korenin pobrano Raziskave: pri korenini strojno izruvanega drevesa je domačin v začetku l. 2021 našel bronasto sulično ost, zatem so na tej lokaciji Primož Kos, Jani Kutin in Nejc Maver aprila 2021 našli v zemlji izruvane korenine še delce bronaste zapestnice in fibule ter lončene črepinje in keramično predilno vretence pa tudi drobce sežganih človeških kosti. Avgusta istega leta je TMT z najditelji opravil pregled lokacije, ob tem so iz korenin pobrano Hrani: TMT. Hrani: TMT. Datacija: halštatska doba, latenska doba, rimska doba (domnevno utrjen poznorepublikanski emporij). Objava: Osmuk 1997; Kos, Žbona Trkman 2009; Fabec, Vinazza 2010; Vinazza 2015; Laharnar, Štular, Mlinar 2015, 244–248, sl. 1–5; Mlinar 2018, 55, sl. 7. Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 429 p Hrani: TMT. Hrani: TMT. d. Kobarid – V Logu (Mlekarna Planika), plano grobišče Lega: grobišče se je razprostiralo ob jugovzhodnem vznožju vzpetine Gradič, na terasi rečnega nanosa na desnem bregu Soče. Koordinate: 391230, 123330, n. v. 225 m. Lega: grobišče se je razprostiralo ob jugovzhodnem vznožju vzpetine Gradič, na terasi rečnega nanosa na desnem bregu Soče. Datacija: kamena doba, mlajše halštatsko obdobje (Sv. Lucija IIb–IIc), srednje/pozno latensko obdobje (LT C1–LT D2), rimska doba, zgodnji srednji vek. g Koordinate: 391230, 123330, n. v. 225 m. Objava: Ciglenečki, Modrijan, Milavec 2011; Božič 2011; Štular 2011a, 407–409, 421–424, sl. 12, 28, 29, 34. Raziskave: l. 1886–1890 in 1892 ter 1903–1904 je Marchesetti izkopal 1100 grobov; ob gradnji mlekarne l. 1953 je bilo uničenih ali nestrokovno izkopanih 10 grobov, l. 1955 je sledilo sondiranje NMS, ki je razkrilo 8 grobov, l. 1979 je GMNG izkopal še 287 grobov. Z zaščitno akcijo l. 2002 je TMT rešil dva grobova, oktobra 2021 je južno od Mlekarne Planika izkopal še en zgodnjehalštatski žgan moški grob, 5 grobov pa je ob arheološkem nadzoru raziskal decembra 2021 in januarja 2022. Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja Lucija IIb/c), zgodnje in pozno latensko obdobje (LT B2 in LT D) ter rimska doba. f. Kobarid – Tonovcov grad (tudi Stari grad), kultno mesto f. Kobarid – Tonovcov grad (tudi Stari grad), kultno mesto Lega: 2 km severno od Kobarida se nad desnim bregom kanjona reke Soče strmo pne hrib, na katerem so ohranjeni naselbinski ostanki in obrambni nasip na zahodnem, severnem in deloma vzhodnem robu platoja na vrhu. Objava: Osmuk 1996; Osmuk 1998; Osmuk 2001; Štular 2011a, 409–411, sl. 13; Nada Osmuk pripravlja s soav toricami monografsko objavo. Koordinate: 390970, 124700, n. v. 410 m. c. Kobarid – Gradič, depo Raziskave: med l. 1993 in 2004 je Iza ZRC SAZU v več kampanjah izvajal arheološke raziskave poznoantičnega cerkvenega kompleksa, vodnega zbiralnika in treh stavb. V ruševinah poznoantične stavbe so bili med drugim odkriti bronasti, železni in stekleni predmeti iz železne dobe, nekaj najdb pa že prej z detektorjem kovin. In terpretirane so kot votivne, kar nakazuje zbir najdb in odsotnost sočasne naselbinske keramike. Lega: natančna lokacija ni znana. Lega: natančna lokacija ni znana. Raziskave: na pobočju vzpetine Gradič je bila naključno odkrita verjetno depojska najdba. Najdbe: omenja se posoda iz debele bronaste pločevine, v njej pa 8 suličnih osti, 4 tulaste, ena plavutasta in ena uhata sekira, poleg tega železna zapestnica, brusni kamen in nekakšno koničasto orodje ali morda sulično kopito (spuntone). Najdbe: iz halštatske dobe so frag. bronaste trortaste fibule in noge certoške fibule X. ali VII. vrste, frag. štule ne govske čelade različice Idrija slovenske vrste, bronasta obročka z izrastki in polovica kroglastega votlega obe ska ter modrozelena steklena jagoda z modro-belimi plastovimi očesci. p Hrani: CMATS. p Hrani: CMATS. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIc). Objava: Marchesetti 1890, XIV; Gabrovec 1976, 50; Guštin, Božič 2021, 491–492. d. Kobarid – V Logu (Mlekarna Planika), plano grobišče g. Kobarid – Za gradom in v reki Soči, posamični najdbi (votivni?) Lega: na desnem bregu reke Soče pribl. 230 m dolvodno od izliva potoka Kozjak je bila ob nizkem vodostaju v rečni strugi najdena železna sulična ost, med kampom Lazar in pobočjem v smeri Tonovcovega gradu pa je bil najden obesek. j Koordinate: 391500, 124420, n. v. 229 m. j j Najdbe: skupaj je bilo doslej odkritih 1413 žganih grobov in sežigališče, večinoma iz halštatske dobe, zgolj nekaj grobov je iz latenske in rimske dobe. Raziskave: naključni odkritji okoli l. 2000; sulično ost je našla Nadja Lazar iz Izole, košaričast obesek pa Edi Lazar iz Kobarida in ga je predal TMT. Hrani: CMATS, TMT, NMS, NHMW. Hrani: CMATS, TMT, NMS, NHMW. Najdba: železna lovorolistna sulična ost z rombičnim presekom in bronast košaričast obesek (t. 1: 13; 3: 3). Datacija: halštatska doba (Sv. Lucija Ia–IIc), latenska doba, rimska doba. 430 Miha MLINAR, Sneža TECCO HVALA 21 Hranita: TMT (obesek) in najditeljica Nadja Lazar, Izola (sulična ost). Robič – Sv. Volar, utrjeno naselje Datacija: obesek sodi v mlajše halštatsko obdobje (Sv. Lucija IIb), sulična ost pa je iz halštatske ali latenske dobe. Lega: v okljuku reke Nadiže pri vasi Robič se na njenem levem bregu dviga hrib s cerkvico sv. Volarja/Hilarija in Tacijana. Okoli vrhnjega platoja, ki se razteza v smeri SZ–JV, so na zahodni in severni strani slabo ohranjeni ostanki obzidja v višini do 1,5 m. Potek obzidja je viden tudi na zahodnem, južnem in tudi delno na vzhodnem robu nekoliko nižjega platoja. Ocenjena površina na selbinskega areala je pribl. 0,7 ha. Objava: Knavs, Mlinar 2007, 84; Štular 2011a, 417, sl. 21; Mlinar 2020b, op. 49; obesek še ni bil objavljen. h. Kobarid – V mevcah, posamična najdba Lega: na vznožju hriba Grmada nad ravnico na levem bregu Soče je bila naključno najdena železna sulična ost. Koordinate: pribl. 391640, 124250, n. v. 280 m. h. Kobarid – V mevcah, posamična najdba Lega: na vznožju hriba Grmada nad ravnico na levem bregu Soče je bila naključno najdena železna sulična ost. g j j j Koordinate: pribl. 391640, 124250, n. v. 280 m. g j p Koordinate: 385085, 123430, n. v. 317 m. Raziskave: odkritje z detektorjem kovin, podatek je bil zabeležen l. 2005. Raziskave: arheoloških raziskav še ni bilo; ob topografskih ogledih je bila na površju najdena prazgodovinska lončenina; posamične kovinske najdbe iz bronaste in železne dobe, pozne antike in zgodnjega srednjega veka pa so izkopali različni iskalci z detektorjem kovin konec 20. 22 Hrani: TMT. Hrani: TMT. Hrani: TMT. Datacija: halštatska ali latenska doba. a. Podbela – Sv. Helena (Na Lupu), delno utrjeno naselje b č l d ž d k j j Lega: ob sotočju Bele in Nadiže se dviga vzpetina s cerkvico sv. Helene na vrhu, ki je izoblikovan v manjši plato in se razteza v smeri SV–JZ. Na severozahodni in jugo vzhodni strani platoja se je v odsekih ohranilo obzidje v višini od 0,1 do 0,3 m in v širini od 1,8 do 3,8 m. V notranjosti so na jugozahodnem delu v konfiguraciji prepoznavni ostanki stavb, severovzhodni del pa je bil poravnan verjetno ob gradnji poznosrednjeveške cerkve ali pozneje. Ocenjena površina naselbinskega areala je pribl. 0,8 ha. Objava: Tratnik 2009, 70–71 (tam navedena lokacija in mesto hrambe sta napačna); Mlinar, Gerbec 2011, 17. 19 Magozd – Jajnkovec, posamična najdba Magozd Jajnkovec, posamična najdba Lega: na majhni ravnici vzdolž levega brega reke Soče je bila ob vznožju hriba Morizna naključno odkrita bronasta fibula. Koordinate: pribl. 391310, 125240, n. v. 269 m. Raziskave: odkritje z detektorjem kovin okoli l. 2005. Koordinate: 380850, 123390, n. v. 363 m. Najdbe: bronasta samostrelna fibula z naprej gledajočo konjsko glavico na zaključku noge (t. 2: 1). Raziskave: arheoloških raziskav še ni bilo; l. 2010 so bili ob topografskih ogledih TMT najdeni opeka, fragmenti lončenine in stekla iz rimske dobe. Ob popotresni obnovi cerkve po l. 1976 je Toni Kramar iz Podbele med izkopom kanala naletel na koščke rimskodobne lončenine in domnevno železnodobni glinast svitek. Posamične kovinske najdbe s tega območja so bile od krite z detektorjem kovin v 90. letih prejšnjega stoletja. Hrani: Kobariški muzej. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb). Š Objava: Mlinar, Gerbec 2011, 17, sl. 5; Štular 2011a, 417, sl. 20. g. Kobarid – Za gradom in v reki Soči, posamični najdbi (votivni?) in v začetku 21. stoletja. Najdbe: železna sulična ost (t. 3: 4). Hrani: TMT. Hrani: TMT. Datacija: halštatska ali latenska doba. Objava: L. Lavrenčič, Topografski zapisnik 2005, arhiv Iza ZRC SAZU; Štular 2011a, 418. Najdbe: iz halštatske dobe sta bronast gumb z zanko in prstan, okrašen z vtisnjenim krožcem in piko ter vrezi ob robovih. i. Kobarid – Mlinsko, reka Soča, posamična najdba (votivna?) i. Kobarid – Mlinsko, reka Soča, posamična najdba (votivna?) Lega: v produ Soče sta bili v bližini današnjega bazena čistilne i. Kobarid Mlinsko, reka Soča, posamična najdba (votivna?) Lega: v produ Soče sta bili v bližini današnjega bazena čistilne naprave pri vasi Mlinsko najdeni železni sulični osti. Koordinate: pribl. 391334, 123463, n. v. 194 m. Hrani: TMT (gumb) in I. Lavrenčič, Potoki (prstan) Lega: v produ Soče sta bili v bližini današnjega bazena čistilne naprave pri vasi Mlinsko najdeni železni sulični osti. Datacija: srednja/mlajša bronasta doba, mlajše halštatsko obdobje (Sv. Lucija IIb), pozna antika, zgodnji srednji vek. naprave pri vasi Mlinsko najdeni železni sulični osti. Koordinate: pribl. 391334, 123463, n. v. 194 m. j Koordinate: pribl. 391334, 123463, n. v. 194 m. Raziskave: naključno odkritje l. 1990. Objava: Osmuk 1997, 9; Bratina 2001, 110; Knavs, Mlinar 2006; Štular 2011a, 401–402, sl. 6; Mlinar, Gerbec, Lahar nar 2014, 13, kat. št. 10 in 22; Mlinar 2018, 53–54, sl. 6. Najdbe: ena sulična ost ima daljši tulec in krajši list z močnejšim sredinskim rebrom, od druge je ohranjen list s trikotnim rebrom (t. 3: 1,2). 22 1 Za posredovane podatke se zahvaljujeva Barbari Nadbath, vodji CPA ZVKDS. 20 20 Trnovo ob Soči – Trnovšček, posamična najdba Lega: ob strmem levem bregu Soče in izteku južnega pobočja Velikega vrha je bila na rečni terasi naključno odkrita bronasta fibula. Koordinate: 389775, 127127, n. v. 288 m. Raziskave: odkritje z detektorjem kovin v začetku 21. stoletja. Najdba: bronasta certoška fibula morda VIII. vrste (?) (t. 2: 13). Hrani: najditelj Primož Kos, Prapetno. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIc). Objava: - Trnovo ob Soči – Trnovšček, posamična najdba Lega: ob strmem levem bregu Soče in izteku južnega pobočja Velikega vrha je bila na rečni terasi naključno odkrita bronasta fibula. Koordinate: 389775, 127127, n. v. 288 m. Raziskave: odkritje z detektorjem kovin v začetku 21. stoletja. Najdba: bronasta certoška fibula morda VIII. vrste (?) (t. 2: 13). Hrani: najditelj Primož Kos, Prapetno. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIc). Objava: - Trnovo ob Soči – Trnovšček, posamična najdba Lega: ob strmem levem bregu Soče in izteku južnega pobočja Velikega vrha je bila na rečni terasi naključno odkrita bronasta fibula. Najdbe: iz halštatske dobe so bronasta certoška fibula XII. vrste, dva narebrena gumba certoške fibule, polovica kroglastega votlega obeska z zanko, obroček z izrastki, bronasti ingoti. Koordinate: 389775, 127127, n. v. 288 m. Raziskave: odkritje z detektorjem kovin v začetku 21. stoletja. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb–IIc), rimska doba, pozna antika. Najdba: bronasta certoška fibula morda VIII. vrste (?) (t. 2: 13). Objava: Ciglenečki, Modrijan, Milavec 2011, 33–52; Štu lar 2011a, 399, sl. 4; Gerbec, Mlinar 2013–2014, 393; Mlinar, Gerbec, Laharnar 2014, 16–17, kat. št. 11–20; Mlinar 2018, 53. 431 Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 25 j g Koordinate: 380377, 123257, n. v. 377 m. , , Raziskave: po odkritju z detektorjem kovin je l. 2021 ekipa Centra za preventivno arheologijo ZVKDS pod vodstvom T. Fabca in T. Nanut v sodelovanju s TMT in NMS opravila preverbo z arheološkimi testnimi jamami. Najdbe: žgani grobovi in posamične najdbe iz mlajšega halštatskega in poznega latenskega obdobja. Hrani: NMS TMT Raziskave: po odkritju z detektorjem kovin je l. 2021 ekipa Centra za preventivno arheologijo ZVKDS pod vodstvom T. Fabca in T. Nanut v sodelovanju s TMT in NMS opravila preverbo z arheološkimi testnimi jamami. a. Srpenica – Ograjenca, plano grobišče Lega: na ravnici ob vzhodnem vznožju Zaglave na desni strani okljukov reke Soče jugovzhodno od vasi Srpenica so bili ob regionalni cesti Kobarid–Bovec odkriti grobovi. g g Koordinate: 385500, 128120, n. v. 372 m. p p j Najdbe: žgani grobovi in posamične najdbe iz mlajšega halštatskega in poznega latenskega obdobja. Raziskave: po naključnem odkritju bronaste situle l. 2003 je TMT opravil strokovni izkop groba, l. 2008 sta bila ob arheološkem nadzoru TMT pri gradbenih delih za vodovod odkrita še dva grobova. g g g j Hrani: NMS, TMT. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIc), pozno latensko obdobje (LT D). g Najdbe: 3 žgani grobovi s pridatki. Objava: T. Fabec, T. Nanut, B. Laharnar, B. Kramberger, T. Leskovar, T. Tolar, K. Kavkler, E. Menart, Poročilo o izvedenih arheoloških raziskavah v Podbeli, 2021 (hrani ZVKDS CPA); objava je v pripravi. Najdbe: 3 žgani grobovi s pridatki. Hrani: TMT. Hrani: TMT. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIa–IIc). Datacija: mlajše halštatsko obdobje (Sv. Lucija IIa–IIc). Objava: Mlinar 2009–2010, 137–153. Objava: Mlinar 2009–2010, 137–153. 23 b. Srpenica – Lanišča, grob b. Srpenica – Lanišča, grob Homec – Na Mlakah, posamične najdbe Lega: na isti ravnici, kjer so bili na ledini Ograjenca l. 2003 in 2008 odkriti žgani grobovi, je bil pribl. 300 m severno od njih, onkraj hudourniškega potoka, najden l. 2016 še en grob. Koordinate: 385350, 128255, n v 375 m Lega: na južnem pobočju Kobariškega stola med manjšima pritokoma Bele je severno od vasi Homec in vzhodno od Sedla viden okop, ki poteka v smeri V–Z v dolžini 108 m; širok je 2,5 m in 1,5 m visok, pod njim so terase, kjer so bile najdene keramične in kovinske najdbe iz več dob. d Raziskave: naključnemu odkritju z detektorjem kovin je l. 2016 sledila arheološka preverba lokacije, ki jo je izvedel TMT. Koordinate: 380874, 124698, n. v. 558 m. Koordinate: 380874, 124698, n. v. 558 m. Raziskave: arheoloških raziskav še ni bilo; najdena opeka in lončenina kažeta na naselbino v rimski dobi, a je bila lahko obljudena že v železni dobi. Posamične kovinske najdbe so bile odkrite z detektorjem kovin v 90. letih prejšnjega stoletja. Najdbe: 1 žgan grob s pridanim orožjem. Hrani: TMT Najdbe: 1 žgan grob s pridanim orožjem. Najdbe: 1 žgan grob s pridanim orožjem. Hrani: TMT. Datacija: zgodnje latensko obdobje (LT B2) Datacija: zgodnje latensko obdobje (LT B2). Objava: Laharnar, Mlinar 2019. Objava: Laharnar, Mlinar 2019. Najdbe: iz halštatske dobe je bronast košaričast obesek s koničnim dnom, zapolnjen s svincem (t. 1: 12). Hrani: TMT 26 p j Hrani: TMT. a. Bovec – Ravelnik (Rabelnik, Rabeljk, Rabelk), delno utrjeno naselje Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb), latenska doba, rimska doba, pozna antika. Lega: vzhodno od Bovca se nad ravnico dviga vzpetina razpotegnjene oblike v smeri SV–JZ, ki ima dva vrha ločena s sedlom. Njena vzhodna pobočja strmo padajo v rečno strugo Koritnice, ki se nedaleč stran steka v reko Sočo. Severni vrh z imenom Rabelnik je oblikovan v plato, ki ga obkrožajo terase z mestoma ohranjenimi ostanki kamnitega nasipa. Ocenjena površina nasel binskega areala je pribl. 3 ha (sl. 6). Objava: Osmuk 1997, 11; Štular 2011a, 398, sl. 3; Mlinar, Gerbec, Laharnar 2014, 18–19, kat. št. 21. Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja b. Podbela – Berjač, plano grobišče1 Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb), rimska doba, zgodnji srednji vek. Lega: okoli 500 m zahodno od naselja pri sv. Heleni (Na Lupu) na levem bregu Nadiže je bilo na bolj ravnem delu pobočja nad staro potjo, ki vodi iz Podbele proti Sedlu, naključno odkrito grobišče. Objava: Osmuk 1985, 297; Štular 2011a, 398–399, sl. 2; Mlinar, Gerbec, Laharnar 2014, 20–21, kat. št. 23 in 24. Objava: Osmuk 1985, 297; Štular 2011a, 398–399, sl. 2; Mlinar, Gerbec, Laharnar 2014, 20–21, kat. št. 23 in 24. 25 2 Za podatek se zahvaljujeva Jani Horvat (Iza ZRC SAZU). 27 Ribčev Laz – cerkev sv. Janeza Krstnika, kultno mesto d šč l ž h d b ž h k Lega: najdišče leži na vzhodnem obrežju Bohinjskega jezera na pomolu med jezerskim izlivom v Savo Bohinjko in njenim levim pritokom Mostnico. 24 Sedlo – Pod cerkvijo, posamične najdbe, grobišče (?) Lega: na južnem pobočju Kobariškega stola, ki se spušča v rečna kanjona Bele in Nadiže, so severovzhodno od vasi Sedlo vidne poljedelske terase; ob južnem robu najširše terase poteka v dolžini 234 m okop, širok 12 m in visok 2 m. g j Koordinate: 390275, 133500, n. v. 519 m. Raziskave: arheoloških raziskav še ni bilo; na jugovzho dnem vznožju in južnem pobočju so bile z detektorjem kovin odkrite predvsem najdbe iz rimske dobe (med njimi bronasta kipca Minerve in Herkula ter deli še treh kipcev), ki kažejo, da je bilo v tistem času ob vznožju hriba tudi svetišče. Iz starejše železne dobe je znan le košček bronaste fibule. Koordinate: 380465, 124750, n. v. 547 m. Koordinate: 380465, 124750, n. v. 547 m. Raziskave: sistematičnih arheoloških raziskav še ni bilo. Po ustnem izročilu naj bi okoli l. 1900 tedanji lastnik zemljišča izkopal večje plano žgano grobišče. Na različnih mestih v bližini cerkve in ob poti so bile z detektorjem kovin v 90. letih prejšnjega stoletja odkrite posamične najdbe iz več dob. Najdbe: bronasta dolgonožna fibula z nogo J-preseka s pestičastim zaključkom, okrašeno s snopi vrezov (t. 2: 5). Hrani: začasno Iza ZRC SAZU. Najdbe: iz halštatske dobe sta bronast obesek in prstan, oba okrašena z vtisnjenimi krožci s piko (t. 1: 10,14). Hrani: zbirka Mazora, Breginj. Najdbe: iz halštatske dobe sta bronast obesek in prstan, oba okrašena z vtisnjenimi krožci s piko (t. 1: 10,14). Hrani: zbirka Mazora, Breginj. Datacija: fibula sodi v mlajše halštatsko obdobje (Sv. Lucija IIa), druge najdbe so iz rimske dobe. oba okrašena z vtisnjenimi krožci s piko (t. 1: 10,14). Hrani: zbirka Mazora, Breginj. Objava: Vuga 1970; Svoljšak 2002; Klavora 2003; Osmuk 2006; Mlinar 2009; Horvat 2018; Mlinar 2018, 53, sl. 5. 432 Miha MLINAR, Sneža TECCO HVALA Hrani: začasno Iza ZRC SAZU.i Hrani: začasno Iza ZRC SAZU.i Hrani: začasno Iza ZRC SAZU.i b. Bovec – Na Raduljah (Dvor), grob Datacija: fibula sodi v mlajše halštatsko obdobje (Sv. Lucija IIa). Objava: - Lega: v oddaljenosti pribl. 3 km zračne črte zahodno od prazgodovinskega naselja na Rabelniku pri Bovcu je bil na terasi nad potokom Gereš pri Dvoru odkrit grob. 30 Koordinate: 387550, 132950, n. v. 450 m. Stara Fužina – Veliki Vegl, posamična najdba Lega: na vzhodnem obrežju Bohinjskega jezera je bila najdena bronasta fibula. Koordinate: pribl. 414135, 127075, n. v. 527 m. Raziskave: naključno odkritje. Najdba: bronasta certoška fibula XIII. vrste (t. 2: 3). Hrani: najditelj Srečo Mikl, Bohinjska Češnjica. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb). Objava: Mertelj 1994–1995. Raziskave: konec 50. let prejšnjega stoletja je TMT izkopal žgan grob z lončeno posodico kot edinim pridatkom. L. 1983 pri terenskem ogledu ZVKD OE NG v nenad zorovanih strojnih izkopih na tem območju niso bile ugotovljene kulturne plasti oz. sledi. Stara Fužina – Veliki Vegl, posamična najdba Lega: na vzhodnem obrežju Bohinjskega jezera je bila najdena bronasta fibula. Koordinate: pribl. 414135, 127075, n. v. 527 m. Raziskave: naključno odkritje. Najdba: bronasta certoška fibula XIII. vrste (t. 2: 3). Najdbe: 1 žgan grob s pridano lončeno posodo. Najdbe: 1 žgan grob s pridano lončeno posodo. Hrani: neohranjeno. Hrani: neohranjeno. Hrani: najditelj Srečo Mikl, Bohinjska Češnjica. Datacija: halštatska doba? Datacija: halštatska doba? Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb). Objava: Mertelj 1994–1995. Objava: Mozetič 1958–1959, 289; Vuga 1974, 96; Osmuk 1985, 210; Svoljšak 2002. 32 Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb). Objava: - Lega: na levem bregu Save Bohinjske, na jugovzhodnem vznožju Rudnice, je bil v vasi Brod pri kopanju vrta odkrit žgan grob (točna lokacija ni znana). Log pod Mangartom, posamična najdba Lega: ob vznožju Kolovrata je bila na ravnici na desni strani Loške Koritnice med njenima pritokoma Predelico in Jereko ob zemeljskih delih odkrita bronasta zapestnica. Lega: ob vznožju Kolovrata je bila na ravnici na desni strani Loške Koritnice med njenima pritokoma Predelico in Jereko ob zemeljskih delih odkrita bronasta zapestnica. Koordinate: pribl. 392402, 141485, n. v. 393 m. R i k klj č dk i j l 2007 Koordinate: 414600, 126645, n. v. 540 m. j p Koordinate: pribl. 392402, 141485, n. v. 393 m. Raziskave: z zaščitnim arheološkim izkopavanjem pod vodstvom D. Josipoviča (podjetje Hemingway) l. 1999 ob cerkvi sv. Janeza Krstnika so bili odkriti ostanki kamnitih suhozidnih temeljev štirioglatega objekta 3 × 4 m, ki je bil odprt proti jezerskemu izlivu na jugozahodni strani. V notranjosti objekta je bila pribl. 0,5 m globoka jama nepravilne oblike, zapolnjena z žganinsko plastjo, ta se je širila tudi severno in južno od objekta. Ugotovljeni sta bili tudi dve manjši okrogli jami (za stojki) – ena ob vzhodnem vogalu, druga pred domnevnim vhodom na jugozahodni strani. V žganinskih plasti so bile najdbe iz mlajšega halštatskega in poznolatenskega obdobja, a največ iz rimske dobe (deli bronastega nakita, železen paličast predmet in nož, številni novci, drobci keramike in kosti). Raziskave: naključno odkritje l. 2007. Najdba: bronasta zapestnica polkrožnega preseka, s prese gajočima koncema in okrašena s snopi prečnih vrezov med rebri (t. 1: 11). Hrani: TMT. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIa). Objava: Mlinar 2008, 142. Objava: Mlinar 2008, 142. Objava: Mlinar 2008, 142. Trenta – V plazeh, sezonska postojanka2 Trenta – V plazeh, sezonska postojanka2 Koordinate: pribl. 417760, 126324, n. v. 512 m. Lega: na planini na sedlu grebena, ki se spušča s Čistega vrha v smeri SV–JZ v globeli Suhega potoka s pritoki, so bili odkriti naselbinski ostanki. Raziskave: naključno odkritje l. 1961, najditelj N. Repinc je pridatke zbral in jih predal muzeju na Jesenicah. Raziskave: naključno odkritje l. 1961, najditelj N. Repinc je pridatke zbral in jih predal muzeju na Jesenicah. Koordinate: 402902, 134850, n. v. 1542 m. Najdbe: odlomki bronaste ovratnice in ogrlica iz rumenih steklenih jagod. Raziskave: l. 2021 je bila okolica grbine pregledana z manj šimi sondami (Iza ZRC SAZU z zunanjimi sodelavci). Najdba: ugotovljena je bila stavba s kamnitimi temelji, južno od nje pa kotanja, zapolnjena z žganino, ta je prekrivala tudi južni temelj stavbe. V kotanji je bilo nekaj drobcev prazgodovinske keramike in košček bronaste trakaste fibule (t. 2: 6). Raziskave: l. 2021 je bila okolica grbine pregledana z manj šimi sondami (Iza ZRC SAZU z zunanjimi sodelavci). Hrani: Gornjesavski muzej Jesenice. Hrani: Gornjesavski muzej Jesenice. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIa). Najdba: ugotovljena je bila stavba s kamnitimi temelji, južno od nje pa kotanja, zapolnjena z žganino, ta je prekrivala tudi južni temelj stavbe. V kotanji je bilo nekaj drobcev prazgodovinske keramike in košček bronaste trakaste fibule (t. 2: 6). Objava: Valič 1960–1961, 228. Trenta – Pod Razorci (nad planino Zapotok), posamična najdba Trenta – Pod Razorci (nad planino Zapotok), posamična najdba Lega: na osrednjem grebenu med rekama Loško Koritnico in Sočo v dolini Trente, je bila v globeli med Nizkim vrhom, Grivo in Skutnikom naključno najdena bronasta fibula. Sočo v dolini Trente, je bila v globeli med Nizkim vrhom, Grivo in Skutnikom naključno najdena bronasta fibula. Koordinate: 398386, 139361, n. v. 1687 m. Najdba: iz halštatske dobe so polovica bronastega krogla stega votlega obeska in bronast trikoten obesek iz dvojne pločevine ter drobci prostoročno izdelane lončenine s primesmi peska. j j Koordinate: 398386, 139361, n. v. 1687 m. j j Koordinate: 398386, 139361, n. v. 1687 m. Raziskave: l. 2008 je Janez Bizjak našel keramične najdbe, sledil je pregled okolice l. 2020 z manjšimi sondami (Iza ZRC SAZU z zunanjimi sodelavci), jeseni 2021 je na tem območju Primož Kos z detektorjem kovin odkril bronasto fibulo. Hrani: GMKR. Hrani: GMKR. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIa/b), pozno latensko obdobje (LT D), rimska doba. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIa/b), pozno latensko obdobje (LT D), rimska doba. Najdbe: bronasta certoška fibula XIII. vrste (t. 2: 4). Hrani: TMT. Objava: Josipovič, Gaspari, Miškec 2012. 32 Brod, grob Objava: Gabrovec 1966b, 243–248, 252–254, sl. 1 in 2; t. 2: 13–23; 3–5; Ogrin 2003, 9–11. 33 a. Jereka – Dunaj, neutrjeno naselje Lega: na koncu severovzhodnega grebena hriba Ščavnica, med Savo Bohinjko in potokoma Jereka in Suhi potok, 2 Za podatek se zahvaljujeva Jani Horvat (Iza ZRC SAZU). Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 433 pribl. 600 m zračne črte proti severovzhodu. Umetna preoblikovanja na teh dveh platojih niso vidna. pribl. 600 m zračne črte proti severovzhodu. Umetna preoblikovanja na teh dveh platojih niso vidna. so bili odkriti sledovi poselitve iz več dob. Naselje ni bilo utrjeno, ocenjena površina domnevnega naselbinskega areala pa je pribl. 2 ha. Koordinate: 420865, 127030, n. v. 554 m. p j p Koordinate: 419980, 128225, n. v. 655 m. Raziskave: na Spodnjem Gradišču je l. 1939 izkopaval W. Schmid in odkril stavbne ostanke. Raziskave: l. 1888/89 so na južnem delu zgornjega platoja potekala izkopavanja pod okriljem Windischgrätza, l. 1937 je Schmid pod najvišjim platojem izkopal stavbne ostanke z velikim ognjiščem iz zloženih lomljencev, omenja tudi pet topilniških jam. L. 1957 je NMS opravil topografski pregled, l. 1959 pa je GMKR izkopal 18 sond po celotnem domnevnem naselbinskem območju. Najdbe: v ruševinah stavbe z zloženimi vogalnimi kamni in ilovnatimi tlemi ter ognjiščem je bil najden hišni omet in lončenina, domnevno iz halštatske dobe. Hrani: neohranjene. Hrani: neohranjene. Datacija: halštatska doba? Objava: Gabrovec 1974, 295. Objava: Gabrovec 1974, 295. Najdbe: na vrhnjem platoju so bili v stavbni ruševini najdeni glinasti svitki in lončenina, certoška fibula V. vrste, bro nast prstan, menda tudi rumena steklena zapestnica in poznolatenska lončenina, kar ni ohranjeno. Na južnem delu so bili odkriti železen prstan, miniaturni železni plugi in črtala, železni žeblji, novci, v sondi na sedlu med Dunajem in Trbežem pa velika količina železne žlindre in železen nož. 36 Lega: na položnem nizkem grebenu jugovzhodno od vzpe tine Dunaj so bili odkriti plani grobovi. Koordinate: 420100, 128090, n. v. 635 m. a. Bohinjska Bistrica – Ajdovski gradec, utrjeno naselje Lega: na desnem bregu Save Bohinjke, blizu izliva njenega pritoka Belca, se dviga vzpetina z dokaj strmimi pobočji in bolj ploskim vrhom, okoli katerega so vidni okopi in terase. Ocenjena površina naselbinskega areala je pribl. 1,5 ha. Raziskave: ob postavljanju daljnovoda je bil l. 1947/48 uničen en grob, dva sta bila odkrita l. 1955 s sondiranjem NMS. Najdbe: 3 žgani grobovi. Koordinate: 420445, 126625, n. v. 580 m. Datacija: mlajše halštatsko obdobje (Sv. Lucija Ic2–IIa). Objava: Gabrovec 1974, 296–298, sl. 3, t. 11: 1–10. Datacija: mlajše halštatsko obdobje (Sv. Lucija Ic2–IIa). Objava: Gabrovec 1974 296 298 sl 3 t 11: 1 10 j j j j Objava: Gabrovec 1974, 296–298, sl. 3, t. 11: 1–10. Raziskave: l. 1849 je izkopaval A. v. Morlot, l. 1936 je W. Schmid raziskal 9 stavb. Te so bile deloma vkopane v skalno osnovo, tla v njih pa umetno poravnana; nekatere stavbe so imele kamnite (suhozidne) temelje in ognjišče s podlago iz kamnov ali pa vglobljeno v naravno skalo. Ob stavbah je bilo odkritih tudi po več jam ovalnih oblik, vkopanih 0,3 m globoko, v njih je bilo veliko žganine, žlindre, kosov železa, žebljev. Med najdbami iz stavb je bilo poleg hišnega ometa, lončenine in svitkov še veliko živalskih kosti in železno orodje ter nekaj malega bronastih predmetov. L. 2003 je GMKR pod vodstvom Mije Ogrin izkopal 7 sond, v njih so bile odkrite stavbne ostaline in sledi metalurških dejavnosti. 35 35 b. Lepence – Na Kremnu, plano grobišče b. Lepence – Na Kremnu, plano grobišče Lega: ob južnem vznožju Spodnjega Gradišča, na levem bregu Save Bohinjke, so bili ob nizki grbini odkriti plani žgani grobovi. p g g Koordinate: 420882, 126890, n. v. 505 m. Raziskave: prvi grobovi so bili izkopani l. 1878, štiri grobove je l. 1906 izkopal Jernej Pečnik, več jih je bilo verjetno uničenih ob gradnji železnice. Hrani: NMS, NHMW in Joanneum Graz. Hrani: NMS, NHMW in Joanneum Graz. Najdbe: grobne celote niso ohranjene, med grobnimi pridatki so svetolucijske, kačaste in certoške fibule, bronasta zapestnica, obročki, diskast in kroglasta votla obeska ter jantarne jagode. Datacija: certoška fibula in bronast prstan sodita v mlajše halštatske obdobje (Sv. Lucija IIb), miniaturni votivni plugi in črtala ter drugo orodje pa v pozno latensko obdobje (LT D) ali rimsko dobo. Objava: Gabrovec 1958–1959; Gabrovec 1966b, 248–249, pril. 1; t. 1; 2: 1–12; Gabrovec 1974, 296, sl. 1; t. 11: 11,12. Hrani: NMS in NHMW. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIa–IIc). Hrani: NMS in NHMW. Objava: Gabrovec 1974, 295–296, sl. 2, t. 9–10. Objava: Gabrovec 1974, 295–296, sl. 2, t. 9–10. b. Jereka – Na sedlu, plano grobišče 34 Bitnje – Krašica, plano grobišče Lega: nasproti Ajdovskega gradca so bili na levem bregu Save Bohinjke ob izlivu njenega pritoka Jereka odkriti plani grobovi na terasi ob skalni grbini. Koordinate: 420350, 126880, n. v. 505 m. Raziskave: l. 1937 je izkopaval W. Schmid. Najdbe: 22 žganih in 3 skeletni grobovi. Med žganimi gro bovi je bil en žarni, dva sta bila brez pridatkov; v enem od skeletnih grobov je bil pridan uhan svetolucijskega tipa, dva pa nista imela pridatkov. Najdbe: stavbe so po načinu gradnje in inventarju iz sta rejše železne dobe, sklenjeno obzidje pa je domnevno iz rimske dobe oz. pozne antike, iz tega časa se omenja tudi nekaj drobnih najdb. Hrani: NMS. Datacija: mlajše halštatsko obdobje (Sv. Lucija Ic2–IIc). Objava: Gabrovec 1974, 287–295, sl. 2, t. 1–8. Datacija: mlajše halštatsko obdobje (Sv. Lucija Ic2–IIc). Objava: Gabrovec 1974, 287–295, sl. 2, t. 1–8. 41 Hrani: neohranjeno. Špeter Slovenov/S. Pietro al Natisone – Sv . Kvirin/S. Quirino (Italija), plano grobišče Datacija: starejše halštatsko obdobje (Sv. Lucija Ic). Objava: Gabrovec 1974, 298. Lega: na prehodu visokogorskega sveta Julijski Alp v Furlansko nižino sta bili na rečni terasi, na levem bregu Nadiže/Natisone, odkriti dve grobišči – eno ob vznožju hriba Roba/Monte Roba, na lokaciji Podvina/ Sottovigna, o katerem je zelo malo znanega, drugo na ravnici vzhodno od Nadiže, na lokaciji Sv. Kvirin/San Quirino ob zahodnem vznožju hriba Barda/Monte Barda. Lončenina (najdena sicer v sekundarni legi na lokaciji Dobje) kaže, da je bilo na hribu Barda morebiti naselje, kovinske najdbe z jugozahodnega pobočja hriba Roba pa nakazujejo, da je bilo na njem v srednjem latenskem obdobju kultno mesto. Objava: Gabrovec 1974, 298. Hrani: NHMW, NMS, GMKR. Hrani: NHMW, NMS, GMKR. a. Lepence – Spodnje Gradišče, neutrjeno naselje (?) Lega: na južnem pobočju, ki se spušča pod strmim skalnim robom v rečno strugo Save Bohinjke, med njenima pritokoma Jereka in Pirašica, sta dva položnejša plato ja, zgornji na n. v. 668 m z imenom Gornje Gradišče, spodnji, 100 m nižje pa Spodnje Gradišče. Slednje je od naselja Ajdovski gradec na nasprotnem bregu oddaljeno Datacija: mlajše halštatsko obdobje (večina keramičnih najdb; bronasta certoška fibula XII. vrste in železna uhata sekira sta iz faze Sv. Lucija IIc), pozno latensko obdobje (LT D), rimska doba ali pozna antika (skle njeno obzidje). Objava: Gabrovec 1966b, 243–248, 252–254, sl. 1 in 2; t. 2: 13–23; 3–5; Ogrin 2003, 9–11. 434 Miha MLINAR, Sneža TECCO HVALA ki jih je razdal. Dva odlomka certoške fibule je Branko Loncner prinesel v GMNG, ta pa je najdbo predal TMT. ki jih je razdal. Dva odlomka certoške fibule je Branko Loncner prinesel v GMNG, ta pa je najdbo predal TMT. b. Bohinjska Bistrica – Osnovna šola, grob Lega: na ravnici ob desnem bregu Save Bohinjske, med njenima pritokoma Bistrico in Belco, je bil ob zaho dnem vznožju Ajdovskega gradca ob gradbenih delih za osnovno šolo najden žgan grob. Najdbe: 1 žgan grob z ohranjenima odlomkoma bronaste certoške fibule (t. 2: 11). j g g Koordinate: 419680, 126230, n. v. 508 m. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIc). Raziskave: naključno odkritje. Objava: Svoljšak 2006, 19; Mlinar, Turk 2016, 9; Mlinar 2018. Najdbe: žgan grob, bronasta svetolucijska fibula s prstanastimi in kroglastimi votlimi obeski. 37 Koordinate: 413190, 113935, n. v. 276 m. Žlan – Groblje, plano grobišče Raziskave: l. 1899 je na tem območju izvedel sondiranje ing. R. Mahnič. Lega: na pobočju manjše vzpetine, ki se dviga na vzhodnem robu ravnice južno od Save Bohinjke, sta bila odkrita dva grobova. Najdbe: en skeletni in 51 žganih grobov, od teh en žarni, 9 jih ni imelo pridatkov. g Koordinate: 417070, 124980, n. v. 571 m. Hrani: NHMW. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIa–IIc), zgodnje latensko obdobje (LT B2), rimska doba. Raziskave: 2 žgana grobova je odkril W. Schmid l. 1938 pod suhozidno stavbno konstrukcijo. zgodnje latensko obdobje (LT B2), rimska doba. Najdbe: v enem so bili poleg žganine ostanki posode, v drugem, pokritem s kamnito ploščo, so bile fibula očalarka, vozlasta ovratnica in frag. lončenine. Objava: Kos 1973, 848–837. Objava: Kos 1973, 848–837. 40 Hrani: GMKR. Hrani: GMKR. Koritnica – Lajišče (Lahovišče), plano grobišče Koritnica – Lajišče (Lahovišče), plano grobišče Lega: v Baški grapi je bilo na rečni terasi na levem bregu Koritnice blizu njenega izliva v Bačo, sredi 19. stoletja odkrito grobišče. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIa). Objava: Valič 1985, 200, sl. 9. Objava: Valič 1985, 200, sl. 9. 38 Vogel – Dolga Planja, sezonska postojanka Lega: najdišče leži v travnati kotanji na Zgornjem Voglu med vrhovoma Orlova glava in Debeli vrh ob smučarski progi. Koordinate: 410952, 123168, n. v. 1680 m. Lončenina (najdena sicer v sekundarni legi na lokaci Raziskave: lokacijo sta odkrila l. 2011 Janez Bizjak in Miran Bremšak, še istega leta je bila opravljena preverba s testnimi sondami, l. 2012 in 2013 pa manjša arheološka izkopavanja pod vodstvom Marije Ogrin (ArheoAlpe) v sodelovanju z odkriteljema in Iza ZRC SAZU. Koordinate: 383150, 109581, n. v. 162 m. Najdbe: odkriti so bili kamniti temelji stavbe iz zgodnjega srednjega veka in kulturna plast z žganino, železovo rudo, živalskimi kostmi in drobci lončenine iz starejše železne dobe, pribl. 6 m južno od stavbe pa je bila v ruši najdena bronasta certoška fibula XI. vrste. V ruši 5,5 m severno od stavbe je bila najdena tudi fibula iz rimske dobe. Raziskave: l. 1818/19 je bilo izkopanih 12 grobov, pozneje je bilo več naključnih odkritij, v l. 1889–1892 je Mar chesetti izkopal 41 grobov za CMATS, l. 1908/09 pa je bilo z raziskavami MAC odkritih 56 grobov. Najdbe: skupaj je zabeleženih 116 žganih grobov, med njimi tudi nekaj žarnih. j j Hrani: MAC in CMATS. j j Hrani: MAC in CMATS. Hrani: GMKR, Muzeji v Bohinju. Hrani: GMKR, Muzeji v Bohinju. Datacija: certoška fibula sodi v mlajše halštatsko obdobje (Sv. Lucija IIc), rimska doba, zgodnji srednji vek. Objava: Ogrin 2020, 63, sl. 3: 2. Datacija: starejše halštatsko obdobje (Sv. Lucija Ia–IIa), srednjelatensko obdobje (LT C). Datacija: certoška fibula sodi v mlajše halštatsko obdobje Datacija: certoška fibula sodi v mlajše halštatsko obdobje (Sv Lucija IIc) rimska doba zgodnji srednji vek Objava: Pettarin 2006a; Pettarin 2006b; Righi 2004, 9–23. Objava: Ogrin 2020, 63, sl. 3: 2. Goljevica – sv. Volbenk, posamični najdbi Datacija: prazgodovina. Datacija: prazgodovina. Objava: Osmuk 1985, 212. Objava: Osmuk 1985, 212. Lega: na hribu s cerkvico sv. Volbenka, ki se dviga na de snem bregu Soče med pritokoma Perivnik in Raztoka, sta bili v vseku kolovoza najdeni železna sekira in kos bronaste pločevine. Rut – V trojah, grob Lega: ob severovzhodnem vznožju hriba Telečnik, ki se dviga nad desnimi pritoki reke Koritnice, je bil ob poti, ki vodi na Rodico, naključno odkrit grob. Koordinate: 414474, 118837, n. v. 741 m. Lega: na ravnici, ki se razteza na levem bregu Nadiže/ Natisone jugovzhodno od Čedada, je bilo odkrito gro bišče tik ob jugozahodnem vznožju vzpetine s cerkvico Madonna delle Grazie, na kateri se domnevata gradišče in kultno mesto. Raziskave: arheoloških raziskav ni bilo. V začetku 70. let 20. stoletja je Viktor Štendler (pr’Pucu) iz Ruta kopal jamo za apno zadaj za zadnjo hišo v vasi (št. 35) na poti proti Rodici. Pri tem je naletel na kamnito ploščo in kup pepela pod njo; v žganini je bilo več bronastih predmetov, Koordinate: 379992, 104177, n. v. 123 m. Koordinate: 379992, 104177, n. v. 123 m. Raziskave: l. 1908/09 je bila nekropola z izkopavanji MAC skoraj v celoti raziskana. 435 Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja Objava: Mihelič, Vinazza 2006, 42. Objava: Mihelič, Vinazza 2006, 42. Najdbe: 287 žganih grobov, med njimi tudi nekaj žarnih. Hrani: MAC. 46 Datacija: mlajše halštatsko obdobje (Sv. Lucija IIa–IIc), zgodnje in srednje latensko obdobje (LT B2–C2). Ajba – Fiščevo, posamični najdbi j p j Lega: na desnem bregu Soče, nasproti vasi Bodrež, na grebenu, ki se dviga med njenima pritokoma pri jezu hidroelektrarne Ajba, sta bili po navedbi najditelja, na ledini Fiščevo odkrita del bronaste fibule in bronast surovec, ki sta ležala 50–100 m narazen. Objava: Pettarin 2006a; Pettarin 2006b. 43 Golo brdo – sv. Marija na Jezeru, utrjeno naselje. Golo brdo – sv. Marija na Jezeru, utrjeno naselje. Lega: na zahodnem robu Goriških brd je na grebenu, ki se spušča z Velikega vrha v zahodni smeri v okljuk reke Idrije, viden kamnit nasip, ki obkroža teraso s cerkvico sv. Marije na Jezeru. Ocenjena površina naselbinskega areala je pribl. 1 ha. Koordinate: pribl. 395222, 107068, n. v. 150 m. Raziskave: naključno odkritje. Raziskave: naključno odkritje. Najdbe: noga bronaste certoške fibule X. vrste (t. 2: 7) in bronast surovec. Koordinate: 384290, 102620, n.v. 189 m. Hrani: najditelj. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIc). Obj G b 2021 137 138 l 9 Raziskave: l. 1976 je bil opravljen topografski ogled ZVKD OE NG; l. 1989 so izkopi za novo cesto razkrili kulturno plast s prazgodovinsko in antično keramiko. V l. 1997/98 je ZVKD OE NG izvedel sondiranja ter l. 2007 izkopavanja, s katerimi so bile ugotovljene plasti in suhozidne konstrukcije iz več dob. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIc Datacija: mlajše halštatsko obdobje (Sv. Lucija IIc). Objava: Gerbec 2021, 137–138, sl. 9. Objava: Gerbec 2021, 137–138, sl. 9. 48 Kanalski Lom – V Glavi (Kamnarjeva glava), delno utr jeno naselje (?) Koordinate: 391237, 104570, n. v. 745 m. Raziskave: arheoloških raziskav še ni bilo; najdbe so bile odkrite z detektorjem kovin. Lega: nad potokom Vogršček se med njegovima pritokoma v osrčju Banjške planote dviga kopasta vzpetina s plato jem na vrhu, ki ga na zahodni in južni strani obdajajo kakovostno grajeni suhi zidovi. Ocenjena površina domnevnega naselja je 0,6 ha. Najdbe: bronasti surovci in ingoti, frag. uhate sekire in srpa, zapestnice in prstani ter frag. lončenine, zvonec. Hrani: najditelj Jože Gabrijelčič, Lig. Koordinate: 403890, 109525, n. v. 580 m. Datacija: pozna bronasta doba (Ha B), iz zgodnje železne dobe so morda del zapestnice in prstani ter frag. lon čenine, zvonec je iz rimske dobe. Raziskave: arheoloških raziskav še ni bilo; na površju so bili ob topografskem ogledu ZVKDS OE NG pobrani drobci prazgodovinske keramike. Objava: Nanut 2018, sl. 4, t. 2–4. Objava: Nanut 2018, sl. 4, t. 2–4. Najdbe: lončenina. Najdbe: lončenina. Najdbe: lončenina. 44 Najdba: bronasta večglava igla (t. 1: 8); fibula je izgubljena. Hrani: TMT (frag. igle). 44 Debenje – sv. Jakob, depo (?), posamične najdbe Datacija: starejše halštatsko obdobje (Sv. Lucija I). Ž Lega: na vrhu grebena s cerkvico sv. Jakoba, ki se spušča v jugovzhodni smeri proti dolini Soče pri Desklah med njenima pritokoma Skalnik in Perivnik, so bile naključno odkrite najdbe iz različnih dob, razpršene na zahodni strani vrha. Objava: Mlinar, Žbona Trkman 2008, 13, kat. št. 5, t. 1: 5. 45 Hrani: ZVKDS OE NG. Hrani: ZVKDS OE NG. Goljevica – sv. Volbenk, posamični najdbi Tolminski Lom – Kal, posamični najdbi Tolminski Lom – Kal, posamični najdbi Lega: na ploskem slemenu jugovzhodnega pobočja Čukle, med dvema pritokoma Vogrščka, ki se spušča proti vasi Tolminski Lom, je bila naključno najdena najprej bronasta fibula, okoli l. 1998 pa še bronasta igla. Najdbe: odkriti so bili stavbni ostanki in obzidje, naselbinska lončenina, novci, kovinski, kamniti in stekleni predmeti. Hrani: začasno ZVKDS OE NG. Koordinate: 403700, 110530, n. v. 680 m. Datacija: pozna bronasta doba, halštatska doba, pozno latensko obdobje (LT D), zgodnje rimsko obdobje, pozna antika. Raziskave: tedanja lastnica zemljišča Ema Winkler je v bližini gospodarskega poslopja s hišno št. 33 našla bronasto fibulo, ki je izgubljena; okoli l. 1998 pa je Danilo Šavli iz Drobočnika, z detektorjem kovin našel odlomek bronaste igle. Objava: Osmuk 1977; Osmuk 1993; Bratina 2001, 28–30; Bratina 2009a, 63–64. 44 Debenje – sv. Jakob, depo (?), posamične najdbe Bodrež – Loga, depo ali kultno mesto (?) Bodrež – Loga, depo ali kultno mesto (?) p Koordinate: 392575, 102895, n. v. 373 m. Koordinate: 392575, 102895, n. v. 373 m. Lega: po primarnih virih (MZK 24, 1898, 111; MZK 27, 1901, 77) je bila na levem bregu Soče pri zaselku Loga ob lokalni cesti naključno odkrita v skalni razpoki (globoki 1 m in široki 0,8 m) žganina, v njej pa lončene črepinje, bronasti in stekleni deli nakita ter železno orodje in orožje. Pri nadaljnjem kopanju v okolici niso naleteli na nobene sledi žganine prav tako ne na arheološke predmete. Lega: po primarnih virih (MZK 24, 1898, 111; MZK 27, 1901, 77) je bila na levem bregu Soče pri zaselku Loga ob lokalni cesti naključno odkrita v skalni razpoki (globoki 1 m in široki 0,8 m) žganina, v njej pa lončene črepinje, bronasti in stekleni deli nakita ter železno orodje in orožje. Pri nadaljnjem kopanju v okolici niso naleteli na nobene sledi žganine prav tako ne na arheološke predmete. Raziskave: l. 2005 sta bila pri topografskem ogledu na sedlu malo nad kapelico, v profilu vseka kolovoza odkrita oglje in kos bronaste pločevine, na površini kolovoza pa železna sekira v sekundarni legi. Najdba: železna uhata sekira (t. 3: 6), bronasta pločevina. Hrani: GMNG. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIc). Raziskave: l. 2005 sta bila pri topografskem ogledu na sedlu malo nad kapelico, v profilu vseka kolovoza odkrita oglje in kos bronaste pločevine, na površini kolovoza pa železna sekira v sekundarni legi. Najdba: železna uhata sekira (t. 3: 6), bronasta pločevina. Hrani: GMNG. Najdba: železna uhata sekira (t. 3: 6), bronasta pločevina. Hrani: GMNG. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIc). Datacija: mlajše halštatsko obdobje (Sv. Lucija IIc). 436 Miha MLINAR, Sneža TECCO HVALA Raziskave: arheoloških raziskav še ni bilo; pri topografskih ogledih Oddelka za arheologijo Filozofske fakultete Univerze v Ljubljani, l. 2007, je bilo pod Gradiščem na terasi z ledinskim imenom Gomila odkritih nekaj frag. domnevno naselbinske keramike iz pozne bronaste in železne dobe ter kosi žlindre. Koordinate: pribl. 397630, 107275, n. v. 115 m. Raziskave: naključno odkritje konec 19. stoletja. Najdbe so bile l. 1898 s posredovanjem R. Mahniča poslane na Dunaj, do l. 1921 jih je hranil dunajski prirodoslovno -zgodovinski muzej, ko so bile razen dveh predane tržaškemu muzeju. Koordinate: 394300, 101450, n. v. 246 m. Levpa – Grad, neutrjeno naselje Lega: hrib nad sotočjem Soče in Avščka ima vrh oblikovan v plato in se imenuje Grad. Na južni strani platoja je izrazit skalni rob, ki prehaja v ravnico, močno preoblikovano zaradi kmetijske rabe. Ocenjena površina domnevno naselbinskega areala je pribl. 0,9 ha. Koordinate: 400140, 106590, n. v. 574 m. Najdbe: bronasta igla z uvito glavico, lončenina, vijčki, kamniti odbitki, del žrmelj, živalske kosti. Hrani: začasno ZVKDS OE NG. Raziskave: l. 1989 so bili s sondiranjem ZVKDS OE NG na vrhnjem platoju odkriti kulturna plast in ostanki stavbe s suhimi zidovi, v njenem zasutju pa lončenina iz rimske dobe. Sondi na južnem delu nista potrdili domnevnega obzidja, so pa bile v premešanih plasteh najdene rimskodobna in prazgodovinska lončenina ter bronasta fibula in zapestnica. Pozneje so bili na pobočjih naključno odkriti še drugi bronasti predmeti iz več dob (zapestnica, del srpa in fibule, kosi pločevine, 2 novca). Datacija: pozna bronasta doba, halštatska doba, rimska doba. Objava: Osmuk 1979, 273; Bratina 2006, 47–49; Bratina 2009b; Gerbec 2018, 70, sl. 12; glej še prispevek P. Bratina v tej publikaciji. Hrani: NHMW in CMATS. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb/c), latenska doba. Koordinate: 397260, 96450, n. v. 351 m. Raziskave: l. 2005 je ob gradbenih posegih ZVKDS OE NG izvedel zaščitna izkopavanja. Odkriti so bili stavbni ostanki suhozidne gradnje ter obzidje, široko 1,8 do 2 m in ohranjeno do višine 0,8 m, ki je imelo zunanjo in notranjo fronto zloženo iz večjih kamnov lomljencev, polnilo pa iz nametanih kamnov. K obzidju so bili dodani prečno na pobočje grajeni podporni zidovi, odkrita sta bila tudi jarek in prazgodovinska kulturna plast/nasutje, ki je radiokarbonsko datirana v starejšo železno dobo (8.–5. st. pr. n. št.). Objava: Guštin 1991, 11–12, sl. 2 (lokacija v objavi je glede na primarne vire napačna), t. 38: 1,2; 39: 8–13,15; 40: 1,6–9,11–14,17,22–26. 50 Bodrež – Loga, depo ali kultno mesto (?) j Najdbe: interpretirane so bile kot grobni pridatki, pa tudi kot velik depo (MZK 27, 1901, 77), a bi lahko šlo za kultno mesto glede na žganino in velik časovni raz pon, ki ga izkazujejo najdbe (od Sv. Lucija IIa do LT D). Med njimi v halštatsko dobo sodi nekaj bronastih fibul (samostrelna trortasta in certoške vrste Ia, VIId,f), prstani z vtisnjenimi krožci in pikami ter snopi vrezov, trakasta spiralna zapestnica, obroček z izrastki, trikotni in košaričasti obeski, morda tudi železna sekira z dvo stranskimi plavutmi, sulična ost in deli bronastih posod. Najdbe: lončenina, žlindra. Najdbe: lončenina, žlindra. Hrani: GMNG. Hrani: GMNG. Datacija: pozna bronasta doba, železna doba. Objava: Svoljšak 1967, 82; Vinazza 2009, 48; Gerbec 2018, 69. Grgar – Grašišče, delno utrjeno naselje Grgar – Grašišče, delno utrjeno naselje Lega: na vzpetini na severozahodnem robu Grgarske kotline, z vodotoki Slatne ob vznožju, je na vzhodnem delu viden izrazit okop, pobočja na jugu, vzhodu in severu so terasirana. Ocenjena površina naselbinskega areala je 1,7 ha. p p Hrani: NHMW in CMATS. 53 Nova Gorica – Sv. Katarina (Kekec), delno utrjeno naselje L tičišč S šk i Vi k d li t Č k G S ( ), j j Lega: na stičišču Soške in Vipavske doline ter Čepovanskega dola se nad Sočo na zahodu in njenima pritokoma na severu in jugovzhodu, dviga vzpetina s kopastim vrhom, ki na zahodni in južni strani prehaja v strma pobočja, na severni strani, kjer je dostop najlažji, pa poteka iz razit okop. V notranjosti so opazne naselbinske terase. Ocenjena površina naselbinskega areala je pribl. 1 ha. Koordinate: 396570, 92560, n v 327 m Najdbe: iz halštatske dobe so bronasta certoška fibula XIII. vrste in odlomki lončenine. Hrani: začasno ZVKDS OE NG 50 Levpa – Grad, neutrjeno naselje 56 Hrani: TMT. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb/c)? Objava: Mlinar et al. 2018, 29, kat. št. 39. Dolenja Trebuša – Sovodenj, posamična najdba Lega: na ravnini severno od grička ob sotočju Trebuščice v Idrijco je bila naključno odkrita železa sekira. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb/c)? Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb/c)? Objava: Mlinar et al. 2018, 29, kat. št. 39. Objava: Mlinar et al. 2018, 29, kat. št. 39. Koordinate: 410596, 106362, n. v. 186 m. Hrani: začasno ZVKDS OE NG. Hrani: začasno ZVKDS OE NG. Datacija: pozna bronasta doba (srp), mlajše halštatsko obdobje – Sv. Lucija IIb (certoška fibula), pozno laten sko obdobje – LT D (lok fibule srednjelatenske sheme), rimska doba (večina keramičnih najdb). Raziskave: l. 1971 in 1976 je GMNG izvedel sondiranja na zahodnem delu obrambnega nasipa. Manjše zaščitne posege ob gradnji so opravili GMNG l. 1984 in l. 1991 ZVKDS OE NG, l. 1999 pa še NMS. Ugotovljenih je bilo več faz utrjevanja. Prazgodovinsko obzidje je ohranjeno do višine pribl. 1 m in je merilo v širino pribl. 3 m. Po najdbah iz plasti, ki so povezane z obzidjem, je bilo to zgrajeno v mlajši/pozni bronasti dobi ter ponovno utr jeno v starejši železni dobi. Na njegovih ruševinah je bil postavljen ožji (pribl. 1,4 m) zid iz lomljencev in vezan z malto, ohranjen do višine 0,5 m, odkrit je bil tudi del stolpa, nekaj najdb pa je iz zgodnjega srednjega veka. Objava: Osmuk 1990, 163–164, sl. 27; Osmuk 1992, 243–244; Gerbec, Vinazza 2018, 86–88, t. 1: 8; Gerbec 2018, 67, sl. 7. 51 Deskle – Gradišče, neutrjeno naselje (?) Lega: na zahodnem robu Banjške planote se jugovzhodno od vasi Deskle, na levem bregu Soče, dviguje hrib Gradišče z ozkim in strmim vrhom. Njegova pobočja se spuščajo v dolino potoka Gomišček na jugu, ki se ob zahodnem vznožju steka v potok Rohot, ta pa v reko Sočo. Na ju gozahodnem pobočju je nekaj teras in sedlo, utrdbenih struktur pa ni opaziti. Ocenjena površina domnevnega naselbinskega areala je pribl. 0,5 ha. Najdbe: iz halštatske dobe so lončenina, omet, koščen držaj, trije medsebojno povezani obročki (dva bronasta in en železen) ter železna paličasta predmeta. 437 Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 55 Objava: Guštin 1991. Objava: Guštin 1991. Objava: Guštin 1991. Gorenja Trebuša – Obenčel, posamična najdba Lega: na severozahodnem bregu hriba Obenčel, med vodotokoma Trebuščica in Jelenk, je bila naključno odkrita bronasta fibula. Koordinate: 412293, 98100, n. v. 880 m. Gorenja Trebuša – Obenčel, posamična najdba Lega: na severozahodnem bregu hriba Obenčel, med vodotokoma Trebuščica in Jelenk, je bila naključno odkrita bronasta fibula. Koordinate: 412293, 98100, n. v. 880 m. Raziskave: odkritje z detektorjem kovin okoli l. 2010. Najdbe: bronasta certoška fibula vrste Xb (t. 2: 12). Hrani: najditelj Primož Kos, Prapetno. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIc). Objava: - 57 Pečine – Vrh gradu, delno utrjeno naselje, kultno mesto Š g j j Lega: na JZ robu Šentviške planote se nad dolino reke Idrijce in grapo njenega hudourniškega pritoka Kosta njevica dviga skalni greben. Na obeh straneh ozkega podolgovatega grebena, usmerjenega SZ–JV, sta vidni terasi in na vzhodnem delu okop. Ocenjena površina naselbinskega areala je pribl. 0,5 ha. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb/c). Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb/c). Objava: - Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja Objava: Marchesetti, 1886, 142, op. 16.; Božič 2011, 265–266, sl. 6.23; Mlinar et al. 2018, 24–26, sl. 3, 19, 20, kat. št. 32–37. Hrani: GMNG. Datacija: mlajša/pozna bronasta doba, halštatska doba, rimska doba, zgodnji srednji vek. Objava: Svoljšak 1990, sl. 2 in 3; Osmuk 1992, 259; Svolj šak 2005. 58 Idrija pri Bači – Na Robu, plano grobišče Idrija pri Bači – Na Robu, plano grobišče 60 Raziskave: odkritje z detektorjem kovin okoli l. 2010, najditelj Martin Levpušček s Seniškega brega. Kneža – Grebljica, posamična najdba Najdba: železna sekira z dvostranskimi plavutmi (t. 3: 10). Hrani: TMT. Lega: ob severnem vznožju Šentviške planote, na levem bregu Bače, nasproti Kneže, je bil v profilu lovske steze naključno najden del bronaste fibule. Datacija: mlajše halštatsko ali pozno latensko obdobje. Objava: - j j Koordinate: pribl. 409603, 113192, n. v. 271 m. Raziskave: naključno odkritje okoli l. 2000, najditelj Primož Lapanja s Slapa ob Idrijci. 61 Koordinate: 408890, 109310, n. v. 588 m. Hrani: NHMW. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb/c), latenska doba (LT B2–LT D), rimska doba. Ponikve – Kračice, posamična najdba Ponikve – Kračice, posamična najdba Lega: na grebenu nad Idrijo pri Bači, ki se spušča v zahodni smeri z Vrha Kračic proti Idrijci in njenima pritokoma, je bila na kolovozu naključno najdena steklena jagoda. Raziskave: odkritje z detektorjem kovin okoli l. 2010. Najdbe: bronasta certoška fibula vrste Xb (t. 2: 12). Hrani: najditelj Primož Kos, Prapetno. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIc). Objava: - Koordinate: 407720, 111400, n. v. 640 m. Raziskave: naključno odkritje l. 2017, najditelj Nejc Maver s Ponikev. Najdba: rumena steklena jagoda z modro-belimi očesci (t. 1: 17). 54 Lega: eponimno grobišče mlajšeželeznodobne idrijske kulturne skupine se je razprostiralo na rečni terasi na desnem bregu Idrijce, blizu njenega pritoka Mlaka na jugovzhodnem robu vasi Idrija pri Bači. Trnovo – Kamni breg, posamična najdba Lega: na zahodnem delu Trnovskega gozda je bila na pobo čju kopaste vzpetine Kamni breg z ozkim grebenom, ki poteka v smeri SZ–JV, naključno najdena železna sekira. Koordinate: 406180, 111030, n. v. 172 m. p j j Koordinate: 404700, 94275, n. v. 916 m. Raziskave: naključnemu odkritju v 70. letih 19. stoletja je sledilo l. 1886/87 izkopavanje J. Szombathyja za dunajski muzej. Raziskave: odkritje z detektorjem kovin l. 1994. Najdbe: železna sekira z dvostranskimi plavutmi (t. 3: 9). Hrani: GMNG. Najdbe: 45 žganih grobov in 2 skeletna, iz starejše železne dobe je 16 grobov. Datacija: halštatska ali latenska doba. Objava: Mlinar, Žbona Trkman 2008, 13–14, t. 1: 6. j Objava: Mlinar, Žbona Trkman 2008, 13–14, t. 1: 6. Hrani: NHMW. Gorski vrh – Jerovca, plano grobišče Gorski vrh – Jerovca, plano grobišče Gorski vrh – Jerovca, plano grobišče Raziskave: arheoloških raziskav še ni bilo; najdbe s tega območja so bile odkrite z detektorjem kovin, njihov značaj pa kaže na kultno mesto. Marchesetti omenja nekje z območja Pečin najdbo trortaste fibule. Lega: grobišče je bilo odkrito v zračni razdalji okoli 2 km severozahodno od Šentviške Gore, v manjši globeli med vzpetinama Dobje (kjer se domneva pripadajoča naselbina) in Vrh ruta, južno od domačij v Jerovci. Koordinate: 411902, 110445, n. v. 790 m. Najdbe: iz halštatske dobe sta bronasta trikotna obeska, polovica kroglastega votlega obeska, del kultne palice in nogi certoških fibul. Raziskave: po naključnem odkritju l. 2007 pri topografskem ogledu s strojem narejenega vseka, so bila izvedena za ščitna izkopavanja poškodovanih grobov v sodelovanju ZVKDS OE NG in TMT. Raziskave: po naključnem odkritju l. 2007 pri topografskem ogledu s strojem narejenega vseka, so bila izvedena za ščitna izkopavanja poškodovanih grobov v sodelovanju ZVKDS OE NG in TMT. 65 Police – Pri cierki (pri cerkvi Marijinega rojstva), naselje (?), plani grobovi (?) Koordinate: 411970, 108683, n. v. 730 m. Raziskave: okoli l. 1840 je bil najden bronast kipec Izide (danes izgubljen); druge kovinske najdbe so bile odkrite z nepooblaščenimi posegi z detektorjem kovin, največ okoli l. 2000, nekaj jih je bilo rešenih ob gozdarskih delih l. 2008. Lega: visoko nad desnim bregom Idrijce leži ravnica z vasjo Police, ki jo omejujejo strma pobočja na zahodu, vzhodu in jugu, kjer se spuščajo proti Idrijci med pritokoma Snoviška grapa in Poličanka. Na jugozahodnem robu Poliškega polja stoji cerkvica Marijinega rojstva, kjer so bile ob zemeljskih delih naključno odkrite naselbinske najdbe, ustno izročilo pa govori o loncih s pepelom. Najdbe: med pribl. 120 posamičnimi najdbami so iz halštatske dobe antropo-ornitomorfni in pločevinast trikotni obesek, trortasta in čolničasta fibula s ptičji ma figuricama, živalska figurica, frag. bronaste situle z votivnim napisom. Tudi značaj najdb iz latenske in rimske dobe govori v prid domnevi o kultnem mestu. Koordinate: 415731, 109414, n. v. 531 m. Raziskave: l. 2003 je Stanko Flego iz Trsta pri gradbenem izkopu ob cerkvi našel v prekopani zemlji predmete iz železne dobe, ki kažejo na naselbino. Različni iskalci z detektorji kovin, med njimi zbiratelj Jože Golja, so našli še nekaj kovinskih predmetov. V ustnem izročilu se je ohranila tudi pripoved o loncih s pepelom, odkritih ob cerkvenem zidu na območju današnjega pokopališča. Hrani: NMS in TMT. Hrani: NMS in TMT. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIa–IIc), latenska doba, rimska doba. Objava: Laharnar, Turk 2017, 167, sl. 164, 189, 191, 193, 196; Mlinar et al. 2018, 17–19, 38–42, sl. 11, kat. št. 28–31, 37; Laharnar 2018b, 367–372, sl. 3: 1,2,5. Najdbe: frag. keramičnega svitka, brusnega kamna in žrmelj, dva bronasta obročka z bradavičastimi izrastki. Najdbe: frag. keramičnega svitka, brusnega kamna in žrmelj, dva bronasta obročka z bradavičastimi izrastki. Hrani: NMS. j Hrani: NMS. 63 Datacija: obročka sodita v mlajše halštatsko obdobje (Sv. Lucija IIa/b). Šentviška Gora – Lipce / Prevala, plano grobišče p , p g Lega: grobišče naj bi se raztezalo na ledini Lipce v močno zakraselem svetu zahodno od ceste med vasema Polje in Šentviška Gora; posamične najdbe so znane tudi vzhodno od ceste na ledini Prevala. Objava: Svoljšak 1984b, 288–289; Flego 2005, 23–34. 66 Koordinate: 412650, 108540, n. v. 605 m. Hrani: TMT in NMS. Datacija: pozna bronasta doba, mlajše halštatsko obdobje (Sv. Lucija IIb/c), srednje/pozno latensko obdobje (LT C/D), rimska doba. Najdbe: 22 žganih grobov, od teh je bil en žarni, 4 niso imeli pridatkov, 4 pa niso bili izkopani. 438 Miha MLINAR, Sneža TECCO HVALA Najdbe: 19 žganih grobov, med pridatki so fibule (trortasti, kačasta, trakasta, certoški X. in XIII. vrste, živalska zgo dnjelatenske sheme), bronast prstan, trakasta in spiralna zapestnica, železna uhata sekira ter modra steklena jagoda z rumeno spiralo, ki je verjetno iz latenske dobe. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIa–IIc). Objava: Laharnar, Mlinar 2013. Objava: Laharnar, Mlinar 2013. Šentviška Gora – Berlotov rob, delno utrjeno naselje (?), kultno mesto Hrani: CMATS. Hrani: CMATS. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIa–IIc), latenska doba (steklena jagoda). Lega: severozahodno od vasi Polje so na kopasti vzpetini, razbrazdani s skalami, škrapljami in brezni, vidne ume tne poravnave in terase z domnevnimi ostanki stavb ter ruševine obzidja. Ocenjena površina domnevnega naselbinskega areala je 0,2 ha. Objava: Mlinar et al. 2018, 12–13, 36–37, kat. št. 13–25; Laharnar 2018b, 372–374. 65 65 Reka – Grad, neutrjeno naselje (?) Lega: na ozkem skalnatem grebenu, ki se strmo spušča v okljuk Idrijce pri vasi Reka, med njenima pritokoma Poličanka in Kozarska, so bile naključno odkrite najdbe iz več dob. Na grebenu je bivalnega prostora zelo malo, na južnem delu so vidne manjše izravnave oziroma terase. Ob njegovem jugovzhodnem vznožju je bilo onkraj potoka Kozarska v 19. stoletju odkrito grobišče iz mlajše železne dobe. Raziskave: okoli l. 1900 je ing. R. Mahnič izkopal 9 žganih grobov, med pridatki naj bi bili lončenina, bronasta situla, fibule in železni ostanki. L. 1962 je TMT pridobil frag. štirih steklenih jagod, ki so bile najdene na njivi na Prevali. Najdbe: iz halštatske dobe je domnevno rumena steklena jagoda z modro-belimi očesci, preostale tri, iz rume nega stekla s spiralnimi modro-belimi očesci, pa so opredeljene v latensko dobo. j Koordinate: 416465, 108915, n. v. 346 m. Raziskave: arheoloških raziskav na samem grebenu še ni bilo; posamične najdbe s tega območja so bile odkrite z detektorjem kovin v 80. in 90. letih 20. stoletja. Hrani: TMT (steklene jagode); najdbe iz l. 1900 odkritih grobov niso ohranjene. Datacija: mlajše halštatsko obdobje? (Sv. Lucija IIb/c?), latenska doba? Najdbe: iz halštatske dobe je trikotni obesek iz dvojne pločevine, morda tudi košček trakaste pločevine, okrašen s tangencialno povezanimi krožci s piko in nizoma pik ob robu. Objave: Mlinar 2006b; Mlinar et al. 2018, 15–17, kat. št. 8–11; Laharnar 2018b, 372–374. 64 Daber – Na Dobcu (Ajdovski britof), plano grobišče 64 Hrani: NMS. Hrani: NMS. 64 Daber – Na Dobcu (Ajdovski britof), plano grobišče Daber – Na Dobcu (Ajdovski britof), plano grobišče Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb/c), pozno latensko obdobje (LT D), rimska doba. Lega: grobišče je bilo odkrito na rahlo dvignjeni terasi ob severnem vznožju razvlečenega grebena Arbišče, med vasema Daber in Zakraj. Objava: Osmuk 1982; Guštin 1991, 25–27; Svoljšak 1994– 1995, t. 4: 23; Božič 1999b, 71–75, sl. 3: 18. j Koordinate: 413755, 108405, n. v. 656 m. Raziskave: l. 1890 je C. Marchesetti izvedel izkopavanja za tržaški muzej. Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 439 Raziskave: arheoloških raziskav še ni bilo; na dveh terasah v osrednjem delu vrha so bile z detektorjem kovin od krite posamične najdbe, med njimi železni surovci, kosi orožja in orodja ter deli oprav pa tudi novci. Raziskave: arheoloških raziskav še ni bilo; na dveh terasah v osrednjem delu vrha so bile z detektorjem kovin od krite posamične najdbe, med njimi železni surovci, kosi orožja in orodja ter deli oprav pa tudi novci. 67 Dolenje Ravne – Gastabil, depo, posamična najdba Dolenje Ravne – Gastabil, depo, posamična najdba j Lega: na vrhu grebena, ki poteka v smeri SV– JZ in se strmo spušča v sotesko Cerknice do sotočja z Idrijco, na zahodu pa v grapo pritoka Idrijce, je bil v bližini kolovoza naključno odkrit prazgodovinski depo razlo mljenih kovinskih predmetov ter v neposredni bližini še fibula iz rimske dobe. Pribl. 320 m proti SV sta bili najdeni živalska fibula zgodnjelatenske sheme in omega fibula iz rimske dobe, okoli 300 m proti JZ pa fibula iz pozne rimske dobe. Najdbe: iz halštatske dobe sta bronasta certoška fibula X. vrste in verjetno tudi železna sulična ost. Najdbe: iz halštatske dobe sta bronasta certoška fibula X. vrste in verjetno tudi železna sulična ost. Hrani: NMS. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIc), pozno latensko obdobje (LT D), rimska doba, pozna antika, zgodnji srednji vek. Objava: Istenič 2015b, 365–377, sl. 5; t. 1: 1,10; 3: 36; Mlinar 2018, 56–57. p Koordinate: 419428, 108381, n. v. 616 m. Raziskave: po nestrokovnem posegu z detektorjem kovin je l. 2007 sledila preverba lokacije, izvedena v sodelovanju ZVKDS OE NG in TMT, ob tem so bili odkriti frag. prazgodovinske lončenine. Koordinate: 425380, 112915, n. v. 758 m. 72 72 Železniki – Štalca, delno utrjeno naselje L d S lšk S d j i i Lega: nad Selško Soro se med njenima pritokoma Češnjico na zahodu in Selnico na vzhodu dviga hrib Štalca, z razpotegnjenim vrhom v smeri SV–JZ. Na njegovem jugovzhodnem pobočju so vidne številne terase, na zahodnem delu pa nasip in jarki. Ocenjena površina naselbinskega areala je pribl. 9 ha. Najdbe: iz halštatske dobe sta živalska fibula zgodnjela tenske sheme in depo bronastih surovcev in ingotov (uhate sekire, paličasti in ploščati ingoti). Datacija: lončenina bi po nekaterih značilnostih lahko bila že iz bronaste dobe, najdbe domnevnega depoja imajo širok časovni razpon (Sv. Lucija Ia–IIa), živalska fibula je iz mlajšega halštatskega obdobja (Sv. Lucija IIc), preostale kovinske najdbe pa iz rimske dobe. Koordinate: 436702, 120983, n. v. 639 m. Raziskave: z arheološkimi testnimi sondami so bile l. 2015 ugotovljene naselbinske in utrdbene strukture ter ostanki metalurške dejavnosti. Objava: Nanut 2018, sl. 2; 3: 1; t. 1. Najdbe: frag. lončenine in svitkov, kamnita utež in brus, kosi železove rude in žlindre, železno sulično kopito. Objava: Nanut 2018, sl. 2; 3: 1; t. 1. 71 71 Žiri – Žirk, utrjeno naselje (?), posamične najdbe h b Ž k d l k Žiri – Žirk, utrjeno naselje (?), posamične najdbe h b Ž k d č l k Lega: na hribu Žirk nad sotočjem Poljanske Sore in Osoj nice pri Žireh so na južnem pobočju kopastega vrha vidne terase in okopi. Na njem so bile naključno odkrite kovinske najdbe iz različnih dob. Ocenjena površina domnevnega naselbinskega areala je pribl. 1 ha. g g j p Koordinate: 421796, 110016, n. v. 450 m. Raziskave: arheoloških raziskav še ni bilo; najdbe s tega območja so bile odkrite v 80. in 90. letih prejšnjega stoletja z detektorjem kovin. Koordinate: 430710, 100405, n. v. 663 m. Najdbe: iz halštatske dobe so bili na spodnji terasi na različnih koncih najdeni trije bronasti trikotni votli obeski, košaričast obesek s koničnim dnom in železna sulična ost ter živalska fibula zgodnjelatenske sheme. Hrani: NMS Raziskave: arheoloških raziskav še ni bilo; posamične najdbe s tega območja je z detektorjem kovin odkril Jože Golja iz Trebenč v 90. letih prejšnjega in začetku 21. stoletja. Najdbe: fibule iz starejše in mlajše železne dobe ter rimske dobe. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb/c), latenska doba (LT B2 in LT D), rimska doba. Hrani: Loški muzej Škofja Loka, NMS, Muzejsko društvo Žiri. Datacija: živalska fibula zgodnjelatenske sheme je iz mlajšega halštatskega obdobja (Sv. Lucija IIc), druge so iz latenske in rimske dobe. Objava: Istenič 2015a, 43–49, sl. 7; t. 1: 1–6; Mlinar 2016, sl. 2; 3: 1; Mlinar 2018, 56. Objava: Mlinar 2018, 57; Nanut 2021, sl. 2: 1. 69 68 Hrani: Loški muzej Škofja Loka. Cerkno – Gradišče, deloma utrjeno naselje, kultno mesto (?) Datacija: mlajše halštatsko obdobje (Sv. Lucija II). 70 70 Železniki – Štalca, delno utrjeno naselje Železniki – Štalca, delno utrjeno naselje Cerkno – Gradišče, deloma utrjeno naselje, kultno mesto (?) Objava: Bogataj et al. 2016; Grahek 2018b, 264–270, sl. 2–4; Mlinar 2018, 58. Lega: vzpetina Gradišče se pne severno nad rečico Cer knico z manjšima pritokoma Zajegrščica in Zapoška ob njenem vzhodnem in zahodnem vznožju in je na vrhu izoblikovana v ozek plato, razpotegnjen v smeri S–J. Na severnem delu, kjer prehaja v sedlo, je viden okop, preostala pobočja so strmejša in terasirana. Površina domnevnega naselbinskega areala je pribl. 0,5 ha. b. Godovič – Jelenšek, plano grobišče Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb/c). Objava: Bratina 1994–1995, 146; Svoljšak 1994–1995, 253; glej še prispevek Bratina, Laharnar, Svoljšak v tej publikaciji. Lega: grobišče se je raztezalo na sedlu severno od gradišča. Koordinate: 430533, 91657, n. v. 795 m. BRATINA, P. 2006, Grgar – Gradišče Grašišče. – Varstvo spomenikov 42, 47–49. ANDRIČ et al. 2020 = M. Andrič, P. Sabatier, W. Rapuc, N. Ogrinc, M. Dolenec, F. Arnaud, U. von Grafenstein, A. 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FORLATI TAMARO, B. 1930, S. Lucia di Tolmino. Nuovi ritrovamenti nella necropoli preistorica. Novaki – Mali Njivč, neutrjeno naselje (?) j j j ( ) Lega: na zahodnem robu hribovja, ki se razteza med rečico Cerkniščico na jugu ter njenima pritokoma Čerinščico na zahodu in Črno na vzhodu, izstopa med Gorenjimi in Dolenjimi Novaki ozek podolgovat vrh Mali njivč, razpotegnjen v smeri S–J, s skalnatimi prepadnimi po bočji. Na samem vrhu je vidnih nekaj manjših izravnav oziroma teras, kjer so bile odkrite kovinske najdbe iz več dob. Ocenjena površina domnevnega naselja je pribl. 0,5 ha. a. Godovič – Jelenšek, utrjeno naselje Lega: na vzhodnem delu ploskega grebena so na vrhu Je lenšek dobro vidne obrambne in naselbinske strukture. Ocenjena površina naselbinskega areala je pribl. 0,8 ha. j p g Koordinate: 430470, 91510, n. v. 817 m. Raziskave: arheoloških raziskav še ni bilo; naselje pa je verjetno sočasno z grobiščem, odkritim v njegovi ne posredni bližini. p Najdbe: - j Hrani: - Koordinate: 425380, 112915, n. v. 758 m. 440 Miha MLINAR, Sneža TECCO HVALA Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb/c)? Raziskave: po naključnih odkritjih l. 1993 z detektorjem kovin so še istega leta sledila arheološka izkopavanja NMS in ZVKDS OE NG. Datacija: mlajše halštatsko obdobje (Sv. Lucija IIb/c)? Raziskave: po naključnih odkritjih l. 1993 z detektorjem kovin so še istega leta sledila arheološka izkopavanja NMS in ZVKDS OE NG. j j j j Objava: Bratina 1994; Mlinar 2018, 57, sl. 8; Laharnar, Turk 2017, sl. 140, 141; glej še prispevek Bratina, Laharnar, Svoljšak v tej publikaciji. Objava: Bratina 1994; Mlinar 2018, 57, sl. 8; Laharnar, Turk 2017, sl. 140, 141; glej še prispevek Bratina, Laharnar, Svoljšak v tej publikaciji. Najdbe: 37 žganih grobov. Hrani: NMS. 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OGRIN, M. 2020, Prve postojanke na Spodnjih bohinjskih gorah – Dolga planja na Voglu. – V: Življenje v Alpah: življenje v Alpah nekoč in danes – od prve obljudenosti visokogorskega sveta pred več tisoč leti do trajnostnega razvoja turizma danes, 59−65, Bohinjska Bistrica, Stara Fužina. RUARO LOSERI, L., E. MONTAGNARI KOKELJ (ur.) 1984, Preistoria del Caput Adriae. Atti del convegno internazionale, Trieste, 19–20 novembre 1983. – Udine. RUPEL, L. 2004, Contributi alla carta archeologica delle Valli del Natisone. – Forum Iulii 28, 55–78. OSMUK, N. 1977, Golo Brdo v Brdih. – Varstvo spome nikov 21, 214. RUTAR, S. 1882, Zgodovina Tolminskega, to je: zgodovinski dogodki sodnijskih okrajev Tolmin, Bolec in Cerkno ž njih prirodoznanskim in statističnim opisom. – Gorica. OSMUK, N. 1979, Grgar. – Varstvo spomenikov 22, 273. SAVNIK, R. (ur.) 1968, Krajevni leksikon Slovenije 1. Za hodni del Slovenije. – Ljubljana. OSMUK, N. 1982, Reka. – Varstvo spomenikov 24, 163–164. OSMUK, N. 1985, Bovec, Kanalski Lom, Sedlo. – Varstvo spomenikov 27, 210–212, 297. SVOLJŠAK, D. 1967, Deskle. – Varstvo spomenikov 12, 82. Š OSMUK, N. 1990, Levpa – Grad. – Varstvo spomenikov 32, 163–164 SVOLJŠAK, D. 1973, Prazgodovinsko grobišče v Tolminu. Prispevek k proučevanju načina pokopa in pogrebnih navad v svetolucijski kulturni skupini / Necropoli preisto rica di Tolmin. – Arheološki vestnik 24 (1975), 397–410. OSMUK, N. 1992, Levpa; Nova Gorica – Sv. Katarina (Kekec). – Varstvo spomenikov 34, 243–244; 259. p OSMUK, N. 1993, Golo Brdo. – Varstvo spomenikov 35, 96. SVOLJŠAK, D. 1981, Kobarid. – Varstvo spomenikov 23, 211–212. OSMUK, N. 1996, Kobarid. – Varstvo spomenikov. Poro čila 37, 46. SVOLJŠAK, D. 1984a, Most na Soči (S. Lucia) e i suoi sistemi di difesa. – V: Preistoria del Caput Adriae. Atti del convegno internazionale, Trieste, 19-20 novembre 1983, 115–118, Udine. OSMUK, N. 1997, Kobarid od prazgodovine do antike. Kratek zgodovinski pregled obdobij in pripadajočih najdišč. – V: Z. Likar, A. Raspet, Ž. Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja – V: L. Ruaro Loseri, E. Mon tagnari Kokelj (ur.), Preistoria del Caput Adriae. Atti del convegno internazionale, Trieste, 19–20 novembre 1983, 91–96, Udine. MLINAR, M. 2021b, Še dve rimski hiši z Mosta na Soči. – Goriški letnik 45, 175–192. MADER, B. 2018, Die Prähistorische Kommission der kaiserlichen Akademie der Wissenschaften 1878–1918. – Mitteilungen der Prähistorischen Kommission 86. MLINAR, M., T. GERBEC 2011, Keltskih konj topòt: najdišče Bizjakova hiša v Kobaridu / Hear the horses of the Celts: The Bizjakova hiša site in Kobarid. Katalog razstave / Exhibition catalogue. – Tolmin. MARCHESETTI, C. 1886, La necropoli di S. Lucia presso Tolmino, scavi del 1884. – Bolletino della Società Adri atica di Scienze Naturali in Trieste 9. g MLINAR, M., B. MUŠIČ 2020, Geofizikalne raziskave v arheologiji: dobre prakse s Tolminskega. – Tolminski zbornik 5, 413–428. MARCHESETTI, C. 1890, Relazione sugli scavi preistorici fatti nel 1889. – Bollettino della Società adriatica di scienze naturali in Trieste 12, XIII–XVII. MLINAR, M., M. TURK 2016, Prapoti skozi praproti: arheološka topografija dolin Tolminke in Zadlaščice. Katalog razstave. – Tolmin. MARCHESETTI, C. 1893, Scavi nella necropoli di S. Lucia presso Tolmino. – Bolletino della Società Adriatica di Scienze Naturali in Trieste 15. MLINAR, M., B. ŽBONA TRKMAN 2008, Banjška planota in Trnovski gozd v luči najnovejših arheoloških najdb. – Goriški letnik 32, 9–22. MARCHESETTI, C. 1903, I castellieri preistorici di Trieste e della regione Giulia. – Trieste. MLINAR, M., T. GERBEC, B. LAHARNAR 2014, Kot nekoč: Breginjski kot v arheoloških dobah. Katalog raz stave. – Tolmin. MARZATICO, F. 1997, I materiali preromani della Valle dell’Adige nel Castello del Buonconsiglio. – Patrimonio storico e Artistico del Trentino I–III, Trento. MLINAR, M., R. KLASINC, M. KNAVS 2008, Zaščitne arheološke raziskave na Mostu na Soči leta 2001: najdišča Maregova guna, Štulčev kuk in Plac / Rescue MERTELJ, M. 1994–1995, Stara Fužina. – Varstvo spo menikov 36, 202. 443 Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja PAVLIN, P. 2014, Starejšeželeznodobni košarasti obeski, okrašeni z vodoravnimi črtami / Early Iron Age basket -shaped pendants with horizontal line decoration. – V: S. Tecco Hvala (ur.), Studia Praehistorica in Honorem Janez Dular, Opera Instituti Archaeologici Sloveniae 30, 341–354 [DOI: https://doi.org/10.3986/9789610503651]. archaeological excavations at Most na Soči in the year 2001: the sites of Maregova guna, Štulčev kuk and Plac. – Arheološki vestnik 59, 189–208. PAVLIN, P. Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja LO SCHIAVO, N. TRAMPUŽ-OREL 1984–1985, Most na Soči (S. Lucia) 2. Szombathyjeva izkopavanja / Die Ausgrabungen von J. Szombathy. – Katalogi in monografije 23. SVOLJŠAK, D. 2018, Posoško železnodobno stavbarstvo / Iron Age architecture in Posočje. – V: Dular, Tecco Hvala (ur.) 2018, 167–194. TOMADIN, V., I. VISENTINI, S. COLUSSA 1989, Castello di Zuccola in Cividale del Friuli. – Premariacco. SVOLJŠAK, D., J. DULAR 2016, Železnodobno naselje Most na Soči. Gradbeni izvidi in najdbe / The Iron Age Settlement at Most na Soči. Settlement Structures and Small Finds. – Opera Instituti Archeologici Sloveniae 33 [DOI: https://doi.org/10.3986/9789612549367]. TOŠKAN, B. 2011, Analiza živalskih kostnih ostankov / Analysis of animal bone remains. – V: M. Mlinar, T. Gerbec 2011, Keltskih konj topòt: najdišče Bizjakova hiša v Kobaridu / Hear the horses of the Celts: The Bizjako va hiša site in Kobarid. Katalog razstave / Exhibition catalogue, 43–50, Tolmin. SVOLJŠAK, D., A. POGAČNIK 2001–2002, Tolmin, pra- zgodovinsko grobišča / Tolmin, the prehistoric cemetery 1–2. – Katalogi in monografije 34–35. TOŠKAN, B., L. BARTOSIEWICZ 2018, Živalski ostanki iz naselbine Most na Soči: vpogled v družbeno komple ksnost železnodobne skupnosti v jugovzhodnoalpskem prostoru / Animal remains from the settlement at Most na Soči: an insight into the social complexity of the Iron Age community in south-eastern Alps. – V: Dular, Tecco Hvala (ur.) 2018, 467–510. ŠMIT, Ž., B. LAHARNAR 2018. Analiza bronastih surovcev iz železnodobne naselbine na Mostu na Soči in grobnih najdb z Mosta na Soči in iz Bohinja / Analysis of raw bronze from the Iron Age settlement Most na Soči and of grave finds from Most na Soči and Bohinj. – V: Dular, Tecco Hvala (ur.) 2018, 321–332. TRATNIK, V. 2009, Idrsko. – Varstvo spomenikov. Poročila 45, 70–71. ŠTULAR, B. 2011a, The use of lidar – derived relief models in archaeological topography. The Kobarid region (Slovenia) case study (Uporaba modelov reliefa pridobljenih z lidarskim snemanjem v arheološki topo grafiji). – Arheološki vestnik 62, 393–432. TURK, P., V. SVETLIČIČ 2018, Nenavadna prazgodovina uhatih sekir (Unusual prehistory of shaft-hole axes). – V: Črešnar, Vinazza (ur.) 2018, 34–38. V: Črešnar, Vinazza (ur.) 2018, 34–38. TURK, P., D. BOŽIČ, J. ISTENIČ 2009, New Pre-Roman inscriptions from Western Slovenia. The archaeological evidence. – V: G. Tiefengraber, B. Kavur, A. Gaspari (ur.), Celtic Studies II, Protohistoire européenne 11, 47–64, Montagnac. ŠTULAR, B. 2011b, Mreža poti / Path network. – V: Ciglenečki, Modrijan, Milavec 2011, 53–64. TECCO HVALA, S. Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja Cimprič (ur.), Kobarid, 9–16, Kobarid. SVOLJŠAK, D. 1984b, Police. – Varstvo spomenikov 26, 288–289. OSMUK, N. 1998, Le sanctuaire protohistorique de Kobarid (Slovénie); Plaquette votive de Kobarid (SI). – Instru mentum 7, 13, 17. SVOLJŠAK, D. 1986, Most na Soči in njegovi obrambni sistemi. – V: Odbrambeni sistemi u praistoriji i antici na tlu Jugoslavije, Materijali 22, 50–54. OSMUK, N. 2001, Kobarid. – Varstvo spomenikov. Poročila 38, 51–52. SVOLJŠAK, D. 1988–1989, Posočje v bronasti dobi (Das Sočagebiet in der Bronzezeit). – Arheološki vestnik 39–40, 367–386. OSMUK, N. 2006, Bovec. – Varstvo spomenikov. Poročila 42, 12–14. 444 Miha MLINAR, Sneža TECCO HVALA SVOLJŠAK, D. 1990, Sv. Katarina nad Novo Gorico. Ar heološka podoba. – Goriški letnik 17, 33–55. TERŽAN, B. (ur.) 1995‒1996, Depojske in posamezne kovinske najdbe bakrene bronaste dobe na Slovenskem / Hoards and individual metal finds from the Eneolithic and Bronze Ages in Slovenia 1‒2, Katalogi in monografije 29‒30. SVOLJŠAK, D. 1994–1995, Reka; Godovič. – Varstvo spomenikov 36, 252–253. Ages in Slovenia 1‒2, Katalogi in monografije 29‒30 SVOLJŠAK, D. 2001, Zametki urbanizma v železnodobni naselbini na Mostu na Soči. –Arheološki vestnik 52, 131–138. TERŽAN, B. 2002, Kronološki oris / Chronological outline. – V: D. Svoljšak, A. Pogačnik, Tolmin, prazgodovinsko grobišča 2 / Tolmin, the prehistoric cemetery 2, Katalogi in monografije 34–35, 85–102. SVOLJŠAK, D. 2002, Arheološka podoba Bovškega. – V: Soški razgovori 1. Zbornik za domoznanstvo Zgodovinske sekcije KD Golobar, 261–277, Bovec. TERŽAN, B. 2016, Stane Gabrovec (1920–2015). – Arhe ološki vestnik 67, 11–20. SVOLJŠAK, D. 2005, L’insediamento fortificato d’altura di Sv. Katarina sopra Nova Gorica. Un castelliere di tipo isontino. – V: G. Bandelli, E. Montagnari Kokelj (ur.), Carlo Marchesetti e i castellieri, 1903–2003, Atti del Convegno internazionale di studi, Castello di Duino (Trieste), 14–15 novembre 2003, Fonti e studi per la storia della Venezia Giulia, Ser. 2, vol. 9, 657–665, Trieste. TERŽAN, B. 2019, Za uvod – kratek oris pretekle spome niškovarstvene službe na Slovenskem. – V: Arheološka dediščina Slovenije od osamosvojitve. Varovanje in pre zentacija, Razprave I. razreda SAZU 38, 9–13. zentacija, Razprave I. razreda SAZU 38, 9–13. TERŽAN, B., N. TRAMPUŽ 1973, Prispevek h kronologiji svetolucijske skupine / Contributo alla cronologia del gruppo preistorico di Santa Lucia. – Arheološki vestnik 24 (1975), 416–460. SVOLJŠAK, D. 2006, Koritnica v arheoloških obdobjih. – V: S. Torkar, K. Kofol (ur.), Baški zbornik. Alpski mladinski raziskovalni tabor Podbrdo 1992, 1993 in 1995, 13–28, Tolmin. TERŽAN, B., F. FEATURES OF THE LANDSCAPE arable flatland is on river terraces, while there are also higher-lying sunny locations of levelled ground that are suitable for habitation. The climate is predominantly alpine with abundant precipita tion, with Mediterranean influences reaching up the Soča Valley as far as to Kobarid. The southern fringes of the Julian Alps lie between the upper reaches of the Soča and the eastern edge of the Friuli Plain; almost half of this area is mountain ous, a fifth hilly. The Nadiža/Natisone carved its way deep into this area. It is one of the warmest Alpine rivers, of a torrential nature and draws waters from several springs. In its upper reaches, it flows towards the southeast down to the right tributary of the Legrada at Logje, where it turns towards the northeast. It again turns southwards at Robič and flows along the gorge between Mounts Matajur (1642 m) and Mija/Monte Mia (1237 m) to S. Pietro al Natisone/Špeter Slovenov, where it enters the Friuli Plain and, after a length of 60 km, discharges into the River Torre, which in turn flows into the Soča/Isonzo in the hinterland of the northern Adriatic. The Posočje Early Iron Age community, also known as the Sveta Lucija cultural group inhabited an area that extended into the Julian Alps in the north. This high-altitude land,1 which includes the areas of Tolminsko, Kobariško, Bovško and Bohinj, is characterised by narrow mountain crests and ridges with sharp peaks separated by deep valleys (Fig. 1). More than half of the surface lies at altitudes above 1000 m asl (Fig. 2), the highest peaks rise over 2000 m above the sea (e.g. Kanin 2587 m, Krn 2244 m, Rombon 2208 m) and the deepest valleys lie in the Soča Basin at around 350 m near Bovec and 152 m near Most na Soči.2 The steep slopes in places form precipices and over hangs, in other places they are gentler. Glaciers and waterways shaped the relief. The floors of valleys and basins are covered with glacial, glacial-fluvial, glacial-lacustrine and slope deposits. Flysch can be found in the Bovec Basin and in Bohinj, elsewhere the underlying geology is composed of carbonate rocks with typical high-altitude karst phenomena. The mountains of Bohinj and Tolminsko once held deposits of limonite and pyrite, though these iron ores were largely exhausted in the past. Surface waters are rare or non extant at higher altitudes. Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 2012, Magdalenska gora. Družbena struktura in grobni rituali železnodobne skupnosti / Social structure and burial rites of the Iron Age community. – Opera Instituti Archaeologici Sloveniae 26. [DOI: https://doi.org/10.3986/9789612546007]. g VALIČ, A. 1960–1961, Brod pri Bohinjski Bistrici – ObLO Radovljica. – Varstvo spomenikov 8, 228. VALIČ, A. 1985, Bohinjska Bistrica. – Varstvo spomenikov 27, 200. TECCO HVALA, S. 2020, The Early Iron Age central place at Most na Soči (NW Slovenia). – V: L. Zamboni, M. Fernández-Götz, C. Metzner-Nebelsick (ur.), Crossing the Alps. Early urbanism between Northern Italy and Central Europe (900–400 BC), 377–391, Leiden. VINAZZA, M. 2009, Deskle. – Varstvo spomenikov. Po ročila 45, 48. VINAZZA, M. 2015, Ostanki starejšeželeznodobne nasel bine na Gregorčičevi ulici v Kobaridu? – Goriški letnik 37/38 (2013–2014), 101–113. TECCO HVALA, S., B. MUŠIČ 2021, Ali je imela nasel bina na Mostu na Soči v železni dobi obzidje? – Goriški letnik 45, 27–50. VUGA, D. 1970, Bovec, Ravelnik. – Varstvo spomenikov 15, 143. VUGA, D. 1974, Bovec. – Varstvo spomenikov 17–19/1, 96–98. TERŽAN, B. 1976, Certoška fibula (Die Certosafibel). – Arheološki vestnik 27, 317–536. 445 Settlement in the Posočje/Sveta Lucija group – new sites and insights 3 Taken from Perko, Orožen Adamič (eds.) 2001, 72–82. 4 Savnik (ed.) 1968, 77 (Planina pri Cerknem). 1 The landscape features are summarised from Perko, Orožen Adamič (eds.) 2001, 54–71. 2 Prior to the creation of the hydropower station dam, the Idrijca discharged into the Soča at 124 m asl (Rutar 1882). 1 The landscape features are summarised from Perko, Orožen Adamič (eds.) 2001, 54–71. 2 Prior to the creation of the hydropower station dam, the Idrijca discharged into the Soča at 124 m asl (Rutar 1882). FEATURES OF THE LANDSCAPE Water mostly comes to the surface in strong karst springs (the Soča/Isonzo, the Nadiža/Natisone, the Savica in Bohinj). In the eastern part, it is Lake Bohinj, in a basin of a glacier-tectonic origin, and the River Sava Bohinjka that collect the waters coming from the Julian Alps, in the western part this role falls to the Soča, which flows along a graben between Tolmin and Kobarid. The Soča has several permanent left tributaries, the longest one being the Idrijca together with the Bača, also prominent are the Tolminka with its picturesque gorges and the Koritnica. In this alpine area, relief is the determining factor with regards to arable land and lines of communication. The bulk of the The mountainous area of the Julian Alps descends towards the southeast to the area of Cerkljansko hribovje (translated as ‘the Cerkno Hills’) between the Rivers Bača in the northwest, the Idrijca in the south, reaching in the southeast to Godovič.3 Just over half of this area lies above the altitude of 600 m, one sixth above 800 m; the highest peak is Mt Porezen (1630 m). The lowest part is the valley of the Idrijca, where the terrain descends to 210–230 m asl. The rivers and streams here are torrential and carved narrow gorges and ravines. The rock composition is varied, with dolomites taking up just over a quarter, limestones under one sixth and the rest are impervious rocks (claystones, siltstones, sandstones, conglomerates). There are minor deposits of copper ore in the surroundings of Cerkno, exploited until the outbreak of World War II.4 The slopes of Cerkljansko are steep and carved by shallow valleys; flatland is scarce. The area has moderate continental climate with abundant Miha MLINAR, Sneža TECCO HVALA 446 precipitation. Favourable climate conditions are on the sunny slopes in the thermal belt between 600 and 800 m asl. ranean. Its surface shows the effects of tectonic faults running in a Dinaric direction. Almost half of the surface is very steep, with gradients between 20o and 45o. Carbonate rocks dominate Banjšice and Čepovanski dol. The central part of the plateau, at an altitude of 700 m, is covered by flysch with marl limestone, conglomerate, brec cia and sandstone; this is also the most extensive piece of flatland. The underlying geology of the Soča Valley is also predominantly marl and flysch with inclusions of limestone breccias. FEATURES OF THE LANDSCAPE The Soča could only carve a very narrow passage through the gorges between Most na Soči and Solkan; the valley widens merely at places where it is fed by more substantial tributaries. Conditions suitable for agriculture are in the area of Kanalski Vrh, Tolminski Lom and Kanalski Lom, where there are several small permanent springs and the flysch soil enables lush grassland; fertile soils also cover the bottom of the Grgar Basin. The plateau boasts abundant rainfall and continental climate, whereas the mild effects of the Mediterranean climate are felt in the Soča Valley and the Grgar Basin. The hilly areas further include Idrijsko hribovje (translated as ‘the Idrija Hills’) lying at the transition from the Julian to the Dinaric Alps.5 This area is delimited with the Idrijca, as the main waterway, and its tributaries of the Bača and Trebuščica. It borders the plateau of Trnovski gozd in the south. It largely consists of levelled karst terrain, into which rivers and streams carved ravines and gullies, thus separating it into individual plateaus that rise steeply above the river valleys. In the north, the plateau of Šentviška planota (roughly 650 m asl) extends between the Rivers Idrijca and Bača; its highest peak reaches 974 m asl, the lowest point, at 156 m asl, is at the confluence of the Idrijca and Bača. Idrijsko hribovje is dominated by carbonate rocks (dolomites and limestones); there are areas of marl and marl slates along the upper reaches of the Idrijca, deposits of mercury and cinnabar around Idrija, as well as surface deposits of iron ore on Šentviška planota that were mined in cer tain places in the past.6 Water supply is limited in the carbonate karst areas, with larger amounts of water coming to the surface in permanent or occasional springs. The highly varied relief in turn brings varied climatic and pedologic condi tions, though continental climate with abundant precipitation prevails. Most flatland can be found on the undulating plateaus, with relatively wide alluvial plains also at the confluences of waterways; in general, however, there is little land suitable for agriculture. y g The mountainous area of Kambreško pogorje,8 with its ridges running in a NE–SW direction, rises towards the west between the Soča Valley and the River Idrija, the latter forming the borderline between Slovenia and Italy. 5 Taken from Perko, Orožen Adamič (eds.) 2001, 342–351. 6 Savnik (ed.) 1968, 400 (Daber); Mlinar et al. 2018. 7 Taken from Perko, Orožen Adamič (eds.) 2001, 312–323. 8 Ib. HISTORY OF ARCHAEOLOGICAL INVESTIGATIONS In the second half of the 19th and the early 20th century – in the time of the Austro-Hungarian Monarchy – the collection and recording of ar chaeological finds was conducted via a network of representatives, correspondents and conserva tors named by the Kaiserlich-Königliche Central- Commission zur Erforschung und Erhaltung der Baudenkmale that was established in 1850 in Vienna and later reorganised on several occasions.12 These collaborators include lawyer Paolo Bizzarro from Gorizia and Enrico Maionica, curator of the museum in Aquileia, both of whom conducted small-scale excavations in the Early Iron Age settlement and cemetery at Most na Soči. There was also senior civil engineer Rudolf Mahnič, who discovered the site at Loga near Bodrež and several contemporary graves at Koritnica and Šentviška Gora, as well as Simon Rutar, historian, geographer and author of a book on the history of the Tolminsko area (1882). Before and during their activities, local parish priests Tomaž Rutar and Alojzij Carli also sought for antiquities at Most na Soči, collecting them in the spirit of Slovenian national awakening. In 1882, a local, Anton Vuga excavated 36 graves at the confluence of the Idrijca and the Soča.13 The plant macroremains surviving in archaeo logical contexts offer a complementary insight into the natural environment and the use of natural resources; they also reflect the natural vegetation, albeit more selectively. They reveal the selection of the tree and shrub species, as well as the seeds and fruit of the plants that people used and cultivated. The analyses of the charcoal samples taken from the Iron Age settlement at Most na Soči have shown that people primarily used fir and oak as structural wood, followed by Scots pine, beech and spruce, while other tree species and beech were used as fuel and for artefact production.10 As for the funerary contexts of the Posočje community, the analyses of the charcoal from the pyres have revealed a predominant share of beech (with the exception of the Tolmin cemetery where not a single sample of beech wood has been recorded), whereas the coniferous tree species are considerably less well Particularly important for the development of prehistoric archaeology was the programme of the Prähistorische Kommission der kaiserlichen Akademie der Wissenschaften established in 1878.14 The archaeological endeavour that left the greatest mark in Posočje is the investigations conducted by Carlo Marchesetti, museum director from Trieste. FEATURES OF THE LANDSCAPE The area comprises Livški Kolovrat with the highest peak at 1243 m, continues south-westwards across a saddle to Kanalski Kolovrat with the highest peak of Korada (812 m), after which it descends to the hilly region of Goriška brda that lies in the Mediterranean part of the Soča Valley, at the fringes of the Friuli Plain. Most of Kambreško pogorje lies at altitudes between 300 and 600 m. Its slopes are gentler, particularly at higher elevations along the main ridge, and steeper in lower-lying areas; terraces are visible at altitudes between 420–460 m. The lowest part is in the west, where terrain descends to the narrow valley of the River Idrija, which only widens at Golo Brdo in Goriška brda. Eastwards, it descends towards the Soča that leaves the hills at Solkan and continues its path along the plain towards the Adriatic Sea. Kambreško pogorje is a meeting point of the Mediterranean and alpine climates. The underlying geology is similar to that of Banjšice, with limited surfaces suitable for agriculture. g South of Most na Soči is the plateau of Banjška planota or Banjšice,7 located between the Idrijca and its tributary the Trebuščica in the east and the Soča in the west, bordering in the south the plateau of Trnovski gozd rising above the Vipava Valley. Banjška planota is separated from Trnovski gozd by a 300–400 m deep and 16 km long dry valley of Čepovanski dol, which ends in the southwest in the Grgar Basin at 300 m asl. In the west, the plateau descends steeply to the Soča Valley be tween Most na Soči and Solkan, with the lowest altitude of 65 m, whereas its highest point is along the eastern edge (up to 1071 m). The landscape here is marked by its position at the junction of the Dinaric Alps, Julian Alps and the Mediter The geomorphology and natural environment certainly played a key role in the settlement and 8 Ib. Settlement in the Posočje/Sveta Lucija group – new sites and insights 447 economy of the region, as well as the communi cation possibilities or lack thereof in the Early Iron Age, when it was inhabited by the Posočje community or the Sveta Lucija cultural group. The recent palynological investigations in the area of Lake Bohinj have provided some indications as to the contemporary vegetation and human impact. FEATURES OF THE LANDSCAPE Because of its great size (318 ha), the lake records changes in the vegetation cover for an area in the circumference of several tens of kilometres, both in the lowland and at higher altitudes.9 The pollen diagram revealed that this part was covered in the past with mixed fir-beech forests composed of fir, spruce, oak and beech. It also showed a minimal human impact that began to increase more dis cernibly from the mid-2nd millennium BC onwards; the impact is visible in the appearance of pollen of cereals, indicators of foraging (narrowleaf plantain) and other ruderal plants growing in areas of human activity. Changes in this time are also perceptible in the forest composition, more precisely in the predominant share of beech, a considerable decrease of fir and an increase in the share of grasses and hazelnut, which grow on treeless land. The ratio between the main tree species (spruce, fir, beech) was created by both climate and man. A substantial decrease of beech occurred in the middle of the 1st millennium BC, when there was a slight increase of hornbeam and the landscape became relatively open due to human intervention. represented due to their inferior calorific values.11 These results also suggest that the contemporary people could potentially largely exterminate a tree species with intentional selection, but also change the landscape through deforestation. 11 Culiberg 2020; Report by M. Culiberg 2021 (kept in the Tolminski muzej in Tolmin). 12 Baš 1955. 13 For a more detailed history of research, see Gabrovec, Svoljšak 1983, 12–24; Gabrovec 1987, 121–123; id. 1999, 145–150; Svoljšak, Dular 2016, 17–24. 14 Gabrovec, Svoljšak 1983, 13; for more details on the commission, see Mader 2018. 9 Taken from Andrič et al. 2020. 10 Motella De Carlo 2018. j 14 Gabrovec, Svoljšak 1983, 13; for more details on the commission, see Mader 2018. 13 For a more detailed history of research, see Gabrovec, Svoljšak 1983, 12–24; Gabrovec 1987, 121–123; id. 1999, 145–150; Svoljšak, Dular 2016, 17–24. 12 Baš 1955. 11 Culiberg 2020; Report by M. Culiberg 2021 (kept in the Tolminski muzej in Tolmin). 12 15 Marchesetti 1903, 88–92, with a topographic map. Our list in this contribution does not include some of the prehistoric hillforts that Marchesetti noted. One of these is Grad (Pavlinčev grad), as the pottery collected there probably dates to the Bronze Age. Another one is Kozlov rob above Tolmin, where the 2020 excavations did reveal a pottery sherd that might date to the Early Iron Age, but it is deemed insufficient evidence of a settlement (Mlinar 2021a, 31). Also insufficient is the evidence from Der near Robič, a site heavily damaged during World War I. 16 Forlati Tamaro 1930, 419–428; Mlinar 2020a, Fig. 3. 17 Gabrovec 1958–1959; id. 1966b; id. 1974. j 19 On the history of the archaeological map of Slove nia and the project, see the contribution by Peter Petru and the preface in Arheološka najdišča Slovenije, 1975 (hereinafter ANSl). HISTORY OF ARCHAEOLOGICAL INVESTIGATIONS In his topographic work, he noted almost all of the known prehistoric settlements in the region, he was also familiar with Ajdovski gradec near Bohinjska Miha MLINAR, Sneža TECCO HVALA 448 Bistrica.15 In 1885–1904, he excavated several cem eteries in the valleys of the Nadiža/Natisone and Soča, as well as on Šentviška planota. He unearthed 41 graves at S. Pietro al Natisone/Špeter Slovenov, 1100 at Kobarid and as many as 3960 at Most na Soči (named Sveta Lucija/S. Lucia prior to 1952). Further 2480 graves at Most na Soči came to light during the excavations by Josef Szombathy, head of the Anthropological-Prehistoric Collection of the Naturhistorisches Museum in Wien; he also excavated the cemetery at Idrija pri Bači. The finds unearthed during these excavations, most extensive excavations of cemeteries to date, served as the foundations for establishing the basic chronologi cal and cultural-historical framework of the Early Iron Age. Already in 1895, Moritz Hoernes, the first professor of prehistoric archaeology at the University of Vienna, recognised the potential of the finds for a chronological division of the Early Iron Age into an early and a late phase. Niko Mozetič, who conducted small-scale field work at Most na Soči and Bovec between 1956 and 1958. Soon afterwards, the museum in Tolmin was joined together with the museum in Nova Gorica (1958), where Drago Svoljšak was employed in 1964. He laid the groundwork and the priorities of the museum’s archaeological department.18 At this time, a state-wide archaeological map project was undertaken as the basis for further settlement studies in the archaeological periods of Slovenia. Almost all Slovenian archaeologists, with the help of students of archaeology, were involved in gathering information on the archaeological finds from published and archival sources. The publication was prepared and edited by a special commission functioning under the auspices of the section for archaeology at the Slovenian Academy of Sciences and Arts. The book titled Arheološka najdišča Slovenije (translated as ‘archaeological sites of Slovenia’) saw the light of day in 1975.19 This lexicon of a publication (incorporating informa tion gathered up to 1965) records 27 Early Iron Age sites in Posočje and Bohinj.20 Concurrently with this project, there were also plans for regional archaeological topographies so that the data gath ered during the field surveys and reconnaissance would be used to upgrade the register of basic archaeological sources and gain information on new sites. 18 On the development of the archaeological depart ment at the Goriški muzej in Nova Gorica, see Kruh 2012. 20 ANSl 1975 (regions of Tolmin, Gorica and Radovljica), 114–127, 164–168. Of these 27 sites, seven are only noted as prehistoric sites, i.e. not necessarily from the Iron Age. 21 https://www.zvkds.si/sl/knjiznica/varstvo-spomenikov. HISTORY OF ARCHAEOLOGICAL INVESTIGATIONS In the areas of Gorica and Tolmin, the latter task was entrusted to Drago Svoljšak (first together with Peter Petru) and Nada Osmuk, the latter conservator of the heritage protection office in Nova Gorica, where she had been employed in 1973. They conducted a series of archaeologi cal field surveys and verified the obtained data through small-scale trial trenching, most frequently reporting their results in the professional journal of Varstvo spomenikov.21 They also carried out numerous archaeological salvage operations, which in some cases turned into exploratory excavations. To mention only the best-known ones related to the Early Iron Age, they unearthed 465 graves in Tolmin, 287 graves in Kobarid, trial trenched Niko Mozetič, who conducted small-scale field work at Most na Soči and Bovec between 1956 and 1958. Soon afterwards, the museum in Tolmin was joined together with the museum in Nova Gorica (1958), where Drago Svoljšak was employed in 1964. He laid the groundwork and the priorities of the museum’s archaeological department.18 At this time, a state-wide archaeological map project was undertaken as the basis for further settlement studies in the archaeological periods of Slovenia. Almost all Slovenian archaeologists, with the help of students of archaeology, were involved in gathering information on the archaeological finds from published and archival sources. The publication was prepared and edited by a special commission functioning under the auspices of the section for archaeology at the Slovenian Academy of Sciences and Arts. The book titled Arheološka najdišča Slovenije (translated as ‘archaeological sites of Slovenia’) saw the light of day in 1975.19 This lexicon of a publication (incorporating informa tion gathered up to 1965) records 27 Early Iron Age sites in Posočje and Bohinj.20 Concurrently with this project, there were also plans for regional archaeological topographies so that the data gath ered during the field surveys and reconnaissance would be used to upgrade the register of basic archaeological sources and gain information on new sites. In the areas of Gorica and Tolmin, the latter task was entrusted to Drago Svoljšak (first together with Peter Petru) and Nada Osmuk, the latter conservator of the heritage protection office in Nova Gorica, where she had been employed in 1973. 28 Teržan, Trampuž 1973; Svoljšak 1973; Kos 1973. 29 Lo Schiavo 1984; Gabrovec 1987, 121–123; id. 1999, 145–150. 30 Preistoria del Caput Adriae 1983; Ruaro Loseri, Montagnari Kokelj (eds.) 1984. 31 Gabrovec, Svoljšak 1983. 32 Teržan, Lo Schiavo, Trampuž-Orel 1984–1985. 33 Montagnari Kokelj (ed.) 1993. 34 Teržan 2019; Brišnik, Kajzer 2019. HISTORY OF ARCHAEOLOGICAL INVESTIGATIONS In 1983, the Early Iron Age finds from Posočje were included in the Preistoria del Caput Adriae exhibition of the museum from Trieste, on the occasion of which an international symposium was also organised.30 In the same year, Gabrovec and Svoljšak published the first volume on Most na Soči in the Katalogi in mono grafije series of the Narodni muzej in Ljubljana,31 which brought an exhaustive presentation of the topography and history of research, as well as a reconstructed plan of the cemetery excavated by Marchesetti and Szombathy. Soon afterwards, Biba Teržan, Fulvia Lo Schiavo and Neva Trampuž-Orel published two volumes presenting the finds from Szombathy’s excavations.32 The Italian colleagues published a reprinted collection of Marchesetti’s treatises in 1993,33 while most of the finds he had excavated and are held at the Triestine museum remain unavailable to the wider public for further analysis. in the settlement on Sv. Katarina (Kekec) above Nova Gorica22 and excavated on Gradič in Ko barid, where they found building remains and a sacred place/sanctuary from the Iron Age and the Roman period.23 By far the best and most widely known, however, are the excavations that Svoljšak and his museum team conducted for a decade in the settlement at Most na Soči, which are as significant as the cemetery investigations. Using modern methods and approaches, as well as meticulous recording, the excavations yielded a wealth of invaluable data on the settlement lay out, architecture, economic activities and material culture of Early Iron Age Posočje.24 y g j The beginnings of another prominent project for the study of the Early Iron Age in Slovenia date back to the 1960s, namely the formation of the Vzhodnoalpski komite / Ostalpenkomitée / Comitato per le Alpi orientali that involved experts from Slovenia, Austria and Italy who organised an international exhibition on situla art (1962). Its programme foresaw the publication of the cemetery and later also settlement at Most na Soči.25 Stane Gabrovec set as a priority the task of publishing the finds from Slovenia kept in museums abroad, with the intention of making it widely available for scientific evaluation, already in a presentation he had in 1963 at the 6th congress of the archaeological association of Yugoslavia, held in Ljubljana. HISTORY OF ARCHAEOLOGICAL INVESTIGATIONS They conducted a series of archaeologi cal field surveys and verified the obtained data through small-scale trial trenching, most frequently reporting their results in the professional journal of Varstvo spomenikov.21 They also carried out numerous archaeological salvage operations, which in some cases turned into exploratory excavations. To mention only the best-known ones related to the Early Iron Age, they unearthed 465 graves in Tolmin, 287 graves in Kobarid, trial trenched Between both world wars, it was only Bruna Forlati Tamaro, conservator of the heritage protec tion service in Trieste, who excavated in Posočje, unearthing four graves in the cemetery at Most na Soči.16 Walter Schmid, museum curator at the Joanneum in Graz and professor of archaeology at the University of Graz, conducted investigations in Bohinj between 1937 and 1939. He excavated at Ajdovski gradec and the associated cemetery at Bitnje, as well as at Spodnje Gradišče near Lepence, Dunaj near Jereka and Žlan.17 To the onset of World War II, there were 25 sites from the Early Iron Age known in Posočje and Bohinj (Fig. 3). A new impetus for archaeological investiga tions came after the Second World War with the establishment of museums in Tolmin (1951) and Nova Gorica (1954), as well as the office of heritage protection service in Nova Gorica (1961); up to then, almost all the archaeological finds recovered in the area were kept in museums outside Yugoslavia. The first archaeologist in the Tolmin Museum was Settlement in the Posočje/Sveta Lucija group – new sites and insights 449 held in the Viennese museum. Using the data from the cemetery at Tolmin, Drago Svoljšak presented the main features of the burial practices and Peter Kos evaluated the graves that Mahnič had exca vated at Koritnica.28 Fulvia Lo Schiavo proposed her chronological framework in 1973 based on Marchesetti’s discoveries at Most na Soči,29 with her periodisation not differing substantially from the chronological framework in use in Slovenian archaeology to this day. 22 Gabrovec, Svoljšak 1983; Svoljšak 1990; id. 2005; Svoljšak, Pogačnik 2001–2002. 23 Osmuk 1997; monographic publication is in preparation. 24 Svoljšak, Dular 2016. 25 Gabrovec, Svoljšak 1983, 7–9; Gabrovec 1999, 145–150. 26 Gabrovec 1964–1965. 27 Teržan 2016. 34 Teržan 2019; Brišnik, Kajzer 2019. HISTORY OF ARCHAEOLOGICAL INVESTIGATIONS In this presentation, he identified the Sveta Lucija group as a specific entity within the south-eastern Alpine Hallstatt culture.26 As professor of prehistoric archaeology at the Faculty of Arts, University of Ljubljana, he was largely able to carry out this task with the help of his students and with a successful collaboration with the Naturhistorisches Museum in Wien.27 Together with his students, he presented the main characteristics and chronological phases of individual Hallstatt cultural groups of the south- eastern Alpine area in 1972, at the international colloquium held in Novo mesto. Their findings were then published in Arheološki vestnik 24, 1973 (1975). In it, Biba Teržan and Neva Trampuž proposed a more detailed chronology of the Sveta Lucija group based on the finds from Most na Soči Another turning point came with the reor ganisation of the heritage protection service and a change in its strategy after Slovenia became an independent state. The role of conservators in the regional offices of the heritage protection institute changed and the emphasis of their work was on the administrative procedures of registering protected areas, determining the conditions for interventions and supervising the archaeological fieldwork. The last of them mainly involves monitoring construc tion and infrastructural interventions, as well as spatial plans, whereas the fieldwork addressing specific archaeological issues is minimal.34 In Posočje, the Nova Gorica office of the heritage protection institute with conservators Nada Osmuk Miha MLINAR, Sneža TECCO HVALA 450 and Patricija Bratina concluded the excavations at Gradič in Kobarid (1997). In collaboration with the Narodni muzej Slovenije, it saved the Early Iron Age cemetery on Jelenšek near Godovič from robbers (1993). It also trial trenched on the prehis toric settlement at Grgar (2005).35 After 2009, the Center za preventivno arheologijo (ZVKDS CPA) trial trenched several new archaeological sites, namely at the foot of Gradič in Kobarid and on Berjač above Podbela in the Breginjski kot area.36 sional meetings and publications. Among the recent publication, we should particularly men tion three monographs on Most na Soči in the Iron Age that are the result of a collaboration of the Goriški muzej, Tolminski muzej, Inštitut za arheologijo ZRC SAZU and a variety of experts. HISTORY OF ARCHAEOLOGICAL INVESTIGATIONS Two of these offer a comprehensive presentation and up-to-date evaluation of the finds and features from the Iron Age settlement on the right bank of the Idrijca, the third one offers a fresh insight into the structure and chronological succession of burial, the burial rite and place of cremation along the northern fringes of the vast cemetery on the left bank of the Idrijca at Most na Soči.38 Another monograph that should be mentioned is a comprehensive and detailed analysis of the Tolmin cemetery in the Katalogi in monografije series of the Narodni muzej Slovenije.39 After 2000, most of the rescue operations in this area are entrusted to the Tolminski muzej, which again became an independent institution and employed an archaeologist (Miha Mlinar). The prominent fieldwork this museum has con ducted includes unearthing building remains at the northern and western edges of the Iron Age settlement at Most na Soči (2001, 2004, 2010) and excavating graves on the lowest terrace on the left bank of the Idrijca (2000–2002 and 2013), with the remains of a habitation layer from the Late Bronze Age found underneath. The museum also unearthed an exceptional cult place with the burial of horses and warrior outfits in Kobarid (2010), where earthworks at the edge of the already investigated cemetery bring to light ever new graves (2002, 2021/22). The recent discoveries at previously unknown archaeological sites further include the burials at Srpenica in the Bovec area (2003), at Jerovca on Šentviška planota (2007) and at Čadrg high above the valley of the Tolminka (2014).37 The museum is gathering data on new sites not only through rescue investigations, which mainly take place in advance of construction and other earthwork interventions, but also from collectors of antiquities and metal-detectorists who have been very active in recent decades. Most of the new data and stray finds come from the latter sources (Fig. 3; 4); the credibility of these data varies, however, as they often come from unknown contexts and even the reported locations are not always reliable. 38 Svoljšak, Dular 2016; Dular, Tecco Hvala (eds.) 2018; Mlinar 2020a. 39 Svoljšak, Pogačnik 2001–2002. 40 Mlinar, Žbona Trkman 2008; Laharnar, Mlinar 2013; Laharnar 2018b; Mlinar et al. 2018. 41 Mlinar, Gerbec, Laharnar 2014; Gerbec, Vinazza 2018; Gerbec 2021. 35 For Jelenšek near Godovič, see the contribution by Bratina, Laharnar, Svoljšak, for Grgar see Bratina, both in this volume. 36 Vinazza 2015; Report by T. Fabec, T. Nanut, B. La harnar, B. Kramberger, T. Leskovar, T. Tolar, K. Kavkler, E. Menart 2021 (kept in the ZVKDS CPA). 37 Mlinar, Klasinc, Knavs 2008; Mlinar 2009–2010; Mlinar, Gerbec 2011; Laharnar, Mlinar 2013; Laharnar, Mlinar 2019; Mlinar 2020a; id. 2020b. 35 For Jelenšek near Godovič, see the contribution by Bratina, Laharnar, Svoljšak, for Grgar see Bratina, both in this volume. 36 Vinazza 2015; Report by T. Fabec, T. Nanut, B. La harnar, B. Kramberger, T. Leskovar, T. Tolar, K. Kavkler, E. Menart 2021 (kept in the ZVKDS CPA). 47 This contribution does not include the sites in the Vipava Valley (see e.g. Bratina 2009–2010) and the south ern fringes of Trnovski gozd, because the lack of evidence prevents us from reliably attributing them to the Early Iron Age and to the Posočje community. Location and size the high-altitude zone.42 All these efforts have contributed to the number of known sites of the Early Iron Age Posočje community rising to 85, some of which are large complexes that comprise the settlements and the associated cemetery and/ or other types of sites in immediate proximity (Fig. 3). The available data show that most of these sites are represented by chance stray finds, while settlements and cemeteries occur in equal shares (Fig. 4). The Iron Age settlements in the area of the Julian Alps are located on elevations lining the valley floors of the main waterways, i.e. the Rivers Soča/ Isonzo, Nadiža/Natisone and Sava Bohinjka, which represent not only a permanent source of water supply, but also the main orientation markers in a mountainous landscape. These inhabited eleva tions (Fig. 5) rise along the skirts of fertile plains. In addition to permanent settlements, there are also two known seasonal camps: one in the Trenta area – V plazeh (No. 29), the other at Dolga planja on Mt Vogel (No. 38), both located above 1500 m asl. In the narrow Soča valley between Most na Soči and Gorica/Gorizia, there are no known Early Iron Age settlements; they are rather located on plateaus. On Banjška planota, for example, they are sited in proximity to permanent springs and areas of fertile soils: Grad near Levpa and presum ably V Glavi near Kanalski Lom (Nos. 50, 48) in the north, Gradišče near Deskle (No. 51) at the west edge, as well as in the Grgar Basin (No. 52) and on Sv. Katarina above Nova Gorica (No. 53) in the southwest. Several settlements are also on the plateau of Šentviška planota (Nos. 57, 62), in its southern part that rises above the River Idrijca, whereas the more remote area of Idrijsko hribovje is for now devoid of known settlements. The settle ment at Štalca near Železniki (No. 70), above the valley of the River Selška Sora, yielded habitation pottery most resembling that from Most na Soči and thus possibly indicating the eastern border of the group’s territory. The easternmost settlement is at Žirk near Žiri (No. 71), the settlement farthest in the southeast is on Jelenšek near Godovič (No. 72a); both lie on narrow ridges similarly as other settlements in Cerkljansko hribovje (Nos. 66, 68, 69). There is no solid evidence of settlement in Kambreško pogorje west of the Soča in this time. Location and size The settlement at Golo Brdo (No. 43), which possibly formed part of the Posočje community, lies in the bend of the border River Idrija at the transition to the Friuli Plain.47 Because the Sveta Lucija cultural group is mainly recognisable as a specific entity in the features of the burial and the grave goods, the settlements located at its border In recent times, topographic work uses airborne laser scanning or LiDAR data that, combined with archaeological registers, digital elevation models and geographic information system or GIS tools, offer a variety of possibilities for spatial analyses. This was the method used to examine and evalu ate the sites in the Kobarid area.43 Geophysical measurements also became an important method in detecting the archaeological potential of a site.44 By including the natural sciences, such as archaeo biology, archaeometallurgy, radiocarbon dating and many others, archaeological investigation is becoming an increasingly interdisciplinary affair. Remarkable progress notwithstanding, there is a lack of targeted research aimed at advancing our knowledge of individual chronological phases and characteristics of the settlement pattern, but also establishing the continuity and turning points of settlement. 42 Horvat 2015–2016; Mlinar, Turk 2016; Horvat 2019. 43 Štular 2011a; Laharnar, Štular, Mlinar 2015. 44 Mlinar, Mušič 2020; Tecco Hvala, Mušič 2021. 45 Mlinar 2018 with earlier literature; Gerbec 2018; Gerbec Vinazza 2018. 46 The catalogue of sites brings data on the geographic location, investigations, small finds and the place of their storage, broad dating and main references. The catalogue numbers are cited in the tables and in the text. HISTORY OF ARCHAEOLOGICAL INVESTIGATIONS The Tolminski muzej regularly informs the interested public of the new discoveries through exhibitions, profes j j With regards to archaeological field survey, we should mention the collaboration between Miha Mlinar and Beatriče Žbona Trkman, late curator at the Goriški muzej in Nova Gorica, in publishing the new finds from Banjška planota and Trnovski gozd, as well as the collabora tion of Mlinar with Boštjan Laharnar from the Narodni muzej Slovenije in presenting the sites on Šentviška planota.40 In outlining the archaeol ogy of Breginjski kot, they were joined by Teja Gerbec, now employed at the Goriški muzej, who also gathered the data on the sites in Kanalski Kolovrat and, together with Manca Vinazza from the Oddelek za arheologijo Filozofske fakultete v Ljubljani, on the sites from the western part of Banjška planota.41 The mountains of Posočje and Bohinj are the subject of a systematic re cording and evaluation of high-altitude sites as part of the project by the Inštitut za arheologijo ZRC SAZU. It is a project conducted by Jana Horvat together with Lucija Lavrenčič, and by Matija Turk in collaboration with the Tolminski muzej, as well as external collaborators of both archaeological and non-archaeological professions who possess an in-depth knowledge of the local environment and have detected most of the new archaeological sites from different periods in Settlement in the Posočje/Sveta Lucija group – new sites and insights 451 46 The catalogue of sites brings data on the geographic location, investigations, small finds and the place of their storage, broad dating and main references. The catalogue numbers are cited in the tables and in the text. 45 Mlinar 2018 with earlier literature; Gerbec 2018; Gerbec Vinazza 2018. 44 Mlinar, Mušič 2020; Tecco Hvala, Mušič 2021. SETTLEMENTS Of the potential settlements mentioned in the sources,45 the catalogue only lists those where there is more or less reliable material evidence of human habitation in the Early Iron Age (Fig. 4; 5).46 There are 24 such sites in the catalogue; most have not yet been archaeologically investigated and human habitation is only indicated by ramparts, terraces and small finds collected on the surface or dug up using a metal detector. Archaeological trial trenching or excavations were conducted on nine of these sites. Miha MLINAR, Sneža TECCO HVALA 452 that are without known associated cemeteries can only tentatively be ascribed to the group. the other.48 In Bohinj, only Ajdovski gradec (No. 36a) was fortified, though the stonework rampart that completely encloses the settlement is believed only to date to the Roman period. Nine settlements had no built defences, suggesting they were either of a different type or different rank. For the time being, the history of research and publication of the settlements discussed here only allows comparisons in location, size and fortification features (Fig. 5). The largest were the settlements in the upper Soča Valley at the confluence with the Idrijca (Most na Soči) and the Koritnica (Bovec), as well as along the natural communication with the valley of the Nadiža/Natisone (Kobarid). By far the largest is Most na Soči, measuring some 13 ha, which makes it up to four times larger than the settlement on Ravelnik in Bovec (3 ha) (Fig. 6) and on Gradič in Kobarid (4 ha); also possibly among the largest is the border settlement at Štalca above Železniki (9 ha) (Fig. 5: Nos. 1a, 18a, 26a, 70). There are six medium-sized settlements, measuring 1 to 2 ha; they are located along the Sava Bohinjka (Nos. 33a, 36a) at the northern border and in the Grgar Basin (No. 52) at the southwestern end of Čepovanski dol that represents a natural com munication across the plateau of Banjška planota to the valley of the Idrijca. Also falling into this size category are Sv. Katarina above Nova Gorica at the junction of the Soča and Vipava Valleys (No. 53), Golo Brdo at the River Idrija on the western border (No. 43) and presumably Žirk near Žiri above the valley of the River Poljanska Sora (No. 71) on the eastern border. 48 Svoljšak 1984a; id. 1986; Gabrovec 1999, 159–161; Mlinar 2018. 49 Svoljšak 1990; id. 2005. 50 Svoljšak 2005, 660, Fig. 3. 51 Osmuk 1977; ead. 1993; Bratina 2001; ead. 2009a. 52 See the contribution by Bratina in this volume. SETTLEMENTS The location and size of these settlements point to an important role of the natural corridors in the choice of settlement location. The settlements in their hinterland are smaller than a hectare, the smallest being those on Šentviška planota (Nos. 57, 62). For five settle ments, it was not possible to estimate their size as there are no artificial modifications visible in relief. The archaeological trial trenching conducted on some of the ramparts have shown that the construction did not differ from that observed at the hillforts of the Notranjska-Kras group. At Sv. Katarina above Nova Gorica, Svoljšak estab lished several phases of fortification (No. 53).49 The first trial trenching, conducted at the north end of the stonework rampart, revealed that an up to 4 m thick wall was constructed on top of the bedrock that had solid inner and outer faces made up of large flat stones assembled in the drystone technique and the core filled with stone rubble. Later, a wall of unworked stones bound with mortar was constructed on top of it in the same width. The layers associated with the first, drystone wall revealed pottery sherds with paral lels from other fortified settlements in the Vipava Valley and hillforts of the Kras. The blackish layer with traces of fire along the wall exterior held several iron, bronze and bone artefacts, as well as clay daub with wattle impressions and hand-built pottery. The finds from the top layers, as well as the mortar-bound wall and defence tower date to the Roman period and the Early Middle Ages. The subsequent trial trenching in the south side clearly revealed three fortification phases. The first and the second walls were separated by a thin layer of earth that suggests an abandonment of the settle ment during its formation. The two walls shared the construction technique and roughly also the width (3.3 and 2.9 m). These prehistoric fortifica tions were reused later for the Roman-period and early medieval forts.50 Fortification features The settlement at the church of St Mary on Jezero above Golo Brdo (No. 43) also yielded cultural layers and drystone constructions from different periods, revealing it was certainly fortified in the Roman period.51 As for the trial trenching on Grašišče at Grgar (No. 52), the results are presented in another contribution in this volume.52 Most settlements were naturally well defended with more or less steep slopes and deep river beds and gorges. Two thirds of the settlements, how ever, show traces of artificial fortification in areas of easier access (Fig. 5). All the large settlements were additionally fortified (Fig. 6). The 2021 trial trenching showed this is also true of the largest settlement, at Most na Soči (Fig. 7), which was previously believed not to have had the need for additional fortification due to its natural protec tion offered by the Rivers Soča and Idrijca, on the one hand, and by the defence posts located at the entrances to the Soča Basin (Nos. 26a, 21, 53), on Settlement in the Posočje/Sveta Lucija group – new sites and insights 453 Sv. Helena near Podbela, Vrh gradu near Pečine, Gradišče in Cerkno and Žirk near Žiri (Nos. 26a, 18a, 21, 22a, 57, 68, 71), reveal a variety of defence systems. They have as yet not been archaeologically investigated and it is unclear whether all ramparts date to the Hallstatt period.55 At Most na Soči (No. 1a), there is a sandy ridge running N–S along the eastern edge of the settle ment from the highest peak towards the riverbed of the Idrijca. In 2021, geophysical measurements and small-scale trial trenching were conducted at two sections of the ridge, 60 m apart, and revealed a stonework rampart (Fig. 7; 8B).53 The unearthed rampart was damaged with two round cuts (probably from World War I) (Fig. 7A: a,b), with the east face surviving relatively well (Fig. 7B). It was dug into the sandy ridge that revealed no cultural remains (Fig. 7C: c). It was constructed of flat stones in places arranged in columns. Beside it was another line of stones, possibly for reinforcement. The west face, towards the interior of the settlement, was in ruins. The core of the wall was filled with small rounded stones. The east and west faces were roughly three metres apart, similarly as on Sv. Katarina above Nova Gorica. 55 Cf. Štular 2011a; Mlinar 2018. 56 Svoljšak, Dular 2016; Dular, Tecco Hvala (eds.) 2018; Tecco Hvala 2020. 57 Subsequent investigations revealed further four buildings from the Hallstatt period (Mlinar, Klasinc, Knavs 2008) and five houses from the Roman period. 58 Svoljšak 1988–1989. 59 Mlinar 2020a, 119–120, Pl. 15A. 60 Svoljšak 2018. Fortification features The construction manner is the same as for the drainage walls of the Iron Age houses in the settlement and for the stonework ramparts of the Notranjska-Kras group. Found on the east side of the ridge, outside the settlement, was a blackish layer of earth with many pieces of charcoal and animal bones; it was deposited on conglomerate that turned into lime in some spots as the consequence of more intense or frequent presence of fire (Fig. 7D, 7E). This layer extended across the sandy ridge and the small finds from it indicate metallurgic activities (pieces of slag and bronze melt, part of a melting pot, semi-finished multi-knobbed pin, piece of a sandstone mould). At the top, the ruins of the rampart in the west and the layer of burnt remains were covered with topsoil with scattered recent and Roman-period finds, as well as grassy turf. The bow fragments of a serpentine fibula, globular two-piece pendants and pottery sherds with plain cordons suggest that the layer of burnt remains in the east, outside the settlement, dates to the Sv. Lucija IIa phase, i.e. the 6th century BC. The radiocarbon analyses of the charcoal and bones corroborate such dating (Fig. 8A).54 The LiDAR-derived images of several fortified 53 Tecco Hvala, Mušič 2021; Report by E. Leghissa 2022 (kept in the archives of the Iza ZRC SAZU). 54 The charcoal sample (VZ 7) from the rampart ruin shows, at the 2 σ calibration, that it more likely (68.6%) dates from 1811 to 1918 cal AD, which would support the supposition it was damaged during World War I. 53 Tecco Hvala, Mušič 2021; Report by E. Leghissa 2022 (kept in the archives of the Iza ZRC SAZU). 54 The charcoal sample (VZ 7) from the rampart ruin shows, at the 2 σ calibration, that it more likely (68.6%) dates from 1811 to 1918 cal AD, which would support the supposition it was damaged during World War I. The architecture and internal layout of settlements The modern excavations that Drago Svoljšak and his team conducted at Most na Soči, and the comprehensive publication of the habitations remains from the right bank of the Idrijca are the most exhaustive, and also the only available source of information on the Early Iron Age architecture, on the urban layout, activities and standard of liv ing in the settlements of Posočje.56 The 4 ha large investigated area in the east part of the modern village revealed the remains of 36 houses from the Early Iron Age, three from the Late Iron Age and ten from the Roman period.57 Traces of a house from the Younger Bronze Age came to light in the western part, closer to the confluence,58 while a modest and more or less contemporary habita tion layer was later found on the other side of the Idrijca, close to its confluence with the Soča.59 j The buildings from the Early Iron Age were constructed according to the established practice characteristic of the Eastern Alpine circle, but they do reveal certain local specifics. The construction pit was dug into the slope on three sides, with the front sides mostly facing south. The walls of the construction pit were lined with drainage walls – this is specific to the Posočje architecture or the Iron Age houses of Posočje, as Drago Svoljšak put it.60 The stone foundations of buildings were slightly removed from the drainage walls and set on levelled ground. They supported a wooden superstructure composed of sleeper beams, posts, braces, tie-beams and cleft planking. The wood species most commonly used for construction The LiDAR-derived images of several fortified settlement or hillforts, such as Ravelnik near Bovec (Fig. 6), Gradič in Kobarid, Sv. The architecture and internal layout of settlements The pottery assemblage from the settlement is marked by a fabric with abundant inclusions of sand, as well as typical forms of jars, pithoi, goblets and situlae, decoration (plain cordons, red-black painting, combing) and a multitude of ceramic rings.63 The remains of slag and bronze melt, casting utensils, ingots and a block of iron indicate that secondary processing of metals and artefact production also took place in the settlement.64 The differences in the quality of construction, f h d d ll f d purposes was hard and resistant oak.61 The buildings better preserved in plan show they ranged from 6 to 28.5 m2 in size. The interior foundations that supported partition walls show they had one, two or three rooms.62 The floors were usually made of beaten earth, one house had stone slabs on the floor and three had wooden floorboards in one of the rooms. Standing apart are the houses entered from the west, with the west side actually completely open, with levelled underlying geology represent ing the floor and a presumably single-pitch roof supported with wooden posts; other buildings had gabled roofs. Differences are also noticeable in interior furnishings that include ceramic plaques decorated with rich geometric designs that rank among the curiosities of the settlement. Some buildings were fitted with hearths made of pebbles and other small stones coated in clay; one building even held a domed oven of marl slabs. Interiors were also fitted with pits. The small round pits in multi-room buildings were usually situated in a small room and presumably served for storage, similarly as globular ceramic containers or silos. The large rectangular pits were mostly found in the single-room buildings, where they took up a substantial part of the functional surface; the finds from these pits suggest they were used in work processes. For one of the large pits, it was possible to postulate a pottery making activity, for several others metallurgic processes. 61 Motella De Carlo 2018. 62 Dular, Tecco Hvala 2018, 14–78. 63 Grahek 2018a. 64 Laharnar 2018a; Šmit, Laharnar 2018; Lamut 2018. 65 The criteria for identifying the function of a build ing are given in Dular, Tecco Hvala 2018, 73–78, and also take into account the qualitative and quantitative differ ences in the types of artefacts, animal and plant remains, quality of construction, size and openness of buildings, their interior furnishings and the number of construction The architecture and internal layout of settlements Volar near Robič, Miha MLINAR, Sneža TECCO HVALA 454 animal bones (including those from the meatiest body parts) and pottery remains in two buildings in the east part as opposed to the quantities from other buildings suggest they were used as a public space intended for common matters;66 in the north eastern part, the range and time span of artefacts that stand out markedly from assemblages in other areas suggest the existence of a cult space.67 The differentiation of buildings according to function, the common infrastructure (such a gravel path found along the workshops in the western part of the excavated area and the drainage ditch that crossed the slope), the shared NW–SE orientation of buildings and their renovation or rebuilding in the same building plots may all be seen as elements of spatial planning and organisation of the Early Iron Age settlement at Most na Soči. purposes was hard and resistant oak.61 The buildings better preserved in plan show they ranged from 6 to 28.5 m2 in size. The interior foundations that supported partition walls show they had one, two or three rooms.62 The floors were usually made of beaten earth, one house had stone slabs on the floor and three had wooden floorboards in one of the rooms. Standing apart are the houses entered from the west, with the west side actually completely open, with levelled underlying geology represent ing the floor and a presumably single-pitch roof supported with wooden posts; other buildings had gabled roofs. Differences are also noticeable in interior furnishings that include ceramic plaques decorated with rich geometric designs that rank among the curiosities of the settlement. Some buildings were fitted with hearths made of pebbles and other small stones coated in clay; one building even held a domed oven of marl slabs. Interiors were also fitted with pits. The small round pits in multi-room buildings were usually situated in a small room and presumably served for storage, similarly as globular ceramic containers or silos. The large rectangular pits were mostly found in the single-room buildings, where they took up a substantial part of the functional surface; the finds from these pits suggest they were used in work processes. For one of the large pits, it was possible to postulate a pottery making activity, for several others metallurgic processes. 66 Cf. Toškan, Bartosiewicz 2018, Tab. 4–7; Grahek 2018a, Fig. 2. 67 Dular, Tecco Hvala 2018, 79–85; Laharnar 2018a, 224–234. 68 Ogrin 2003. 69 Gabrovec 1958–1959; id. 1966b, 262; id. 1974, 306–307. 70 Grahek 2018b, 267–268. phases on the same plot, all of which are analysed in detail and discussed in the monograph by Dular, Tecco Hvala (eds.) 2018. Having said that, we should note the differ ences with regards to the interpretation of buildings that Teržan proposes in the introduction and brief overview of the Sveta Lucija Hallstatt cultural group in this volume of Arheološki vestnik. Her interpretation is based on a single type of finds (scarce remains of slag and melts), the association of which with a particular building is in several cases questionable. 66 Cf. Toškan, Bartosiewicz 2018, Tab. 4–7; Grahek 2018a, Fig. 2. phases on the same plot, all of which are analysed in detail and discussed in the monograph by Dular, Tecco Hvala (eds.) 2018. Having said that, we should note the differ ences with regards to the interpretation of buildings that Teržan proposes in the introduction and brief overview of the Sveta Lucija Hallstatt cultural group in this volume of Arheološki vestnik. Her interpretation is based on a single type of finds (scarce remains of slag and melts), the association of which with a particular building is in several cases questionable. 67 Dular, Tecco Hvala 2018, 79–85; Laharnar 2018a, 224–234. 70 Grahek 2018b, 267–268. 63 Grahek 2018a. 65 The criteria for identifying the function of a build ing are given in Dular, Tecco Hvala 2018, 73–78, and also take into account the qualitative and quantitative differ ences in the types of artefacts, animal and plant remains, quality of construction, size and openness of buildings, their interior furnishings and the number of construction 61 Motella De Carlo 2018. end of the Middle Bronze Age (bronzo recente) and the beginning of the Hallstatt period; the finds are kept in the MAC (Tomadin, Visintini, Colussa 1989). 77 E.g. from the hillfort on Pavlinčev grad (see Note 15). 78 Cf. Svoljšak 1990, Pl. 2–3 and Grahek 2018a, 249–306, Fig. 28. The architecture and internal layout of settlements The pottery assemblage from the settlement is marked by a fabric with abundant inclusions of sand, as well as typical forms of jars, pithoi, goblets and situlae, decoration (plain cordons, red-black painting, combing) and a multitude of ceramic rings.63 The remains of slag and bronze melt, casting utensils, ingots and a block of iron indicate that secondary processing of metals and artefact production also took place in the settlement.64 g Sources mention similar buildings and pits unearthed in the settlements in the Bohinj area, where Walter Schmid excavated for the Joanneum between the two world wars, but the results were not comprehensively published. On Ajdovski gradec near Bohinjska Bistrica (No. 36a), he found nine buildings sunken into the bedrock. Some had stone foundations and hearths made of stones or sunken into the ground. Unearthed next to the buildings were oval pits that served metallurgic purposes. The trial trenching that the Gorenjski muzej con ducted there in 2003 also revealed buildings and traces of metallurgic activities similar to those of Posočje.68 Moreover, Schmid excavated similar building remains, a hearth of neatly arranged stones and five smelting pits at Dunaj near Jereka (No. 33a).69 Analogous remains also came to light at Štalca above Železniki (No. 70).70 The differences in the quality of construction, interior furnishings and recovered small finds in dicate a distinction between residential buildings and workshops.65 The extraordinary amounts of Settlement in the Posočje/Sveta Lucija group – new sites and insights 455 Grašišče near Grgar, Sv. Katarina and Gradišče near Deskle on the plateau of Banjška planota (Nos. 50–53), on Golo Brdo above the vally of the River Idrija (No. 43), as well as on Vrh gradu near Pečine (No. 65) and several other presumed prehistoric sites on the plateau of Šentviška planota that otherwise lack convincing material evidence from the Early Iron Age.77 This also indicates that the land hosting human habitations in the Iron Age had in part already been cultivated before. In almost all the settlements discussed in this contribution, the chronologically diagnostic finds show habitation in the Late Hallstatt period (Nos. 1a, 18a, 21, 22a, 26a, 33a, 36a, 50, 57, 62, 65, 66, 68, 69, 70, 71, 72a), even on Sv. Katarina above Nova Gorica (No. Chronology Establishing the beginning and the duration of a settlement, as well as the (dis)continuity in habitation would require research excavations in practically all the discussed settlements, but also on all potential locations suitable for habitation,71 and even more so comprehensive publications of the results of the investigations conducted thus far. Of the best and most extensively investigated settlement at Most na Soči (No. 1a), the excavated eastern part dates entirely to the Late Hallstatt period (6th–4th centuries BC). Albeit in a more modest extent, it was also inhabited later, in the Late La Tène period, i.e. the late 2nd and the 1st century BC (three houses),72 and again more intensely in the Roman period (fifteen houses). The remains of the four buildings investigated in 2001 at Maregova guna in Most na Soči, in the westernmost part of the habitation area close to the confluence of the Idrijca and the Soča, also date to the Late Hallstatt period (6th and 5th centuries BC).73 The location of the settlement from the previous, i.e. Early Hallstatt period, has as yet not been established.74 The earliest habitation traces, which were unearthed in the westernmost part of the promontory and on the lowest terrace of the Idrijca close to its confluence with the Soča, date to the Late Bronze Age and show a different type of construction, using posts and wattle-and-daub walls.75 This indicates that the settlement was likely inhabited neither continuously nor equally intensely through the periods, but also that it reached its maximum extent in the Late Hallstatt period. A clearer insight can be gained from the associ ated cemeteries, but these were only found at seven settlements: Most na Soči, Kobarid and Podbela in upper Posočje (Nos. 1b, 18d, 22b), Jereka, Lepence and Bohinjska Bistrica in Bohinj (Nos. 33b, 35b, 36b), as well as Godovič (No. 72b). In the Late Bronze Age, settlements also existed on Sv. Volar, an elevation above Robič in the Ko barid area (No. 21), on the Zuccola castle hill on the right bank of the Nadiža/Natisone north of Cividale del Friuli/Čedad,76 on Grad near Levpa, j 76 The 1989 archaeological excavations of the medieval Castle Zuccola yielded pottery sherds dating between the 74 Indirect evidence that the settlement was inhabited in the Early Hallstatt period comes from the graves of the Sv. Lucija I phase found on the left bank of the Idrijca (cf. Marchesetti 1886; id. 1893; Teržan, Lo Schiavo, Trampuž-Orel 1984–1985). The architecture and internal layout of settlements 53), as can be inferred from the pottery reminiscent of that from the settlement at Most na Soči in fabric, certain forms and primar ily the decoration of plain applied cordons and combing.78 The Early Hallstatt phase, which has been positively identified in several cemeteries (Fig. 9), is markedly less clear in the settlements. Most of these went on to be populated in the subsequent periods – La Tène and/or Roman pe riods, Late Antiquity and the Early Middle Ages. Having said that, the continuity from the Early to the Late Iron Age has as yet not been confirmed in any habitation context, where finds from the Late La Tène period predominate with rare exceptions, indicating decline in habitation in the Middle La Tène period. ; ; , , p ) 75 Svoljšak 1988–1989; Mlinar 2020a, 119–120, Pl. 15A. 73 Mlinar, Klasinc, Knavs 2008, 203. 71 Stray finds came to light by chance at certain loca tions suitable for habitation, such as Vrsno – Strničelo (Strenčl), Trnovo ob Soči – Trnovšček, Debenje – sv. Jakob, Goljevica – sv. Volbenk, Tolminski Lom – Kal, Dolenja Trebuša – Sovodenj (Fig. 11: No. 14, 20, 44, 45, 47, 56). 72 Dular 2018 79 There is no data available on some of the cemeteries/ graves unprofessionally excavated in the past that would allow us to positively attribute them to the Early Iron Age, e.g. the cemetery at Pod Cerkvijo near the village of Sedlo (Osmuk 1985, 297) and 36 urn graves that A. Vuga excavated at Most na Soči, on the promontory at the confluence of the Idrijca and the Soča (Marchesetti 1886, 97). Allegedly, there are several other potential cemetery locations, e.g. at Doblar, Modrej (ANSl, 117, 124), Libušnje near the cem eterial church of St Lawrence, at Smast (Knific et al. 2021, 20, 22), Police (Flego 2005), Krnice/Šance above Poljubinj/ Žabče (Mlinar, Turk 2016, 28–29) and Pečine (Mlinar et al. 2018, 26). These data, however, are not archaeologically verified, hence their dating is unclear. Bovec. Other funerary finds in the wider area are men tioned at the foot of Mt Rombon, at Kasarne, Bisli along the road to Plužna, and at Ograjnice in Bovec – Kaninska vas (Svoljšak 2002, 272, 276), which would indicate the existence of another settlement besides that on Ravelnik. CEMETERIES Although the number of known cemeteries and settlements from the Hallstatt period is equal, two thirds of these are not mutually interconnected. The ratios according to landscape units are the following: almost twice as many cemeteries in Miha MLINAR, Sneža TECCO HVALA 456 Jelenšek near Godovič (No. 72b) lies on a saddle close to the settlement. At much higher altitudes, small groups of graves were excavated near Krn (No. 13) and in Čadrg (No. 8), an isolated grave also in Rut (No. 39). Their location is associated either with the pathways connecting Posočje and Bohinj or with the proximity of iron ore deposits. There are no habitation traces known in their vicinity, which suggests they belonged to unfortified settlements. On Šentviška planota, the cemetery at Jerovca (No. 61) may have belonged to a posited settlement on the elevation of Dobje, where stonework ramparts and terraces are visible, but not yet archaeologically verified. The graves at Lipce/Prevala (No. 63) could be associated with a small settlement on Berlotov rob near Šentviška Gora. There are no observable habitation traces in the vicinity of the cemetery at Na Dobcu near Daber (No. 64). comparison with settlements are known in the high-altitude area (cf. Fig. 5 and 9), the same goes for Idrijsko hribovje with Šentviška planota, no cemeteries and six settlements have been recorded on Banjška planota and Kanalski Kolovrat, a single cemetery has been noted in Cerkljansko.79 80 Marchesetti (1903, 90) mentions a prehistoric settle ment on Kozlov rob. See Note 15. 84 Marchesetti 1886; id. 1893, 133–141; also see Ber gonzi et al. 1981; Boiardi 1983. Location and size Similar as settlements, the largest burial grounds are also known in the upper valleys of the Soča/ Isonzo and Nadiža/Natisone (Fig. 9), but they were excavated to different degrees and their size is a matter of conjecture. The largest excavations were conducted at Most na Soči, Kobarid and Tolmin, as well as Dernazzacco near Gagliano and S. Pietro al Natisone/Špeter. The Early Iron Age cemetery at Most na Soči (No. 1b) extended across the terraced slope on the opposite bank of the Idrijca to the settlement. At Kobarid, the cemetery was located on the river terraces of the Soča at the foot of the settlement on Gradič (No. 18d). The necropolis in Tolmin (No. 5) spread across the river terrace above the confluence of the Soča and Tolminka, at the foot of the isolated hill of Kozlov rob, on which contem porary habitation has not been reliably identified, i.e. the associated settlement has as yet not been found.80 The grave unearthed at Na Raduljah in Bovec (No. 26b) was located on a terrace along a stream, but the distance of 3 km as the crow flies shows that it obviously did not belong to the set tlement on Ravelnik.81 Other known cemeteries 82 Svoljšak 1973; Teržan, Trampuž 1973; Boiardi 1983, 164–166; Gabrovec 1987, 138–141. 83 See Teržan in this volume; for Kobarid, see the contribution by Kruh. 81 The cemetery associated with the settlement on Ravelnik could be located on a nearby site known under the toponym of Gomilce (Na Gomilcah – translated as ‘Little Barrows’) in Mala vas, in the southeastern part of 79 There is no data available on some of the cemeteries/ graves unprofessionally excavated in the past that would allow us to positively attribute them to the Early Iron Age, e.g. the cemetery at Pod Cerkvijo near the village of Sedlo (Osmuk 1985, 297) and 36 urn graves that A. Vuga excavated at Most na Soči, on the promontory at the confluence of the Idrijca and the Soča (Marchesetti 1886, 97). Allegedly, there are several other potential cemetery locations, e.g. at Doblar, Modrej (ANSl, 117, 124), Libušnje near the cem eterial church of St Lawrence, at Smast (Knific et al. 2021, 20, 22), Police (Flego 2005), Krnice/Šance above Poljubinj/ Žabče (Mlinar, Turk 2016, 28–29) and Pečine (Mlinar et al. 2018, 26). These data, however, are not archaeologically verified, hence their dating is unclear. 80 Marchesetti (1903, 90) mentions a prehistoric settle ment on Kozlov rob. See Note 15. 81 The cemetery associated with the settlement on Ravelnik could be located on a nearby site known under the toponym of Gomilce (Na Gomilcah – translated as ‘Little Barrows’) in Mala vas, in the southeastern part of Main features of the mortuary practices Flat cremation burial in a simple pit with the cremated remains and pyre ashes strewn across the bottom, stone slab as grave cover and marker and the absence of weapons among grave goods are the main distinguishing features that reflect the originality and specificity of the Sveta Lucija group within the Hallstatt culture.82 The two largest burial grounds with the longest span – at Most na Soči and Kobarid (Fig. 9) – hold the greatest potential for different multifaceted analyses;83 it is a potential not fully explored, as the finds and data have as yet not been wholly published. In addition to describing 2950 graves and a small selection of finds excavated up to 1892, Marchesetti also wrote a synthetic overview of the structure and the rite of burial.84 He stated that the human bone remains were only rarely thor oughly cremated, some only barely. The patches of burnt remains and burnt earth with an abundance of charcoal, pottery sherds and pieces of bronze 81 The cemetery associated with the settlement on Ravelnik could be located on a nearby site known under the toponym of Gomilce (Na Gomilcah – translated as ‘Little Barrows’) in Mala vas, in the southeastern part of Settlement in the Posočje/Sveta Lucija group – new sites and insights 457 The characteristics of the cemetery and graves, as well as burial rite parallel to these revealed by the early excavations were also observed during the recent investigations at the northern fringes of the vast cemetery, the only difference is that one site (Repelc) revealed Late Hallstatt and later burials – from the La Tène and Roman periods – as well as an early medieval pit. In addition to cremations, there was an inhumation burial, from the Roman period. The recent investigations also unearthed a cremation site (Fig. 10), as well as a cemetery drystone wall.87 Only three of the altogether 87 graves at the Pucarjev rob and Repelc sites were urn burials; two of these held the remains of adult women with precious goods, the third one an adult person of indeterminable sex. The amount of cremated bones in graves varied greatly, from 1 to 523 g. Two cremation burials were associated with unburnt horse bones and teeth. Main features of the mortuary practices Upon cremation, the bones were col lected and transferred to the grave pit together with the wood charcoal of the pyre. The grave pits varied in size and depth, they usually held the remains of a single individual, exceptionally two (in five cases). The pits were covered by one or more slabs and stones of different rocks (limestone, marls, conglomerate). Only a third of the burials stood out from the usual practice. These include five inhumations, with parts of human skeletons found inside, and two burials of horse skeletons. Just under one tenth (9.5%) are urn burials. Only 13.2% of grave pits were not covered by a slab or stones, in some cases one slab covered two graves. There were also roughly thirty groups (1%) covered by a cairn, the largest among them (10 × 5 m) covering 41 burials (25 adults and 16 children). In 58 graves (2%), the walls of the pit were lined with stones or the stone slabs were arranged to form a cist; 21 of these held urns. There were also lines of stones in the necropolis that must have been placed there with a different purpose (possibly as boundary markers), as no burials were found underneath. Using an estimate of the biological age of the deceased, Marchesetti attempted to outline the demographic profile of the population. He recorded 26% children, the graves of which were mostly without goods. Of the graves of the adult population, less than a tenth had no grave goods, 14% only held pottery. The pieces of weapons or tools represent a 2.3% share of the grave goods, but were often found beside or on top of the grave and some date to the La Tène period. The burial rite in the Tolmin cemetery (No. 5) is not substantially different. Of the 465 investigated graves, none had a stone cist and one held an urn burial that the anthropological analysis and the pin among the grave goods show to be that of a man. It has a higher share of graves with pottery as the sole grave good (30%) and a lower share of graves without goods (10%). The occurrence of weapons and tools is similar (2.2%). Most of these are knives found together with both typically male and female goods. One bronze spearhead was found in a grave and another one was deposited between the graves, as was a razor. Main features of the mortuary practices Weapons and tools came to light in the graves from the La Tène period, one knife was placed in an urn grave (anthropologically identified as the burial of a woman) from the Late Hallstatt period. At the Repelc site, pieces of weapons and tools were also found at the cremation site and in a mixed layer that covered the graves. indicate that the deceased were cremated in their costumes at specific spots inside or outside the necropolis. Upon cremation, the bones were col lected and transferred to the grave pit together with the wood charcoal of the pyre. The grave pits varied in size and depth, they usually held the remains of a single individual, exceptionally two (in five cases). The pits were covered by one or more slabs and stones of different rocks (limestone, marls, conglomerate). Only a third of the burials stood out from the usual practice. These include five inhumations, with parts of human skeletons found inside, and two burials of horse skeletons. Just under one tenth (9.5%) are urn burials. Only 13.2% of grave pits were not covered by a slab or stones, in some cases one slab covered two graves. There were also roughly thirty groups (1%) covered by a cairn, the largest among them (10 × 5 m) covering 41 burials (25 adults and 16 children). In 58 graves (2%), the walls of the pit were lined with stones or the stone slabs were arranged to form a cist; 21 of these held urns. There were also lines of stones in the necropolis that must have been placed there with a different purpose (possibly as boundary markers), as no burials were found underneath. Using an estimate of the biological age of the deceased, Marchesetti attempted to outline the demographic profile of the population. He recorded 26% children, the graves of which were mostly without goods. Of the graves of the adult population, less than a tenth had no grave goods, 14% only held pottery. The pieces of weapons or tools represent a 2.3% share of the grave goods, but were often found beside or on top of the grave and some date to the La Tène period. indicate that the deceased were cremated in their costumes at specific spots inside or outside the necropolis. 87 Mlinar 2020a. 85 Graves Sz 592 and 2304: Teržan, Lo Schiavo, Tram puž-Orel 1984–1985, 120, 363, Pl. 51; 52A; 242B. 86 These estimates use the database that Sneža Tecco Hvala had created using the data published in Teržan, Lo Schiavo, Trampuž-Orel 1984–1985. 86 These estimates use the database that Sneža Tecco Hvala had created using the data published in Teržan, Lo Schiavo, Trampuž-Orel 1984–1985. 85 Graves Sz 592 and 2304: Teržan, Lo Schiavo, Tram puž-Orel 1984–1985, 120, 363, Pl. 51; 52A; 242B. Dating its maximum extent in the Late Hallstatt, and in the investigated areas revealed no traces from the previous period.91 The earliest Iron Age burials (from the Sv. Lucija I phase) concentrate in the upper valleys of the Soča/Isonzo and Nadiža/Natisone, more precisely at Tolmin, Most na Soči, Kobarid, Ladra (Pl. 1: 1–6) and S. Pietro al Natisone/Špeter (Fig. 9); this area may be seen as the early settlement core. There are no known burials on the terri tory of the Posočje community from the period preceding this phase. At the transition to the Late Hallstatt period, new burials occur at Srpenica in the Bovec area (No. 25a), at Koritnica and on Šentviška planota (Nos. 40, 61, 64) (Fig. 9). The beginnings of the cemeteries in Bohinj (Nos. 32–37) also date to this time.92 In the final phases of the Early Iron Age (Sv. Lucija IIb–IIc), burial thus also began in the more remote and high-altitude areas (Nos. 8, 13, 22b, 39, 58, 63, 72b), which indicates an expansion of habitation into the side valleys and high mountains. p g p At Most na Soči and Kobarid (Nos. 1b, 18d), burial continued uninterruptedly into the Late Hallstatt period (Sv. Lucija II), whereas it stopped at Tolmin (No. 5). Only few finds are known from later periods at S. Pietro al Natisone/Špeter (No. 41).88 At that time, burial just began at Dernaz zacco (No. 42), and continued into the La Tène period. This was also a time when Most na Soči, in the upper Soča valley, witnessed a rise and peak of prosperity; estimations show that the greatest number of burials dates to the 6th and 5th cen turies BC (Sv. Lucija IIa and IIb). The majority of urn burials and stone cists dating to the Late Hallstatt period also indicates structural changes taking place at this time.89 This is supported by a rough comparison with the Early Hallstatt Tol min cemetery (No. 5), roughly 5 km away, which revealed a single urn burial and no stone cists. Furthermore, there are noticeable differences in the grave goods: the Tolmin cemetery included approximately 10% graves without grave goods, the Most na Soči cemetery twice as much (roughly 23%), which suggests an increase in graves without goods in the Late Hallstatt period. The cemeter ies at Koritnica and Jerovca (Nos. g 96 Svoljšak, Pogačnik 2001–2002. A bronze spearhead was also found in the Ladra cemetery (No. 16; Pl. 1: 6). 88 At S. Pietro al Natisone/Špeter, a Certosa fibula, as well as an iron socketed and a shaft-hole axe are known from the Late Hallstatt period (Pettarin 2006a, Cat. No. 174, 393, 588; ead. 2006b). 98 Guštin 1991, Pl. 23: 6,7,11,15; 24: 15,16; 25: 1,7,8; 26: 1,2; 27: 11,12. y 97 See the contribution by Bratina, Laharnar, Svoljšak in this volume. p ( 95 Guštin 1991, 30–33, Fig. 7 and 21. Main features of the mortuary practices Szombathy’s excavation of the cemetery paint a similar picture. Of the 2478 graves he had excavated, less than one tenth (7.5%) were urn burials, over a quarter (27%) had no grave goods, pottery as the sole good occured in 16%. Also similar is the share of graves lined with stones or graves with stone slabs arranged to form a cist (1.4%), eleven of them holding urns. An ustrinum in two spots is reported. Two graves held inhumation burials, one of these a horse and its tack.85 Weapons or tools, in most cases knives, are noted in 65 graves (2.6%) or in their immediate vicinity.86 The same characteristics and structure of buri als has been observed in the smaller cemeteries at S. Pietro al Natisone/Špeter and Dernazzacco in the valley of the Nadiža/Natisone (Nos. 41, 42), at Koritnica (No. 40), Jerovca on Šentviška planota (No. 61) and Bitnje in Bohinj (No. 34). They include the odd urn burial, at Koritnica and Bitnje also few inhumations. The graves at Čadrg in Tolminsko (No. 8), Krn in Kobariško (No. 13) and Jelenšek near Godovič (No. 72b) have a markedly higher share of burials with weapons; this share is also higher at Koritnica (11%). 458 Miha MLINAR, Sneža TECCO HVALA 94 A piece of an iron wire fibula of the Middle La Tène construction was found at Jerovca, at the nearby spring, but this is insufficient evidence of burial continuing into the La Tène period (Laharnar, Mlinar 2013, Fig. 11: 3). 92 Grave 2 from Jereka and Grave 5 from Bitnje (Ga brovec 1974, Pl. 4: 5,6; 11: 1–7) may date towards the end of the Early Hallstatt period. g , , ; ; 90 Teržan, Trampuž 1973; Bergonzi et al. 1981, 214–215, 226–228, 246–248; Boiardi 1983, 165–166; ead. 1984; also see Teržan in this volume. 93 Guštin 1991, Fig. 1; Božič 1999a, 203, Fig. 1; also see Mlinar, Gerbec 2011, 23; Mlinar 2020a, 120–121; id. 2020b. 91 Dular, Tecco Hvala 2018, 109–130; Dular 2018. 88 At S. Pietro al Natisone/Špeter, a Certosa fibula, as well as an iron socketed and a shaft-hole axe are known from the Late Hallstatt period (Pettarin 2006a, Cat. No. 174, 393, 588; ead. 2006b). 89 Bergonzi et al. 1981, 187–188, Tab. 23; 24; 26. 90 Teržan, Trampuž 1973; Bergonzi et al. 1981, 214–215, 226–228, 246–248; Boiardi 1983, 165–166; ead. 1984; also see Teržan in this volume. 89 Bergonzi et al. 1981, 187–188, Tab. 23; 24; 26. Stray finds The artefacts found by chance over the last three decades further advance our knowledge of the set tlement in the region. They significantly raise the number of known archaeological sites (Fig. 3; 4; 11). Given that most came to light with the help of metal detectors, however, the locations of these sites are not always reliable, the circumstances of the find most often not documented and, when left unverified by archaeologists, the context unclear. As such, they are difficult to identify in terms of character and significance (whether they are funerary, habitation or votive finds, or even lost items). The archaeological verification of certain locations has revealed the existence of graves (Fig. 9: Nos. 8, 13, 16 (Pl. 1: 1–6), 22b, 25a,b, 39 (Pl. 2: 11), 72b). Some artefacts, found on elevations with ramparts or terraced slopes, could originate from habitation contexts (Fig. 5: Nos. 21, 22a, 26a (Pl. 2: 5), 66, 68, 69, 71). y y g A different significance may be ascribed to the finds from the gravel banks of the River Soča (Nos. 4, 18g,h,i). They are iron spearheads and an axe (Pl. 3: 1–4,8),106 while a Certosa fibula (Pl. 2: 3) came to light on the eastern shore of Lake Bohinj at Stara Fužina (No. 30). The close correlation between the sites of the Posočje com munity and sources of water may suggest that the people here believed in miraculous powers and performed purification or votive rituals in their proximity.107 This hypothesis then leads to the possibility of the finds from the immediate proximity of riverbeds and lake may be associated with votive offerings and worshipping. Having said that, water sources were primarily linked to the daily water supply. Moreover, the river valleys not squeezed between steep slopes also served as lines of communication that provided easiest passage through the mountainous landscape. We should therefore, first and foremost, see these stray finds Most stray finds have been recorded in the immediate vicinity and in the hinterland of Ko barid and Tolmin (Fig. 11). Parts of the costume (fibulae, pin, finger ring) and weapons/tools (axe) even came to light above 1000 m asl, on high-altitude pastures and along paths towards alpine pastures (Nos. 6, 7, 10, 11, 12, 28; Pl. 1: 7,9; 2: 2,4,10; 3: 6). Given their setting, we may relate them to transhumance or ore collection, as well as paths leading to Bohinj. Dating 40, 61), which are later in date, lend support to this observation – their share of graves without goods is higher (roughly 18 to 22%) compared with Tolmin. In contrast, most of the richest graves at Most na Soči dates to this very time, the goods in which include imports that even imply a greater social stratification.90 Artefacts and raw materials im ported from distant lands also came to light in the settlement at Most na Soči, which reached Almost all the cemeteries in the upper valleys of the Soča and Nadiža/Natisone, as well as the Cerkljansko area, revealed burials or finds from the La Tène period93 except at Tolmin (No. 5), Jerovca (No. 61)94 and Jelenšek (No. 72b). La Tène graves even predominate at Idrija pri Bači (No. 58); they are largely located in the south-eastern part of the cemetery, whereas those from the Hallstatt period lie in the north-western part.95 A comparison of the structure of burials in the Tolmin necropolis as the representative of the early development phase of the Posočje com munity (Sv. Lucija I) with the burials from the late phase (Sv. Lucija IIc) shows greatest differ ences in the occurrence of weapons. The Tolmin cemetery only yielded two (bronze) spearheads,96 whereas the cemetery at Jelenšek, for example, revealed weapons in almost half of the inves tigated graves,97 the Hallstatt-period graves at Idrija pri Bači included 37.5% such graves,98 the cemetery at Čadrg had weapons in three of the four investigated graves, one of them dating to Settlement in the Posočje/Sveta Lucija group – new sites and insights 459 centuries BC.101 A similar site has been recorded at V plazeh in the Trenta area (No. 29), at 1542 m asl, where drystone foundations of a building were found, as well as a depression filled with burnt remains that included sherds of prehistoric pottery and a piece of a band fibula from the 6th century BC (Pl. 2: 6).102 An ingot of copper alloy was collected somewhere on the hill of Kobilnik, in the Tolmin area (No. 7), though not together with the Certosa fibulae found on the same hill (Pl. 2: 2).103 Similarly, the ingot found in the area of Fiščevo near Ajba along the Soča (No. 46) is also believed to have been found some 50–100 m away from a Certosa fibula (Pl. 102 We thank Jana Horvat (Inštitut za arheologijo ZRC SAZU) for the information and the drawing of the fibula. For the typo-chronological identification, see e.g. Tecco Hvala 2012, 243–244, Note 1029 with references to the examples from the cemetery at Most na Soči. Dating 2: 7).104 The bronze ingots found at the church of St Jacob above Debenje (No. 44), in Kambreško pogorje, may also date to the Early Iron Age.105 the La Tène period.99 This increase in weaponry in graves may mirror the turbulent times that marked the end of the Hallstatt and transition to the La Tène period.100 101 Ogrin 2020, 63, Fig. 3; for the typo-chronological identification, see Teržan 1976. 107 Similarly as elsewhere in the south-eastern Alps, the Soča and its gravelly banks revealed pieces of weapons from the Late Bronze Age that can also be interpreted as votive finds (cf. Gerbec, Mlinar 2011). 106 The iron axe was found during sand separation near Volče, with the sand originating from the Soča gravel between Idrsko and Tolmin (No. 4; Pl. 3: 8). 99 Mlinar 2020b. 100 Teržan, Trampuž 1973, 440. 105 Nanut 2018, Fig. 4; Pl. 2–4. y 103 Mlinar, Turk 2016, 23–24, Cat. No. 22 and 24. 104 Gerbec 2021, 137–138. p y 103 Mlinar, Turk 2016, 23–24, Cat. No. 22 and 24. 104 Gerbec 2021, 137–138. , , , 104 Gerbec 2021, 137–138. Stray finds We have already mentioned the seasonal camp in the mountains of Bohinj, i.e. at Dolga Planja on Mt Vogel (No. 38), located at 1680 m asl; the site revealed the remains of a building from the Early Middle Ages and a layer of burnt remains with iron ore, animal bones and pottery sherds from the Early Iron Age, the turf in the immediate vicinity also revealed a Type XI Certosa fibula from the 5th/4th Miha MLINAR, Sneža TECCO HVALA 460 as indicators of pathways and the exploitation of natural resources, i.e. ordinary human activities in the hinterland of settlements. recovered at Log pod Mangartom (No. 27; Pl. 1: 11), is a rare example of its kind in the region,113 and has parallels from Notranjska and among the bracelets typical of the Dolenjska costume from the late 7th to the 5th century BC.114 Also not usual for the Posočje costume is the bronze crossbow fibula with a forward-facing horse head (Pl. 2: 1) that came to light on Jajnkovec on the left bank of the Soča near Kobarid (No. 19); an identical example was found in the Kobarid cemetery, a similar one with a ram head on the foot terminal at Most na Soči.115 Such fibulae are known from the Dolenjska cultural circle in the 5th century BC, where they formed part of the male costume.116 With regards to the examples from Posočje, we should mention two similar fibulae from neighbouring regions, one from Podgora at the River Poljanska Sora and the other from Gurina in Kärnten, Austria.117 Other stray finds of fibulae (Fig. 11) belong to the Certosa fibulae of Types VI (No. 12; Pl. 2: 10), X (Nos. 46, 14, 55, 20; Pl. 2: 7,8,12,13), XI (No. 60; Pl. 2: 9) and XIII (Nos. 7, 28, 30; Pl. 2: 2–4), which were worn by men in the final phases of the Early Iron Age, similarly as weapons, Type X even later.118 Dating to the last phase is the animal fibula of the Early La Tène construction (No. 67), which is most likely a product of a local workshop, as similar examples are limited with rare exceptions to the area of the Posočje community, where they were presumably made using the contemporary southern Alpine Early La Tène fibulae as models.119 The stray finds comprise pieces of costume (fibulae, pins, bracelets, finger rings, pendants, glass beads) and weapons (axes and spearheads). 117 Gabrovec 1966a, 34, 38–39, Map 3 and list of sites with references. On the connection with the Carinthian or Frög/Breg group, see the contribution by Gleirscher in this volume. 113 Similar fragments are known from Repelc in Most na Soči (Mlinar 2020a, 127, Pl. 29: 4,5) and from the cem eteries along the Nadiža/Natisone (Pettarin 2006a, Pl. 20: 298,317; 21: 328,330; ead. 2006b). 118 Teržan 1976; also cf. Teržan, Lo Schiavo, Trampuž-Orel 1984–1985, Pl. 7B; 27F; 29D; 39B; 47D; 50B; 124A; Mlinar 2020b, Fig. 5 and 8; Guštin 1991, Pl. 23: Grave 30; 25: Grave 37; 26: Grave 40. 114 For Notranjska, see Guštin 1979, Pl. 22: 8–22; 66: 24–26,28,29; for Dolenjska, see e.g. Tecco Hvala 2012, Fig. 109: 6 (Variant IIb), 294, 298, Notes 1270–1273. ( ) 115 Marchesetti 1903, Pl. 18: 5; also see Mlinar 2020a, 122–123, Pl. 42: 1. 116 Tecco Hvala 2012, 262–263, Fig. 99: 5,6. 119 Nanut 2021, 85–90, cf. Fig. 1 and 6 (maps), and Fig. 7. 111 For Krn, see the contribution by Laharnar, Mlinar (Pl. 4: 1) in this volume, for Dernazzacco, see Pettarin 2006a, Pl. 26: 451; ead. 2006b. 112 Laharnar 2018a, 213; Mlinar 2020a, 115; also see Pavlin 2014. 108 Teržan, Trampuž 1973, 438, App. 1; Teržan 2002, 89 (pins of Types III and V after Pogačnik). Also cf. Most na Soči: Teržan, Lo Schiavo, Trampuž-Orel 1984–1985, Pl. 14G: 1; 37G; 43C: 1; 75E: 1; 95B: 1; 101B: 1; 102B: 1; 112E: 3; 119A: 1; 126R: 2. 110 Nanut 2021, 87–89. 108 Teržan, Trampuž 1973, 438, App. 1; Teržan 2002, 89 (pins of Types III and V after Pogačnik). Also cf. Most na Soči: Teržan, Lo Schiavo, Trampuž-Orel 1984–1985, Pl. 14G: 1; 37G; 43C: 1; 75E: 1; 95B: 1; 101B: 1; 102B: 1; 112E: 3; 119A: 1; 126R: 2. 109 Božič 2011, 248; Laharnar 2018a, 213–214; Mlinar 2020a, 135; also see Laharnar, Mlinar (Pl. 6 and 7) in this volume. 110 Nanut 2021, 87–89. 111 For Krn, see the contribution by Laharnar, Mlinar (Pl. 4: 1) in this volume, for Dernazzacco, see Pettarin 2006a, Pl. 26: 451; ead. 2006b. 112 Laharnar 2018a, 213; Mlinar 2020a, 115; also see Pavlin 2014. 109 Božič 2011, 248; Laharnar 2018a, 213–214; Mlinar 2020a, 135; also see Laharnar, Mlinar (Pl. 6 and 7) in this volume. 110 N t 2021 87 89 Stray finds The earliest among them are two bronze multi- knobbed pins (Pl. 1: 7,8), such as are characteristic of the Sv. Lucija Ib male costume in the Tolmin cemetery.108 One of the pins was found below Zeleni vrh high above Tolminske Ravne (No. 11) and the other at Tolminski Lom on Banjšice (No. 47), in an area that reportedly yielded a bronze fibula, now lost. All other finds date to the Late Hallstatt period (Sv. Lucija II), some could also be from the La Tène period. Parts of the female costume were the beads of yellow glass with layered blue-white eyes (Fig. 11: Nos. 9, 17; Pl. 1: 15–17), such as occur in the Sv. Lucija IIb and IIc phases, widely distributed in the 5th and 4th centuries BC.109 Two of the finger rings (Nos. 6, 24; Pl. 1: 9,10) are decorated with ring-and-dots and stripes of transverse incisions, an ornamentation common on the jewellery of the Posočje community and probably made in the local workshops in Sv. Lucija IIb and IIc.110 The same decoration can also be found on the pendant from Sedlo (No. 24; Pl. 1: 14), possibly representing a stylised female figure, and most closely resembling two pendants from Krn, in the Kobarid area, and from Dernazzacco, in the valley of the Nadiža/Nati sone, respectively.111 Anthropomorphic pendants are not common in the Posočje community, more frequent are the basket-shaped examples known from the Late Hallstatt graves and the settlement at Most na Soči.112 The stray finds discussed here were found in the Kobarid area (Nos. 15, 18g, 23; Pl. 1: 12,13,18). The bronze bracelet with overlap ping ends and decorated with stripes of incisions, y These finds indicate that activities in the moun tainous hinterland were mainly in the male do main. The stray finds also include iron shaft-hole Settlement in the Posočje/Sveta Lucija group – new sites and insights 461 axes (Nos. 2, 4, 10, 45; Pl. 3: 5–8);120 they have parallels in the funerary contexts at Most na Soči, Srpenica and Čadrg, occuring together with Type X Certosa fibulae, at Koritnica in the combination with the above-mentioned animal fibulae of the Early La Tène construction.121 They were also in use in the Late La Tène period, but in a slightly different form – with a hammer-like butt.122 It is more challenging to date the stray finds of iron winged axes (Nos. 9, 54, 56; Pl. 130 Nanut 2016; ead. 2018. Similar fragments of bronze shaft-hole axes, raw bronze, bar-shaped and flat ingots were also found in the settlement near the church of St Helen (Na Lupu) above Podbela, though it is not known whether they were found together and could hence be interpreted as a hoard (cf. Mlinar, Gerbec, Laharnar 2014, 30–31). 121 Teržan, Trampuž 1973, 440, Fig. 4: Map 3, App. 1; also see Kos 1973, Pl. 5: 1; 11: 1; Teržan, Lo Schiavo, Trampuž-Orel 1984–1985, Pl. 7B: 2; Laharnar, Mlinar 2019; Mlinar 2020b, Fig. 5. 120 Found in an area rich in iron ore, the shaft-hole axe from the Kal below Mt Tolminski Migovec may have been used as a tool (cf. Mlinar, Turk 2016, 25). Stray finds 3: 9–11),123 which at Idrija pri Bači (No. 58) occur in the graves from the Late Hallstatt and La Tène periods.124 There, they were found together with both weapons and tools, hence their function is ambiguous – they may have been used as weapons and/or tools. Much earlier ones are known from warrior graves in Dolenjska (at the very beginning of the Iron Age).125 In the community of central Friuli, they occur in graves together with serpentine fibulae from the late 7th and early 6th centuries BC.126 In Notranjska (Tržišče) and Friuli (Porpetto), they are also known from hoards dating to the middle and second half of the 6th century BC.127 finds are not published in their entirety) relate a large situla-like vessel containing iron weapons (one winged, one shaft-hole and four socketed axes, eight spearheads and a pointed tool or spear butt, a whetstone and an iron bracelet). The hoard from Rubbio shared a similar location on the slope of a hill and also held socketed axes. The parallel with the Porpetto hoard is in the deposition in a container, as well as winged axes, of which as many as 23 came to light at Porpetto.129 y g A hoard of a very different composition was unearthed with the help of a metal detector at Gastabil near Dolenje Ravne, in Cerkljansko (No. 67). In a small depression next to a natural rocky ridge, broken pieces of bar-shaped and flat bronze ingots were found together with bronze shaft-hole axes with high lead content. The latter spread at the transition from the 2nd to the 1st millennium BC from their area of origin on the Apennine Peninsula to western and central Slovenia, where they were in use all to the 6th century BC.130 Similar finds were found by chance at Kanalski Kolovrat, near the church of St Jacob above Debenje, lying in a depression on the west terrace just below the summit of the hill (No. 44).131 131 Nanut 2018. 132 Mitheilungen der k.k. Central-Commission (MZK) 24, 1898, 111 and MZK 27, 1901, 77; Guštin 1991, 11–12, Pl. 38–40. 122 E.g. Guštin 1991, Pl. 6: 2; 10: 11; 14: 4 etc.; also cf. Turk, Svetličič 2018 and the contribution by Laharnar, Mlinar in this volume (Pl. 13: 5; 14). 123 The iron axe from Javorca, winged and with a saddle-shaped transition to the blade, is similar to the finds in Trentino that are seen as Early Iron Age forms ( f M ti 1997 C t N 1986) 120 Found in an area rich in iron ore, the shaft-hole axe from the Kal below Mt Tolminski Migovec may have been used as a tool (cf. Mlinar, Turk 2016, 25). 121 Teržan, Trampuž 1973, 440, Fig. 4: Map 3, App. 1; also see Kos 1973, Pl. 5: 1; 11: 1; Teržan, Lo Schiavo, Trampuž-Orel 1984–1985, Pl. 7B: 2; Laharnar, Mlinar 2019; Mlinar 2020b, Fig. 5. 122 E.g. Guštin 1991, Pl. 6: 2; 10: 11; 14: 4 etc.; also cf. Turk, Svetličič 2018 and the contribution by Laharnar, Mlinar in this volume (Pl. 13: 5; 14). 123 The iron axe from Javorca, winged and with a saddle-shaped transition to the blade, is similar to the finds in Trentino that are seen as Early Iron Age forms (cf. Marzatico 1997, Cat. No. 1986). 124 E.g. Guštin 1991, Pl. 2: 3; 15: 5; 26: 2. 125 Tecco Hvala 2012, 111–114, Fig. 46: 1. 126 See the contribution by Vitri, Corazza (Fig. 2; 18; 20) in this volume. 127 Guštin, Božič 2021, 499–501, 505–508, Fig. 1 and 2: D. 128 Ib. 20) in this volume. 127 Guštin, Božič 2021, 499–501, 505–508, Fig. 1 and 2: D. 128 Ib. 129 Ib. 131 Nanut 2018. (cf. Marzatico 1997, Cat. No. 1986). 124 E.g. Guštin 1991, Pl. 2: 3; 15: 5; 26: 2. 125 Tecco Hvala 2012, 111–114, Fig. 46: 1. 126 See the contribution by Vitri, Corazza (F 129 Ib. 130 Nanut 2016; ead. 2018. Similar fragments of bronze shaft-hole axes, raw bronze, bar-shaped and flat ingots were also found in the settlement near the church of St Helen (Na Lupu) above Podbela, though it is not known whether they were found together and could hence be interpreted as a hoard (cf. Mlinar, Gerbec, Laharnar 2014, 30–31). 131 Nanut 2018. 132 Mitheilungen der k.k. Central-Commission (MZK) 24, 1898, 111 and MZK 27, 1901, 77; Guštin 1991, 11–12, Pl. 38–40. Cult-offering places A hoard of weapons similar to those from Rubbio, along the lower reaches of the Isonzo/Soča close to its confluence of the River Vipacco/Vipava, and from Porpetto along the Tagliamento, was found buried somewhere on the slope of Gradič at Kobarid (No. 18c), though it is a century and a half later (4th century BC) than the two.128 The reports (the Primary sources report that the roadworks con ducted towards the end of the 19th century on the left bank of the Soča, somewhere near the hamlet of Loga north of Bodrež, revealed a deep crevice filled with ashes that included pottery sherds, pieces of bronze vessels and jewellery, as well as iron weapons and tools (No. 49). The artefacts have a broad span, from the Late Hallstatt period (Sv. Lucija II), represented by several types of fibulae, pendants, finger rings, a bracelet, part of a handle and attachment of a situla, possibly also an iron winged axe, to the Late La Tène period represented by characteristic iron tools.132 The sources also note Miha MLINAR, Sneža TECCO HVALA 462 that there were no ashes or finds in the vicinity of the spot, suggesting we are not dealing with a cemetery. The natural setting would rather speak in favour of the interpretation as a hoard, as the primary source already proposed. However, hoards usually do not include ashes, hence the find might also be a cult place. of a small settlement (0.5 ha). The spectre of the recovered small finds also indicates a cult-offering place of a long duration, from the 6th to the 1st century BC, with a piece of a bronze sickle of an earlier date (11th/10th centuries BC);135 the most revealing are a bronze sceptre and a rectangular offering plaque of silver with an inscription in the Venetic script. A special offering place located among rocky faces, karren and abysses is presumed to have existed at Berlotov rob on Šentviška planota (No. 62). Cult-offering places Roughly 120 artefacts have been collected there through unprofessional detectorist activi ties over the last decades, and a bronze statuette of the goddess Isis was reportedly found there in the mid-19th century, but has since been lost.133 These finds also show a wide span – from the Late Hallstatt to the beginning of the Roman period – and include pendants, fibulae, iron tools and axes, as well as parts of bronze vessels similar to those from Bodrež. The site is heavily damaged, but we can still notice artificially levelled terrain and ter races that might have been used for habitation. A rough estimate of the surface suggests it would have been a very small settlement (0.2 ha), whereas the wealth and exceptional character of the finds are unusual for habitation context, suggesting that the interpretation of the site as a cult-offering place of a long duration seems more plausible. Among other things, this is supported by the pendants that might be seen as apotropaic, such as the anthropo- ornitomorphic pendant from the 5th/4th centuries BC with iconographic parallels in the depictions of the Mistress of the Animals, also in the silver disc from the late 2nd or the 1st century BC believed to show the phases of the moon (similar ones were also unearthed at Bodrež). Another piece of cor roborating evidence is the rim of a bronze situla with a Venetic inscription, as well as the now missing statuette of Isis.134 Another such site is known on the plateau of Šentviška planota: Vrh gradu near Pečine (No. 57) located just over 3 km west of Berlotov rob. The site lies on the edge of the plateau, where the terrain descends steeply towards the Idrijca and the ravine of a torrential stream. The narrow rocky ridge shows artificial terraces with traces of buildings and the rampart A similar offering plaque of bronze came to light during the investigations of the fortified Late Antique settlement on Tonovcov grad near Kobarid (No. 18f), where the excavations of three churches, a cistern and residential buildings from the Late Antiquity also brought to light modest prehistoric remains. 133 Božič 2011, 265; Laharnar, Mlinar 2014; Laharnar, Turk 2018, Fig. 164; 188–190; 191; 193; 195; 196; Mlinar et al. 2018, 17–19, Fig. 11, Cat. No. 28–31, 37, 42–44, 46–48, 50, 51, 55–59, 61, 66–68, 71, 73, 75–77, 79–83. 134 Laharnar, Mlinar 2014; Laharnar, Turk 2018, 166–168; also see Turk, Božič, Istenič 2009, 57–59. 135 Božič 1999b; Turk, Božič, Istenič 2009, 57–59; Božič 2011, 256–258; Laharnar, Mlinar 2014; Laharnar, Turk 2018, Fig. 192; Mlinar et al. 2018, 24–26, Cat. No. 32–36, 38, 40, 45, 52–54, 60, 62–65, 69, 70, 72, 74, 84–86, 92–97, 100–111. 135 Božič 1999b; Turk, Božič, Istenič 2009, 57–59; Božič 2011, 256–258; Laharnar, Mlinar 2014; Laharnar, Turk 2018, Fig. 192; Mlinar et al. 2018, 24–26, Cat. No. 32–36, 38, 40, 45, 52–54, 60, 62–65, 69, 70, 72, 74, 84–86, 92–97, 100–111. 136 Božič 2011, 239–260, Fig. 6.2. 137 Osmuk 1997; ead. 1998; Božič 2011, 262, Fig. 6.17: 1,8. The monograph is in preparation. We thank Nada Os muk and co-authors for allowing us to examine the finds. 138 Toškan 2011. 137 Osmuk 1997; ead. 1998; Božič 2011, 262, Fig. 6.17: 1,8. The monograph is in preparation. We thank Nada Os muk and co-authors for allowing us to examine the finds. 133 Božič 2011, 265; Laharnar, Mlinar 2014; Laharnar, Turk 2018, Fig. 164; 188–190; 191; 193; 195; 196; Mlinar et al. 2018, 17–19, Fig. 11, Cat. No. 28–31, 37, 42–44, 46–48, 50, 51, 55–59, 61, 66–68, 71, 73, 75–77, 79–83. 136 Božič 2011, 239–260, Fig. 6.2. 134 Laharnar, Mlinar 2014; Laharnar, Turk 2018, 166–168; also see Turk, Božič, Istenič 2009, 57–59. Cult-offering places Jewel lery pieces consisted of one bronze fibula and four different bracelets (bronze ribbed, bronze knobbed, hollow bronze with relief decoration, one iron). Further finds include pottery sherds, a decorated iron plaque, pieces of iron slag and bronze melt. The burial was covered and bordered by stones. Parallels for the finds come from the central European Celtic area and the burial may be presumed to have been a one-time ritual act of a military nature.139 In the valley of the Nadiža/ Natisone, other cult places with Celtic weapons from the Middle La Tène period are presumed on Monte Barda in S. Pietro al Natisone/Špeter (No. 41), also in the hillfort on Madonna delle Grazie with the cemetery at Dernazzacco at its foot (No. 42), as well as at the church of St Matthias near Costne – Grimacco / Hostne – Garmak, where a bronze statuette and a Celtic silver coin came to light.140 In the Cerkljansko area, notes on the find of a (now missing) statuette and chance finds of bronze pendants from the Late Hallstatt period, as well as an iron sickle, hoe and spearhead from the Late La Tène period in addition to finds from the Roman period, suggest that a pre-Roman cult place probably existed on Gradišče in Cerkno (No. 68). In Posočje, surviving inscriptions in the Venetic script also point to cult places.141 y It would be unusual for Most na Soči, with the largest settlement and cemetery in Posočje, not to have sacred places. One of these has been identified underneath the walls of a Roman building com plex in the north-eastern part of the settlement. Although the Roman walls largely destroyed the earlier remains, there did survive a roughly a metre wide strip of burnt remains that reached 75 cm deep to the underlying geology. It was delimited in the northwest and northeast by lines of stones that suggested a quadrangular plan (6 × 4 m). The fill did not appear to be layered and contained, in spite of its limited extent, over 200 artefacts of a composition different from the assemblages recovered from any other building. The artefacts were predominantly cut into pieces and exposed to fire. 142 Josipovič, Gaspari, Miškec 2012. 143 Svoljšak, Dular 2016, 71–74, Fig. 57–60; Pl. 26; 27 (House 6/Phase 2); Dular, Tecco Hvala, 2018, 79–85; Laharnar 2018a, 224–234, Fig. 7–9; Motella De Carlo 2018, 379; Grömer et al. 2018; Toškan, Bartosiewicz 2018, 491, Tab. 11. 144 Svoljšak, Dular 2016, 67–70, Fig. 50–56; Dular, Tecco Hvala 2018, 78, Fig. 74; Svoljšak 2018, Fig. 11. 139 Mlinar, Gerbec 2011. 140 Rupel 2004, 67–69. 141 See Turk, Božič, Istenič 2009, 57–59; also see the contribution by Luka Repanšek in this volume. Cult-offering places Further finds include pottery sherds, a decorated iron plaque, pieces of iron slag and bronze melt. The burial was covered and bordered by stones. Parallels for the finds come from the central European Celtic area and the burial may be presumed to have been a one-time ritual act of a military nature.139 In the valley of the Nadiža/ Natisone, other cult places with Celtic weapons from the Middle La Tène period are presumed on Monte Barda in S. Pietro al Natisone/Špeter (No. 41), also in the hillfort on Madonna delle Grazie with the cemetery at Dernazzacco at its foot (No. 42), as well as at the church of St Matthias near Costne – Grimacco / Hostne – Garmak, where a bronze statuette and a Celtic silver coin came to light.140 In the Cerkljansko area, notes on the find of a (now missing) statuette and chance finds of bronze pendants from the Late Hallstatt period, as well as an iron sickle, hoe and spearhead from the Late La Tène period in addition to finds from the Roman period, suggest that a pre-Roman cult place probably existed on Gradišče in Cerkno (No. 68). In Posočje, surviving inscriptions in the Venetic script also point to cult places.141 A special offering place of a long duration was recently unearthed in Bohinj, on the eastern interior a 50 cm deep pit filled with burnt remains. The fill held finds from different periods: two pendants from the Late Hallstatt period, pieces of costume, pottery and coins from the La Tène and Roman periods. The finds suggest that the place was in use from the 5th century BC to the late 4th century AD.142 humerus, though its location suggests it may not be linked or contemporary with the horse burial. The finds also include several pieces of horse gear (two iron bits and half of the third, four small and three large bronze strap distributors). The iron objects comprise eight spearheads, five swords in their scabbards, several parts of two-piece chains for sword suspension and seven halves of two-piece shield bosses, none of which fitted together; in one case two different halves were even placed side by side. The shields were not deposited complete, but only their metal parts. The weapons were not ritually damaged, neither were the iron scythe or sickle and a knife. Cult-offering places In addition to the plaque, the ruins of one of the Late Antique buildings re vealed broken pieces of Late Hallstatt and La Tène costume, pendants, a piece of a Negova helmet, as well as a scabbard chape and handguard of a sword.136 Other offering plaques and similar finds of a wide span were found at the west edge of the settlement on Gradič in Kobarid (No. 18b),137 more precisely in the southern part of the investigated terrace where a sanctuary of supra-regional sig nificance stood in the Roman period. Roughly 20 m north, the remains of a Late Hallstatt building were found that held typical household items, had two drainage walls, foundation stones and floors paved with unworked stones. A special place of a completely different character lies at the south foot of the settlement on Gradič (No. 18e). It is the burial of horse skeletons and Early La Tène warrior outfits from the transition from the 4th to the 3rd century BC. The horse skeletons of six or seven animals were without skulls with the exception of one (its skull was crushed) and only half of the skeletons was recovered of two animals. The bones were found in more or less anatomical position and partially overlapped; they belonged to riding horses of the small-bodied ‘western’ type aged between 7 and 13 years. Also found were the bones and horns of other animals (goats, sheep, cattle, pig and chamois),138 as well as a human 138 Toškan 2011. Settlement in the Posočje/Sveta Lucija group – new sites and insights 463 humerus, though its location suggests it may not be linked or contemporary with the horse burial. The finds also include several pieces of horse gear (two iron bits and half of the third, four small and three large bronze strap distributors). The iron objects comprise eight spearheads, five swords in their scabbards, several parts of two-piece chains for sword suspension and seven halves of two-piece shield bosses, none of which fitted together; in one case two different halves were even placed side by side. The shields were not deposited complete, but only their metal parts. The weapons were not ritually damaged, neither were the iron scythe or sickle and a knife. Jewel lery pieces consisted of one bronze fibula and four different bracelets (bronze ribbed, bronze knobbed, hollow bronze with relief decoration, one iron). 140 Rupel 2004, 67–69. 144 Svoljšak, Dular 2016, 67–70, Fig. 50–56; Dular, Tecco Hvala 2018, 78, Fig. 74; Svoljšak 2018, Fig. 11. Cult-offering places Most were parts of bronze jewellery, pendants, small glass beads, several worked and unworked red coral pieces, parts of bronze vessels and very little pottery, as well as three Norican silver coins found in the north corner. In addition to wood charcoal, the fill also held charred grains, either scattered or in clumps (that gave the impression of a cake), hazelnuts and walnuts, a piece of fab ric and animal bones, of which only parts of the skull and feet are represented.143 In the vicinity stood a building from the Late Hallstatt period with floors of stone slabs in the main room and an Attic skyphos, such as were used in religious rituals, found in the adjacent room.144 A similar pit with burnt remains, surrounded with a ring of stones, also came to light on the lowest terrace, close to the confluence of the Idrijca and Soča, at the northern edge of the cemetery at Most na Soči (Fig. 10B). It was roughly 12 m2 large and around 30 cm deep. It held bits of cremated human bones, as well as scattered unburnt animal bones and an abundance of artefacts (pieces of bronze A special offering place of a long duration was recently unearthed in Bohinj, on the eastern shore of the lake (No. 31). Excavations next to the church of St John the Baptist, surrounded by three bodies of water (lake outflow and the Rivers Sava Bohinjka and Mostnica), revealed an area (roughly 3 × 4 m) enclosed with a drystone wall and in its Miha MLINAR, Sneža TECCO HVALA 464 jewellery, iron weapons, glass beads and pottery sherds) from the Late Hallstatt, Late La Tène and Early Roman periods.145 well as modest traces of occasionally used camps are evidence of the contemporary human presence also in the high mountains.148 The most revealing indicators of permanent settlement at the beginning of the Iron Age are cemeteries (Fig. 9 and 12). The Early Hallstatt graves from Tolmin and S. Pietro al Natisone/Špeter indicate small local communities and, considering the number of burials, a 150 to 200-year span of burial; the grave goods do not point to any major social differences.149 For the neighbouring Most na Soči and Kobarid, it is as yet not possible to estimate the size of the population and its social structure in the Early Hallstatt period. 145 Mlinar 2020a, 121, 143–145, Fig. 40–42; Pl. 36–41. 146 Svoljšak 1988–1989, Fig. 7; Tomadin, Visintini, Colussa 1989; Mlinar, Gerbec, Laharnar 2014, 24 (map), 27–29; Gerbec 2018; Gerbec, Vinazza 2018; Gerbec 2021. 147 Svoljšak 1988–1989, Fig. 3–5, App. 1; 2; Pl. 5: 3–6,8,10; 6: 1,4–10,12,13; 7: 1,4–6; 8: 1–5,8; Mlinar 2020a, Cult-offering places The rough estimate of the graves with the fibulae, torques and pins characteristic of the Sv. Lucija I phase150 sug gests that Most na Soči had more inhabitants than Tolmin, but less than it had in the Late Hallstatt period,151 when there were greater differences in the burial rite and grave goods as indicators of greater social stratification. In the Late Hallstatt period, Most na Soči developed into the main regional centre with early urban features and a production of metal goods and pottery, as well as exchange of goods that went beyond the scope of household economy and self-sufficiency.152 The growing need for raw materials and natural resources likely caused a more intense exploitation of the mountainous hinterland all the way to Bo hinj, which brought about new small settlements, probably also hamlets and seasonal camps (Fig. 12). We can presume that the community in the 6th and 5th centuries BC witnessed a great integration and affirmation of its own cultural identity, but also more intense contacts with other cultural entities. The circumstances of discovery and the structure of finds are essential in interpreting such sites, as they help us understand the nature of rituals prac tised at a place, be it worshipping natural forces, fertility and sources of life in a particular natural setting (e.g. Berlotov rob), venerating ancestors as could be inferred for the offering places within cemeteries (e.g. Most na Soči – Repelc), venerating divine patrons of local communities in the case of such places within settlements (e.g. Kobarid – Gradič, Most na Soči – settlement) or practis ing martial cults at battlefields. In the absence of ancient literary sources that would shed light on the matter, all the above-mentioned possibilities are merely conjecture. What we can safely presume is that the life of the prehistoric communities, in a time prior to scientific and historical notions, was suffused with myths, folk beliefs and customs. 151 Until 1892, there were at least 1343 excavated graves with fibulae of Late Hallstatt forms that men also wore instead of pins. 152 See Notes 63, 64, 89–91 and also Gabrovec 1987, 141–150. 119–120, Fig. 23–27; Pl. 15A. 148 Teržan (ed.) 1995–1996 (stray finds: Cat. No. 61, 66, 146, 216, 221, 234 and Addendum 1–3; hoards: Cat. No. 11, 17, 18 and 30); for water finds, see Gerbec, Mlinar 2011; for high-altitude sites, see Horvat 2019, 5–6, Fig. 4, Tab. 1. 149 See in this contribution the chapter on cemeteries. 150 Based on the identification of the leading types for individual chronological phases as proposed in Teržan, Trampuž 1973, there were at least 1150 graves with Early Hallstatt fibulae, pins and torques among the graves that Szombathy and Marchesetti excavated until 1892. See Marchesetti 1886, 114–125; id. 1893, 141–169; for Szom bathy’s excavations, see Teržan, Lo Schiavo, Trampuž-Orel 1984–1985. 119–120, Fig. 23–27; Pl. 15A. SETTLEMENT PATTERN AND LINES OF COMMUNICATION The collected data, as well as the investigation results and publications allow us to provide the following outline of the settlement of the Early Iron Age Posočje community. The earliest core of permanent settlement formed along the natural lines of communication that led from the Friuli Plain or the hinterland of the northern Adriatic to the high mountains of the Alps. The available data is too limited to permit any conjectures as to the appearance of the first permanent settlements. In the Younger and Late Bronze Ages, we presume the existence of fortified settlements or hillforts on the plateau of Banjšice, on the elevations along the periphery of the valley of the Nadiža/Natisone and at the entrance to the valley of the border River Idrija.146 At Most na Soči, there are the remains of an earthfast post-constructed building on the right bank of the Idrijca and a modest habitation layer on its left bank.147 Rare hoard and stray finds, as Settlement in the Posočje/Sveta Lucija group – new sites and insights 465 Soriška planina and onwards to Bohinj has as yet not been confirmed through small finds, but it seems very likely.157 The trade and freight routes led partly along riv ers, turning towards the plateaus when stumbling upon gorges and ravines (Fig. 13). The Iron Age sites at Tolmin, Volarje and Ladra show that the main line of communication that connected the two key sites of the Posočje community, namely Most na Soči and Kobarid, led along the left bank of the Soča. It then forked at Kobarid. The north branch crossed the Soča and continued along the right bank towards the Bovec Basin, across the Predel/Predil Pass (1156 m) to the area of Tarvi sio/Trbiž/Tarvis and Kärnten, Austria. The west branch continued past the hillfort on Sv. Volar above Robič and along the Nadiža/Natisone towards Friuli.153 The main communication southwards, towards the Gorica/Gorizia area and further on to the Adriatic Sea, probably led from Most na Soči along the west edge of Banjška planota to the fortified settlement on Sv. 153 The route along the western slopes of Gradič and Tonovcov grad to Kobarid avoided the ravine of the Soča above Kobarid (cf. Štular 2011b, Fig. 1.41), it again came close to the Soča at Trnovo and led roughly along the route of the modern-day road between Srpenica and Bovec; it is more difficult to reconstruct the route further on past Kluže and Log pod Mangrtom to the Predel/Predil Pass. 157 The site of Štalca near Železniki indicates the line of communication with the valley of Selška dolina (cf. Grahek 2018b; Mlinar 2018). Evidence corroborating this communication may be seen in the material used for a quern found in the settlements at Most na Soči, i.e. quartz conglomerate, the closest deposits of which lie in the area of Cerkno, as well as Poljanska dolina and Selška dolina (Dular, Tecco Hvala 2018, 94). 155 This communication is indicated by the Iron Age sites on Šentviška planota (cf. Mlinar et al. 2018) and the absence of sites in the narrow gorge between Dolenja Trebuša and Reka near Cerkno. 156 The routes between Posočje and Bohinj probably also led across other passes or peaks of the mountains of Bohinj and Tolmin such as Škrbinsko sedlo, Dobrenjska Planja and Tolminski Migovec (Mlinar, Turk 2016, 9–14). 154 Mlinar, Žbona Trkman 2008, Fig. 1. 158 Gabrovec 1987, 144–145; Guštin 1991, 51–52; Pl. 22: 1 (Grave 25 from the IIc phase). 159 Turk, Božič, Istenič 2009, 57–59; Laharnar, Turk 2018, Fig. 158–160, 164; also see Repanšek 2020 in this volume. Translation: Andreja Maver SETTLEMENT PATTERN AND LINES OF COMMUNICATION Katarina above Nova Gorica at the junction with the Vipava Valley.154 The eastward route, towards Pannonia and the Balkans, ran along the valley of the Idrijca to Slap ob Idrijci, where it ascended to Šentviška planota and only came back down to the Idrijca Valley at Reka near Cerkno, continuing along the Zala gorge to Godovič.155 The other eastward route led along the upper reaches of the Bača and near Koritnica climbed to Rut, continuing across the Vrh Bače Pass to Bohinj.156 A branch forked at Koritnica to cross the Bača and continue across Cerkljansko hribovje and Poljanska dolina eastwards. The section between Koritnica and Petrovo Brdo past The rise and prosperity of the 6th and 5th cen turies BC was followed by times of turmoil and changes in the 4th century BC, which finally led to the decline of the centres at Most na Soči and the disintegration of the community. There was a marked decrease in the size of the population, which retreated to the side valleys and higher altitudes. The funerary and offering rituals see a greater presence of weapons that include elements of the central European Celtic armament and intentional deformation. The glorification of the military status may also be perceived in the bronze statuette of a man donning a Negova helmet, found at Idrija ob Bači, which is the earliest sculpture in the round showing a realistic and individualised image of a man in the area of the Posočje community.158 This is also the time of the earliest evidence of writing here, occurring on bronze situlae as offering ves sels from Grad near Reka and Berlotov rob and written in the Venetic script.159 Further, targeted archaeological investigations and comprehensive publications of the sites already investigated will certainly offer a more detailed insight into the processes taking place during the Iron Age within the Posočje community. They will also help answer the numerous questions that must for now remain open. Translation: Andreja Maver 466 Miha MLINAR, Sneža TECCO HVALA T. / Pl. 1: 1–6 Ladra – Na goricah (št. / No. 16); 7 Tolminske Ravne – Zeleni vrh (št. / No. 11); 8 Tolminski Lom – Kal (št. / No. 47); 9 Zatolmin – Zavrh (št. / No. 6); 10,14 Sedlo – Pod cerkvijo (št. / No. 24); 11 Log pod Mangartom (št. / No. 27); 12 Homec (št. / No. SETTLEMENT PATTERN AND LINES OF COMMUNICATION 23); 13 Kobarid – Za gradom (št. / No. 18g); 15 Koseč (št. / No. 17); 16 Zatolmin – Javorca (št. / No. 9); 17 Ponikve – Kračice (št. / No. 59); 18 Smast (št. / No. 15). 4,5 keramika / ceramic; 15–17 steklo / glass; ostalo bron / other bronze. M. / Scale 4,5 = 1:3; ostalo / other = 1:2. Miha MLINAR, Sneža TECCO HVALA 466 T. / Pl. 1: 1–6 Ladra – Na goricah (št. / No. 16); 7 Tolminske Ravne – Zeleni vrh (št. / No. 11); 8 Tolminski Lom – Kal (št. / No. 47); 9 Zatolmin – Zavrh (št. / No. 6); 10,14 Sedlo – Pod cerkvijo (št. / No. 24); 11 Log pod Mangartom (št. / No. 27); 12 Homec (št. / No. 23); 13 Kobarid – Za gradom (št. / No. 18g); 15 Koseč (št. / No. 17); 16 Zatolmin – Javorca (št. / No. 9); 17 Ponikve – Kračice (št. / No. 59); 18 Smast (št. / No. 15). 4,5 keramika / ceramic; 15–17 steklo / glass; ostalo bron / other bronze. M. / Scale 4,5 = 1:3; ostalo / other = 1:2. T. / Pl. 1: 1–6 Ladra – Na goricah (št. / No. 16); 7 Tolminske Ravne – Zeleni vrh (št. / No. 11); 8 Tolminski Lom – Kal (št. / No. 47); 9 Zatolmin – Zavrh (št. / No. 6); 10,14 Sedlo – Pod cerkvijo (št. / No. 24); 11 Log pod Mangartom (št. / No. 27); 12 Homec (št. / No. 23); 13 Kobarid – Za gradom (št. / No. 18g); 15 Koseč (št. / No. 17); 16 Zatolmin – Javorca (št. / No. 9); 17 Ponikve – Kračice (št. / No. 59); 18 Smast (št. / No. 15). 4,5 keramika / ceramic; 15–17 steklo / glass; ostalo bron / other bronze. M. / Scale 4,5 = 1:3; ostalo / other = 1:2. 467 Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 467 467 Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja T. / Pl. 2: 1 Magozd – Jajnkovec (št. / No. 19); 2 Zadlaz-Čadrg – Kobilnik (št. / No. 7); 3 Stara Fužina – Veliki Vegl (št. / No. 30); 4 Trenta – Pod Razorci (št. / No. 28); 5 Bovec – Ravelnik (št. / No. 26a); 6 Trenta – V plazeh (št. / No. 29); 7 Ajba – Fiševo (št. / No. SETTLEMENT PATTERN AND LINES OF COMMUNICATION 46); 8 Vrsno – Strničelo (št. / No. 14); 9 Kneža – Grebljica (št. / No. 60); 10 Selišče – Mrzli vrh (št. / No. 12); 11 Rut – V trojah (št. / No. 39); 12 Gor. Trebuša – Obenčel (št. / No. 55); 13 Trnovo ob Soči – Trnovšček (št. / No. 20). Vse bron / all bronze. M. / Scale = 1:2 (po / from: 3 Mertelj 1994–1995; 7 Gerbec 2021). ra Fužina – Veliki Vegl p j p j drg – Kobilnik (št. / No. 7); 3 Stara Fužina – Veliki Vegl R l ik (št / N 26 ) 6 T t V l h (št / N 29) T. / Pl. 2: 1 Magozd – Jajnkovec (št. / No. 19); 2 Zadlaz-Čadrg – Kobilnik (št. / No. 7); 3 Stara Fužina – Veliki Vegl (št. / No. 30); 4 Trenta – Pod Razorci (št. / No. 28); 5 Bovec – Ravelnik (št. / No. 26a); 6 Trenta – V plazeh (št. / No. 29); 7 Ajba – Fiševo (št. / No. 46); 8 Vrsno – Strničelo (št. / No. 14); 9 Kneža – Grebljica (št. / No. 60); 10 Selišče – Mrzli vrh (št. / No. 12); 11 Rut – V trojah (št. / No. 39); 12 Gor. Trebuša – Obenčel (št. / No. 55); 13 Trnovo ob Soči – Trnovšček (št. / No. 20). Vse bron / all bronze. M. / Scale = 1:2 (po / from: 3 Mertelj 1994–1995; 7 Gerbec 2021). Miha MLINAR, Sneža TECCO HVALA 468 , T. / Pl. 3: 1,2 Kobarid – Mlinsko, reka Soča (št. / No. 18i); 3 Kobarid – Za gradom, reka Soča (št. / No. 18g); 4 Kobarid – V mevcah (št. / No. 18h); 5 Tolminske Ravne – Kal (št. / No. 10); 6 Goljevica – sv. Volbenk (št. / No. 45); 7 Bača pri Modreju – Senica (št. / No. 2); 8 Idrsko-Tolmin, reka Soča (št. / No. 4); 9 Trnovo – Kamni breg (št. / No. 54); 10 Dolenja Trebuša – Sovodenj (št. / No. 56); 11 Zatolmin – Javorca (št. / No. 9). Vse železo / all iron. M. / Scale = 1:4 (po / from: 1,2 Tratnik 2009; 6 Mihelič, Vinazza 2006). T. / Pl. 3: 1,2 Kobarid – Mlinsko, reka Soča (št. / No. 18i); 3 Kobarid – Za gradom, reka Soča (št. / No. SETTLEMENT PATTERN AND LINES OF COMMUNICATION 18g); 4 Kobarid – V mevcah (št. / No. 18h); 5 Tolminske Ravne – Kal (št. / No. 10); 6 Goljevica – sv. Volbenk (št. / No. 45); 7 Bača pri Modreju – Senica (št. / No. 2); 8 Idrsko-Tolmin, reka Soča (št. / No. 4); 9 Trnovo – Kamni breg (št. / No. 54); 10 Dolenja Trebuša – Sovodenj (št. / No. 56); 11 Zatolmin – Javorca (št. / No. 9). Vse železo / all iron. M. / Scale = 1:4 (po / from: 1,2 Tratnik 2009; 6 Mihelič, Vinazza 2006). Poselitvena slika posoške/svetolucijske skupine – nova najdišča in spoznanja 469 Miha Mlinar Tolminski muzej Mestni trg 4 SI-5220 Tolmin miha.mlinar@tol-muzej.si Miha Mlinar Tolminski muzej Mestni trg 4 SI-5220 Tolmin miha.mlinar@tol-muzej.si Sneža Tecco Hvala Znanstvenoraziskovalni center SAZU Inštitut za arheologijo Novi trg 2 SI-1000 Ljubljana snezana.tecco-hvala@zrc-sazu.si ORCID: 0000-0003-4419-1331 Slikovno gradivo: Sl. 1, 12, 13: (izdelava: Drago Valoh). – Sl. 6: (izdelava: Edisa Lozić). – Sl. 7: (foto: Davor Pečar). – Sl. 8B: (Branko Mušič). – T. 1: 1–6; 3: 7 (Jerca Brečić). – T. 1: 8 (Matevž Lavrinc). – T. 1: 7,11–13,15,18; 2: 2,4–6,12,13; 3: 5 (Tamara Korošec). – T. 1: 9,10,14,16; 2: 8,10,11; 3: 8 (Teja Gerbec). – T. 2: 9; 3: 9 (Romana Vidmar). – T. 3: 3,4,11 (Natalija Grum). – T. 3: 10 (Manca Omahen). Slikovno gradivo: Sl. 1, 12, 13: (izdelava: Drago Valoh). – Sl. 6: (izdelava: Edisa Lozić). – Sl. 7: (foto: Davor Pečar). – Sl. 8B: (Branko Mušič). – T. 1: 1–6; 3: 7 (Jerca Brečić). – T. 1: 8 (Matevž Lavrinc). – T. 1: 7,11–13,15,18; 2: 2,4–6,12,13; 3: 5 (Tamara Korošec). – T. 1: 9,10,14,16; 2: 8,10,11; 3: 8 (Teja Gerbec). – T. 2: 9; 3: 9 (Romana Vidmar). – T. 3: 3,4,11 (Natalija Grum). – T. 3: 10 (Manca Omahen). Illustrations: Figs. 1, 12, 13: (elaborated by: Drago Valoh). – Fig. 6: (elaborated by: Edisa Lozić). – Fig. 7: (photo: Davor Pečar). – Fig. 8B: (Branko Mušič). – Pls. 1: 1–6; 3: 7 (Jerca Brečić). – Pl. 1: 8 (Matevž Lavrinc). – Pls. 1: 7,11–13,15,18; 2: 2,4–6,12,13; 3: 5 (Tamara Korošec). – Pls. 1: 9,10,14,16; 2: 8,10,11; 3: 8 (Teja Gerbec). – Pls. 2: 9; 3: 9 (Romana Vid mar). – Pl. 3: 3,4,11 (Natalija Grum). – Pl. 3: 10 (Manca Omahen). Illustrations: Figs. 1, 12, 13: (elaborated by: Drago Valoh). – Fig. 6: (elaborated by: Edisa Lozić). – Fig. Slikovno gradivo: Sl. 1, 12, 13: (izdelava: Drago Valoh). – Sl. 6: (izdelava: Edisa Lozić). – Sl. 7: (foto: Davor Pečar). – Sl. 8B: (Branko Mušič). – T. 1: 1–6; 3: 7 (Jerca Brečić). – T. 1: 8 (Matevž Lavrinc). – T. 1: 7,11–13,15,18; 2: 2,4–6,12,13; 3: 5 (Tamara Korošec). – T. 1: 9,10,14,16; 2: 8,10,11; 3: 8 (Teja Gerbec). – T. 2: 9; 3: 9 (Romana Vidmar). – T. 3: 3,4,11 (Natalija Grum). – T. 3: 10 (Manca Omahen). SETTLEMENT PATTERN AND LINES OF COMMUNICATION 7: (photo: Davor Pečar). – Fig. 8B: (Branko Mušič). – Pls. 1: 1–6; 3: 7 (Jerca Brečić). – Pl. 1: 8 (Matevž Lavrinc). – Pls. 1: 7,11–13,15,18; 2: 2,4–6,12,13; 3: 5 (Tamara Korošec). – Pls. 1: 9,10,14,16; 2: 8,10,11; 3: 8 (Teja Gerbec). – Pls. 2: 9; 3: 9 (Romana Vid mar). – Pl. 3: 3,4,11 (Natalija Grum). – Pl. 3: 10 (Manca Omahen). Članek je deloma nastal v okviru projekta Na stiku med Alpami in Mediteranom – kontinuiteta in prelomnice (J6-1802), ki ga je sofinancirala Javna agencija za raziskovalno dejavnost Republike Slovenije. j p j p p (J ), ki ga je sofinancirala Javna agencija za raziskovalno dejavnost Republike Slovenije. The authors acknowledge that the article is partially a result of the project (At the contact between the Alps and Medi terranean – continuity and turning points J6-1802), which was financially supported by the Slovenian Research Agency. The authors acknowledge that the article is partially a result of the project (At the contact between the Alps and Medi terranean – continuity and turning points J6-1802), which was financially supported by the Slovenian Research Agency.
https://openalex.org/W2972174564	https://europepmc.org/articles/pmc6718421?pdf=render	English	null	The marginal cells of the Caenorhabditis elegans pharynx scavenge cholesterol and other hydrophobic small molecules	Nature communications	2,019	cc-by	20,835	1 Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada. 2 The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON M5S 3E1, Canada. 3 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, ON M5S 3M2, Canada. 4 Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON M5S 1A8, Canada. 5 Department of Neuroscience, Albert Einstein College of Medicine, New York, NY 10461, USA. 6 Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3E1, Canada. 7 Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON M5S 3E1, Canada. 8 Department of Biochemistry, University of Toronto, Toronto, ON M5S 3E1, Canada. Correspondence and requests for materials should be addressed to P.J.R. (email: peter.roy@utoronto.ca) ARTICLE The marginal cells of the Caenorhabditis elegans pharynx scavenge cholesterol and other hydrophobic small molecules A lack of select sterols, which are relatively insoluble and rare in the wild, is likely one factor that contributes to Caenorhabditis existing in the dauer state outside of the laboratory6. Mechanisms that maximize the absorption of these essential and scarce nutrients must therefore be essential to the overall success of the species. We investigated whether the crystals and spheres are a response of the worm to the wactives or whether the objects are likely to be the wactives themselves. We found that one of the crystallizing wactives, wact-469, ﬂuoresces yellow-green (Supple- mentary Fig. 1), while no other crystallizing wactive that we investigated in detail ﬂuoresces using our detection methods. We found that crystals that form in response to wact-469 ﬂuoresce yellow-green, but crystals that form in response to other wactives like wact-190 do not ﬂuoresce (Fig. 1b). Similarly, the wactive wact-43 ﬂuoresces blue, and results in blue ﬂuorescent spheres in the animal, whereas other non-ﬂuorescent wactives like wact-209 do not produce ﬂuorescent spheres (Fig. 1b). We conclude that the crystals and spheres are at least partly, if not entirely, composed of the respective wactive compound. p Caenorhabditis is a ﬁlter-feeder7,8. Its feeding organ, called the pharynx, has a threefold symmetry with three muscles divided by three sets of marginal cells, all radially oriented with respect to the central lumen (see ﬁgures for schematics)7. The ﬁrst step of the feeding cycle is that the radially aligned muscles of the pharynx contract, thereby opening the central lumen and creating negative pressure that sucks the bacterial suspension from the environ- ment into the lumen. The pharynx then relaxes in an anterior to posterior wave, concentrating the suspended bacterial cells cen- trally within the lumen. Because of increasing pressure, the excess liquid escapes to the environment in a posterior-to-anterior manner via channels within the marginal cells9. The microbes are then macerated in the posterior pharynx and passed on to the neighboring intestine via peristaltic movements7. We investigated whether there are basic physicochemical features of the wactives that correlate with crystallization and sphere formation. We found that both crystal-forming and sphere-forming wactives are less hydrophilic (and have fewer hydrogen bond donors, which is related to hydrophilicity) on average than the wactives that do not generate unusual objects (Fig. 1d). This trend is consistent with the molecules precipitating out of solution when concentrated within the animal. The marginal cells of the Caenorhabditis elegans pharynx scavenge cholesterol and other hydrophobic small molecules Muntasir Kamal1,2, Houtan Moshiri1,2, Lilia Magomedova3, Duhyun Han2,4, Ken C.Q. Nguyen5, May Yeo1,2, Jessica Knox1,2, Rachel Bagg1,2, Amy M. Won2,6,7,8, Karolina Szlapa1,2, Christopher M. Yip2,6,7,8, Carolyn L. Cummins 3, David H. Hall 5 & Peter J. Roy1,2,4 The nematode Caenorhabditis elegans is a bacterivore ﬁlter feeder. Through the contraction of the worm’s pharynx, a bacterial suspension is sucked into the pharynx’s lumen. Excess liquid is then shunted out of the buccal cavity through ancillary channels made by surrounding marginal cells. We ﬁnd that many worm-bioactive small molecules (a.k.a. wactives) accu- mulate inside of the marginal cells as crystals or globular spheres. Through screens for mutants that resist the lethality associated with one crystallizing wactive we identify a presumptive sphingomyelin-synthesis pathway that is necessary for crystal and sphere accumulation. We ﬁnd that expression of sphingomyelin synthase 5 (SMS-5) in the marginal cells is not only sufﬁcient for wactive accumulation but is also important for absorbing exogenous cholesterol, without which C. elegans cannot develop. We conclude that sphingomyelin-rich marginal cells act as a sink to scavenge important nutrients from ﬁltered liquid that might otherwise be shunted back into the environment. 1 Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada. 2 The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON M5S 3E1, Canada. 3 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, ON M5S 3M2, Canada. 4 Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON M5S 1A8, Canada. 5 Department of Neuroscience, Albert Einstein College of Medicine, New York, NY 10461, USA. 6 Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3E1, Canada. 7 Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON M5S 3E1, Canada. 8 Department of Biochemistry, University of Toronto, Toronto, ON M5S 3E1, Canada. Correspondence and requests for materials should be addressed to P.J.R. (email: peter.roy@utoronto.ca) 1 NATURE COMMUNICATIONS \| (2019) 10:3938 \| https://doi.org/10.1038/s41467-019-11908-0 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 T he survival of life in the wild is dependent on enduring the boom and bust cycles of nutrient availability. This is especially true for the free-living nematode Caenorhabditis elegans that must survive extreme population bottlenecks because of limited nutrients1. Under ideal conditions, C. The marginal cells of the Caenorhabditis elegans pharynx scavenge cholesterol and other hydrophobic small molecules elegans will undergo embryogenesis and develop through four larval stages called L1–L4 to reach reproductive maturity in as little as 3 days2. Upon nutrient deprivation, overcrowding, or excessive tempera- ture, the animal will enter an alternative stress-resistant long-lived dispersal state called dauer instead of L33. Extensive sampling of nematodes in the wild reveals that C. elegans is most often found in the dauer state, unlike other free-living nematodes outside of the Caenorhabditis genus that are found to be actively pro- liferating1. Hence, Caenorhabditis seems particularly sensitive to stress, including nutrient deprivation. T animals incubated in some compounds have unusually dark pharynxes when visualized at ×100 magniﬁcation12–14. We investigated this phenomenon further by incubating synchro- nized ﬁrst larval stage (L1) wild-type worms in 238 compounds from our wactive library at a concentration of 30 μM for 48 h (Fig. 1a). The wactive library is a collection of 627 compounds that we previously found to have bioactivity in C. elegans12–14. The 238 compounds that we surveyed are from plates 1 and 2 from the wactive library, which are enriched for potent bioactives, plus additional wactives that our group had working stocks of during the survey. g y We visualized the worms at ×400 magniﬁcation using differential interference contrast (DIC), which allows clear visualization of unusual objects, and polarized light (birefringent) microscopy, which allows for the detection of objects containing molecules that are arrayed in a regular pattern (i.e., are crystalline)15. We found that 38 (16%) wactives accumulated as birefringent crystals and another 33 (14%) accumulated as non- birefringent globular spheres (henceforth referred to as spheres) (Fig. 1a–c). Of the 115 molecules that kill worms at a concentration of 30 μM, 66 (57%) form crystals or spheres in at least 25% of the animals (Fig. 1 in Source Data File). The only cells in which we found these unusual objects are within the corpus (anterior half) of the pharynx. C. elegans is a sterol auxotroph4. In the laboratory, C. elegans’ food (E. coli) is supplemented with cholesterol, but other select fungal, plant, and animal sterols will also sufﬁce5. The worm modiﬁes the sterols for use in signaling and, unlike vertebrates, the sterols may be a structural component within the membranes of only a limited number of cells4. Depending on the extent and timing of sterol withdrawal, C. elegans will either enter the dauer state or fail to develop and perish5. The marginal cells of the Caenorhabditis elegans pharynx scavenge cholesterol and other hydrophobic small molecules One trend that may distinguish the crystal-forming compounds from those that form spheres is that the former have a greater topological polar surface area (PSA) on average (Fig. 1d). A key determinant of whether a membrane protein will crystallize in X-ray crystallographic studies is its topological PSA; more exposed polar groups increase the likelihood that a membrane protein will crystallize easily16. This raises the possibility that the crystallizing wactives are associating with the plasma membrane (PM) and coming out of solution in crystalline form due to their relatively large PSA. g g p Here we report our discovery that select hydrophobic mole- cules visibly accumulate in the marginal cells of the C. elegans pharynx. No other tissue visibly accumulates hydrophobic small molecules. Through a forward genetic screen, we identiﬁed sev- eral components of a presumptive sphingomyelin (SM) synthesis pathway, at least one of which is speciﬁcally expressed in the pharynx, that are necessary for this accumulation. We ﬁnd that the expression of the presumptive terminal enzyme of this pathway (sphingomyelin synthase 5 (SMS-5)) speciﬁcally in the marginal cells is sufﬁcient for wactive accumulation. These observations led us to the ﬁnding that the marginal cells play an important role in the absorption of cholesterol, which is consistent with previous work showing that the pharynx accu- mulates sterols and other natural products10,11. Together, our observations indicate that the marginal cells scavenge nutrients from ﬂuid that would otherwise be discarded by the worm. This nutrient-scavenging mechanism may be important in allowing Caenorhabditis to survive periods of limited nutrient availability. To probe these trends further, we tested a set of ten newly purchased molecules whose physicochemical properties fall on one side of the trend toward crystallization and another set of ten newly purchased molecules whose physicochemical properties fall on the other side of the trend (away from object formation). Of the ten molecules with crystal-like physicochemical properties, two crystallized in the anterior pharynx. By contrast, none of the molecules with non-object-like physicochemical properties formed objects in the pharynx. That 20% of the molecules with crystal-like physicochemical properties form crystals is an NATURE COMMUNICATIONS \| (2019) 10:3938 \| https://doi.org/10.1038/s41467-019-11908-0 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 The gray shape represents all results and the thickness indicates ho mmon that value is within the dataset; a white circle indicates the median; a red circle indicates the mean; the center thick line represents one standard i i h hi li h d d d f d i i Th b b h l h h i iﬁ f h diff d Control wact-209 wact-190 wact-469 wact-43 t Viability b c 10 µm * Birefringence Fluorescence DIC b a c DIC DIC Birefringence 200 250 300 350 400 450 500 –1 0 1 2 3 4 0 2 4 6 8 20 40 60 80 100 –6 –4 –2 0 0 2 4 6 8 LogSw (solubility in water) 152 Mass (Da) Hydrogen bond donors Hydrogen bond acceptors Topological polar surface area (Å2) Rotatable bonds d cr sp no cr sp no cr sp no cr sp no cr sp no cr sp no 0.08 • • • • • • • • • • • • 0.40 <0.01 0.01 0.03 0.45 <0.01 0.55 <0.01 <0.01 0.90 0.06 • • • • Crystal-forming (<25%) Mixed-crystals predominate Sphere-forming (>25%) Sphere-forming (<25%) Mixed-spheres predominate Culture thriving Culture struggling Culture dead/arrested Crystal-forming (>25%) d d 200 250 300 350 400 450 500 Mass (Da) cr sp no • • • 0.90 0.06 LogSw (solubility in water) Fig. 1 Wactives can form objects in the marginal cells of the anterior pharynx. a A chart showing our survey of the effects of 238 wactives on C. elegans L1 animals. Each row is a distinct wactive. The ﬁrst column indicates whether a wactive accumulates as a crystal, sphere, or neither at a concentration of 30 μM. The second column indicates whether the wactive also kills/arrests worm development at a concentration of 30 μM. The percentages in the legend refer to the percentage of animals harboring the indicated objects. See “Methods” for details. b Examples of the accumulation of crystals and spheres in the anterior pharynx of young adults treated for 24 h to allow easy visualization of the channels. The ﬁrst row shows differential interference contrast images; the second row shows whether the objects are birefringent (and therefore crystalline); and the third row shows whether the object ﬂuoresces. White arrows indicate the channels, blue arrows indicate crystals, and orange arrows indicate spheres. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 ARTICLE 200 250 300 350 400 450 500 –1 0 1 2 3 4 0 2 4 6 8 20 40 60 80 100 –6 –4 –2 0 0 2 4 6 8 Control a wact-209 wact-190 wact-469 wact-43 Object Viability LogSw (solubility in water) 152 wactives that do not form objects (<2%) b Mass (Da) Hydrogen bond donors Hydrogen bond acceptors Topological polar surface area (Å2) Rotatable bonds d c cr sp no cr sp no cr sp no cr sp no cr sp no cr sp no 0.08 • • • • • • • • • • • • 0.40 <0.01 0.01 0.03 0.45 <0.01 0.55 <0.01 <0.01 0.90 0.06 10 µm * 38 crystallizing wactives 15 mixed wactives 33 sphere- forming wactives Birefringence Fluorescence DIC • • • • Crystal-forming (<25%) Mixed-crystals predominate Sphere-forming (>25%) Sphere-forming (<25%) Mixed-spheres predominate Culture thriving Culture struggling Culture dead/arrested Crystal-forming (>25%) g. 1 Wactives can form objects in the marginal cells of the anterior pharynx. a A chart showing our survey of the effects of 238 wactives on C. elegans imals. Each row is a distinct wactive. The ﬁrst column indicates whether a wactive accumulates as a crystal, sphere, or neither at a concentration of 30 μM e second column indicates whether the wactive also kills/arrests worm development at a concentration of 30 μM. The percentages in the legend refer e percentage of animals harboring the indicated objects. See “Methods” for details. b Examples of the accumulation of crystals and spheres in the anteri arynx of young adults treated for 24 h to allow easy visualization of the channels. The ﬁrst row shows differential interference contrast images; the secon w shows whether the objects are birefringent (and therefore crystalline); and the third row shows whether the object ﬂuoresces. White arrows indicate t annels, blue arrows indicate crystals, and orange arrows indicate spheres. The white asterisk indicates where the micrograph was spliced to ensure annel is in the correct focal plane. The scale is the same for all panels. c A schematic of C. elegans (courtesy of WormAtlas). The anterior pharynx is boxe Violin plots of the physical–chemical properties of molecules that crystallize (cr) (n = 38 distinct molecules), form spheres (sp) (n = 33 distinct olecules), or fail to form objects in the animal (no) (n = 152 distinct molecules). Results Wactives accumulate as crystals and spheres in the pharynx. Observations from our previous drug screens revealed that 2 NATURE COMMUNICATIONS \| (2019) 10:3938 \| https://doi.org/10.1038/s41467-019-11908-0 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 The white asterisk indicates where the micrograph was spliced to ensure channel is in the correct focal plane. The scale is the same for all panels. c A schematic of C. elegans (courtesy of WormAtlas). The anterior pharynx is boxed. d Violin plots of the physical–chemical properties of molecules that crystallize (cr) (n = 38 distinct molecules), form spheres (sp) (n = 33 distinct molecules), or fail to form objects in the animal (no) (n = 152 distinct molecules). The gray shape represents all results and the thickness indicates how common that value is within the dataset; a white circle indicates the median; a red circle indicates the mean; the center thick line represents one standard of deviation; the thinner line represents the second standard of deviation. The number above each plot shows the signiﬁcance of the difference compared to the “no-object” data using the nonparametric two-tailed Mann–Whitney U test. Source data for a and d are provided in the Source Data ﬁle 3 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 enrichment of 147-fold over what is expected by random chance (Supplementary Table 1). enrichment of 147-fold over what is expected by random chance (Supplementary Table 1). media. Instead, it is more likely that soluble small molecules accumulate in the tissue of the anterior pharynx, precipitate out of solution, and form crystals or spheres therein. To examine the location of crystals in greater detail, we inspected the location of crystals using transmission electron microscopy. In cross-sections of wild-type animals incubated with wact-190, we found unusual structures that are consistent with the crystals we see with light microscopy. The unusual objects are located in association with the PM of the marginal cells and extend into the cytoplasm (Fig. 3d–g). In transverse sections of wild-type animals incubated with wact-190, we also observe crystal-like objects in the marginal cells. In addition, we see areas of clear material and/or separation of the PM from the luminal cuticle of the marginal cells (Supplementary Fig. 5). These observations are consistent with the idea that the marginal cells have unique properties that facilitate the absorption of small molecules with the physicochemical properties described above. Wactive crystals may contribute to the death of C. elegans. The majority of wactives that form crystals and spheres at 30 μM also induce developmental arrest and/or death of L1 larvae at that concentration (Fig. 1a). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 These data are consistent with the idea that crystal formation may contribute to the death or arrest of the nematode by disrupting PM integrity and that spheres are simply a visible manifestation of the accumulation of select small molecules. Sequencing the genomes of the 46 mutants revealed 29 alleles of pgp-14, whose contribution to small molecule accumulation will be discussed elsewhere. Sequencing also revealed 12 strains that have mutations in 3 different components of a predicted de novo SM biosynthetic pathway (Fig. 5a). Sphingomyelin is one of the four major phospholipids that constitute the metazoan PM. Sphingomyelin and phosphatidylcholine are the major lipids in the outer leaﬂet of the PM, while phosphatidylserine and phosphatidylethanolamine are the major lipids within the inner leaﬂet18,19. The marginal cells act as a sink for select wactives. To inves- tigate the dynamics of crystal formation, we performed a time- course analysis of wact-190 crystal formation. We found that small birefringent objects can be seen as early as 30 min after incubation with wact-190 (Fig. 3a–c). By 48 h, the pharynx lumen of many animals is entirely ﬁlled with wact-190 crystals. Five of the wact-190-resistant strains have a mutation (including three early non-sense alleles) in sms-5 (a.k.a. W07E6.3), which is a gene predicted to encode an integral membrane SMS and the terminal enzyme in the pathway20 (Figs. 4 and 5a). SMS catalyzes the production of SM from phosphatidylcholine and ceramide on the outer leaﬂet of the PM21. Another ﬁve wact-190-resistant strains have a mutation (including two nonsense alleles) in sptl-2, which encodes a serine palmitoyltransferase. Two strains have a mutation (including one nonsense allele) in ttm-5, which is predicted to encode a sphingolipid delta(4)-desaturase. Both SPTL- 2 and TTM-5 are predicted to function upstream of SMS-5 in the production of SM21. y y y Early in the time course, wact-190 crystals accumulate near the channels of the anterior pharynx (Figs. 3a–c and 1b). The channels are formed by specialized cells called marginal cells and are part of the ﬁltering mechanism by which the animal feeds on bacterial cells within its environment. The area of crystal and sphere formation, together with the animals’ ﬁlter-feeding strategy, raised the possibility that crystals might be ﬁltered from the media, get trapped in the channels, and seed the growth of larger crystals. Three observations argue against this. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 To better understand the relationship between object formation and lethality/arrest, we performed a dose–response analysis on a small subset of wactives that form crystals or spheres (Fig. 2a). We collected synchronized ﬁrst larval stage animals (L1s) and inspected the pharynxes and other tissues at higher magniﬁcation after 48 h of incubation in the wactives. In parallel, we performed our standard 6-day viability assay14. We found a strong correlation between the presence of objects in the anterior pharynx and the associated lethality/arrest (an average R2 of 0.964). We also asked whether the lethality associated with crystal-forming wactives can be avoided if the animals are removed from the wactive. We found that the lethal effect of the wactives can be prevented for most of the animals if they are removed from the wactive during the ﬁrst 2 days of co-incubation with the molecule; thereafter, most animals do not recover from wactive exposure (Supplementary Fig. 2). SMS-5 functions cell-autonomously in the anterior pharynx. To better understand the accumulation of crystals in the anterior pharynx, we performed a forward genetic screen to isolate mutants that resist the lethal effects of the crystal-forming wact- 190. We reasoned that, if crystal formation is related to wact- 190’s lethality, mutants that resist its lethality would also resist crystal formation. We screened 1.3 million randomly mutagen- ized wild-type F2 genomes and isolated 46 mutants that resist the lethal effects of wact-190 (Fig. 4; Fig. 4 in Source Data File). A screen of 200,000 mutant F1 genomes did not yield wact-190- resistant mutants, suggesting that the resistant mutants isolated in the F2 screens are likely recessive. Many of the object-forming wactives are structurally diverse but have crystal or sphere formation in common (Fig. 2a). These observations raised the possibility that the objects themselves are contributing to the death or arrest of the animal. We reasoned that the crystals might perforate the membrane of the anterior pharynx and lead to death. We have no similar hypothesis about the spheres. We incubated the animals in the crystal-forming wactive wact-190 and sphere forming wact-34 and tested whether a membrane impermeable dye permeates the pharynx. We found that wact-190-exposed worms allow the permeation of Evans Blue dye17 (Fig. 2b, c, e). By contrast, wact-34 failed to allow Evans Blue dye entry (Fig. 2d, e). NATURE COMMUNICATIONS \| (2019) 10:3938 \| https://doi.org/10.1038/s41467-019-11908-0 \| www.nature.com/naturecommunications ARTICLE ARTICLE spectrometric analysis (Fig. 5e). The sms-5 null mutant also resists Evans Blue dye penetration into the anterior pharynx when co- incubated with wact-190 (Fig. 2e). We next investigated the tissues in which sms-5 plays a role in small molecule accumulation. We used a fosmid-based reporter in which SMS-5 is tagged with yellow ﬂuorescent protein (YFP) on its 0 1 2 3 4 5 6 a Animals with objects (%) Animals alive (#) Sphere-forming wactives Crystal-forming wactives b wt control c wt wact-190 wt wact-34 d e wact-190 wact-390 wact-469 wact-2 wact-34 0 0.5 3.8 15 12 0 0 0 0 0.5 3.8 15 12 0 0.5 3.8 15 12 wact-128 wact-220 wact-124 wact-140 20 µm wt + wact-190 Fold change in EBD signal intensity relative to DMSO negative control (1) wt + wact-34 sms-5(0) + wact-190 * 100 50 0 100 50 0 100 50 0 >100 50 0 >100 50 0 >100 50 0 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-1190 a A i l ith bj t (%) A i l li (#) Sphere-forming wactives Crystal-forming wactives wact-190 wact-390 wact-469 wact-2 wact-34 0 0.5 3.8 15 12 0 0 0 0 0.5 3.8 15 12 0 0.5 3.8 15 12 wact-128 wact-220 wact-124 wact-140 100 50 0 100 50 0 100 50 0 >100 50 0 >100 50 0 >100 50 0 a ng wactives wact-190 wact-390 wact-469 100 50 0 >100 50 0 Fig. 2 Wactive-induced lethality and object formation are correlated. a Crystal and sphere formation in the pharynx was analyzed after a 48-h incubation of L1 larvae in the respective wactive and is indicated in blue. The lethality induced by the respective wactive, assayed after 50 L1s were incubated with the molecules for 6 days, is indicated in red. Animals in each well were counted if not over-grown with worms; otherwise, the well was scored as 100 animals. Low numbers of animals (see wact-190 or wact-128, for example) are invariably arrested L1s. The twofold serial dilution dose of the wactive, in micromolar, is indicated at the bottom of the three columns of graphs. The small molecule structure is illustrated below each graph. b–e An analysis of the ability of Evans Blue dye (EBD) to penetrate the anterior pharynx. L1-stage worms were incubated with control (1% dimethyl sulfoxide (DMSO) only) or 60 µM of the indicated compound for 24 h. ARTICLE Worms were then incubated in EBD for 4 h. b–d We see an enrichment of EBD signal in the anterior pharynx of worms treated with the crystal- forming wact-190 but not with the sphere-forming wact-34 or control. The scale is the same for all panels. For each pair of images, the differential interference contrast image is on the left, and the ﬂuorescent image showing EBD signal is on the right. e Quantiﬁcation of the EBD signal intensity of wact-190-treated worms relative to the DMSO negative control treated worms. Asterisk indicates a signiﬁcant difference between the control and the wact-190-treated sample (p < 0.05) using Student’s T test. Means and standard error are derived from n = 3 independent biological trials with at least six animals analyzed per sample per trial. Error bars represent standard error of the mean (SEM) for all graphs in this ﬁgure. Source data for a and e are provided in the Source Data ﬁle a Crystal-forming wactives Crystal-formin wact-2 wact-128 wact-220 100 50 0 >100 50 0 phere-forming wactives wact-34 0 0.5 3.8 15 12 0 0 0 0 0.5 3.8 15 12 0 0.5 3.8 15 12 wact-124 wact-140 100 50 0 >100 50 0 Sphere-forming wactives Animals with objects (%) clear enrichment in the anterior half (corpus) relative to the posterior half (isthmus and terminal bulb) (Supplementary Fig. 6). Animals mosaic for the transgene show SMS-5::YFP expression in the marginal cells (Fig. 5f, g). b wt control c wt wact-190 wt wact-34 d 20 µm wt wact-34 d d g ( g g) Wild-type animals accumulate wact-190 crystals speciﬁcally in the anterior marginal cells. We therefore tested whether the expression of SMS-5 speciﬁcally in the marginal cells was sufﬁcient to rescue the mutant’s resistance to wact-190 crystal formation. We used a promoter (Supplementary Fig. 7) to express an SMS-5::FLAG::mCherry fusion protein (pPRHZ1138) speciﬁ- cally in the marginal cells of the anterior pharynx. For simplicity, we refer to this transgenic fusion protein as SMS-5(MC) henceforth. We found that expression of SMS-5(MC) can rescue the sms-5 null phenotypes, including its resistance to both wact- 190’s lethality and crystal formation (Fig. 5d). Together, these data indicate that SMS-5 functions in the anterior pharynx to make the marginal cells especially sensitive to the accumulation of select small molecules. NATURE COMMUNICATIONS \| (2019) 10:3938 \| https://doi.org/10.1038/s41467-019-11908-0 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 First, the birefringent crystalline objects that can been seen early in the time-course experiment are not found in the lumen of the channels but are instead associated with the cytoplasmic face of the basal lamina of the marginal cells (see the 30-min time point in Fig. 3). Second, crystals accumulate in the pharynx of young larvae that hatch within the parent’s uterus and are not exposed to the external media (Supplementary Fig. 3). Third, 0 out of 17 molecules that precipitate in the media and fail to perturb the worm’s development lead to crystal formation (Supplementary Fig. 4). Together, these observations argue against the idea that marginal cell-associated crystals are seeded from crystals in the At each step of the SM synthesis pathway, there are multiple paralogs within the C. elegans genome that are predicted to perform the same biochemical role (Fig. 5a). We investigated independently derived deletion alleles of the paralogs including the genes we identiﬁed in our genetic screen. In every case, only the publicly available deletion allele of the respective mutant gene identiﬁed in our screen resisted the effects of wact-190 (Fig. 4). This suggests that either the speciﬁc expression pattern or the biochemical activity of the respective paralog identiﬁed in our screen is important for mediating sensitivity to wact-190. The sms-5 null mutant resists wact-190’s crystal formation (Fig. 5b–d), wact-190-associated developmental arrest/lethality (Fig. 5d), and the accumulation of wact-190 in the worm as revealed by mass 4 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 ARTICLE 0 1 2 3 4 5 6 e wt + wact-190 Fold change in EBD signal intensity relative to DMSO negative control (1) wt + wact-34 sms-5(0) + wact-190 * e SMS-5 contributes to SM abundance in the pharynx. Sphin- gomyelin facilitates a dense packing of lipids within a mem- brane22. Consequently, SM abundance is correlated to a resistance to solubilization by detergents23,24. We therefore tested whether sms-5 mutants are more sensitive to detergents com- pared to wild-type animals. As predicted, we observed that the sms-5 mutant is hypersensitive to detergents compared to wild- type controls. Furthermore, the expression of SMS-5 from the anterior marginal cells was sufﬁcient to rescue this hypersensi- tivity to the detergents (Fig. 6a, b). Fold change in EBD signal intensity relative to DMSO negative control (1) spectrometric analysis (Fig. 5e). The sms-5 null mutant also resists Evans Blue dye penetration into the anterior pharynx when co- incubated with wact-190 (Fig. 2e). We next investigated the relative abundance of SM in the anterior pharynx using a protein probe called GFP-NT-Lysenin that binds to clustered SM (Supplementary Fig. 8)25,26. In wild- type ﬁxed worms, we observed an enrichment of GFP-NT- Lysenin in tissues surrounding the pharynx, in the buccal cavity, and in the channels of the anterior marginal cells (Fig. 6c). The sms-5 null mutant shows a signiﬁcant decrease of ﬂuorescent signal in the anterior pharynx (Fig. 6d, e). We infer that the We next investigated the tissues in which sms-5 plays a role in small molecule accumulation. We used a fosmid-based reporter in which SMS-5 is tagged with yellow ﬂuorescent protein (YFP) on its C-terminus (pPRHM1051). We observed SMS-5::YFP to be expressed in only two tissues: the spermatheca and the pharynx. In the pharynx, SMS-5::YFP is expressed in the muscle and marginal cells, with an enrichment at cell–cell junctions, and a 5 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 Each pair of images shows the procorpus (anterior quarter) of the pharynx. The white arrows indicate channels; green arrows indicate select crystals. The scale is the same for all panels. c Quantitative analysis of wact-190 crystal formation over time. Synchronized ﬁrst-stage (L1) worms of the indicated genotype were incubated in liquid with 60 μM wact-190 for the indicated time period and crystals were identiﬁed by their birefringence. Error bars represent standard error of the mean (SEM) and n = 3 independent biological samples. Source data are provided in the Source Data ﬁle. d–f Transmission electron micrographs (TEMs) of pharynx cross-sections of the indicated genotype and wactive treatment. The channels are indicated with white arrows; presumptive crystals with green arrows; marginal cell plasma membranes with fuchsia arrows. See Supplementary Fig. 5 for transverse sections. g A schematic of the pharynx illustrating the approximate area of the cross-sections imaged by TEM. The marginal cells are indicated in fuchsia. Image adapted from WormAtlas with permission Fig. 3 A microscopic analysis of wact-190 crystal formation. a A schematic of the pharynx with the three channels indicated with white arrows; the central lumen and grinder are in black. The procorpus of the anterior pharynx, which is shown in b, is boxed in orange. b Qualitative analysis of wact-190 crystal formation over time. Synchronized fourth-stage (L4) animals were grown in 60 μM of wact-190 in liquid culture for the time indicated at the top of each column. The top images show differential interference contrast micrographs; the corresponding bottom images are taken with polarized ﬁlters to visualize birefringence. Each pair of images shows the procorpus (anterior quarter) of the pharynx. The white arrows indicate channels; green arrows indicate select crystals. The scale is the same for all panels. c Quantitative analysis of wact-190 crystal formation over time. Synchronized ﬁrst-stage (L1) worms of the indicated genotype were incubated in liquid with 60 μM wact-190 for the indicated time period and crystals were identiﬁed by their birefringence. Error bars represent standard error of the mean (SEM) and n = 3 independent biological samples. Source data are provided in the Source Data ﬁle. d–f Transmission electron micrographs (TEMs) of pharynx cross-sections of the indicated genotype and wactive treatment. The channels are indicated with white arrows; presumptive crystals with green arrows; marginal cell plasma membranes with fuchsia arrows. See Supplementary Fig. 5 for transverse sections. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 0 20 40 60 80 100 0 0.5 1 2 4 6 24 48 Wild type sms-5(ok2498) 0.5 h 1 h 2 h 6 h 24 h 48 h Birefringence DIC a C b d Wild type + wact-190 mc1 g Wild type control sms-5(ok2498) + wact-190 e f Time (h) Worms with wact-190 crystals (%) c 10 µm 2.5 µm 1 µm 2.5 µm pic analysis of wact-190 crystal formation. a A schematic of the pharynx with the three channels indicated with whi are in black. The procorpus of the anterior pharynx, which is shown in b, is boxed in orange. b Qualitative analys me. Synchronized fourth-stage (L4) animals were grown in 60 μM of wact-190 in liquid culture for the time indicat mages show differential interference contrast micrographs; the corresponding bottom images are taken with polariz h pair of images shows the procorpus (anterior quarter) of the pharynx. The white arrows indicate channels; green a e is the same for all panels. c Quantitative analysis of wact-190 crystal formation over time. Synchronized ﬁrst-sta e were incubated in liquid with 60 μM wact-190 for the indicated time period and crystals were identiﬁed by the ndard error of the mean (SEM) and n = 3 independent biological samples. Source data are provided in the Sourc 0.5 h 1 h 2 h 6 h 24 h 48 h Birefringence DIC a C b 10 µm b a 2 h 0 20 40 60 80 100 0 0.5 1 2 4 6 24 48 Wild type sms-5(ok2498) Time (h) Worms with wact-190 crystals (%) c g g d Wild type + wact-190 2.5 µm c d Time (h) sms-5(ok2498) + wact-190 f 1 µm Wild type control e 2.5 µm Fig. 3 A microscopic analysis of wact-190 crystal formation. a A schematic of the pharynx with the three channels indicated with white arrows; the central lumen and grinder are in black. The procorpus of the anterior pharynx, which is shown in b, is boxed in orange. b Qualitative analysis of wact-190 crystal formation over time. Synchronized fourth-stage (L4) animals were grown in 60 μM of wact-190 in liquid culture for the time indicated at the top of each column. The top images show differential interference contrast micrographs; the corresponding bottom images are taken with polarized ﬁlters to visualize birefringence. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 Source data and homozygous mutation information for the relevant strains are provided in the Source Data ﬁle wt 1.5 1.0 0.5 0 sms-5(ok2498) sms-5(tr433) f g e 10 µm * * Relative abundance of wact-190 by mass-spec s e g g resistant wactives, the majority of which are structurally distinct. Detailed analyses with two resistant wactives show that SMS-5 expression speciﬁcally in the anterior marginal cells (SMS-5(MC)) rescues this resistance (Fig. 7b, c). Like wact-190, we measured the accumulation of two other resistant wactives by mass spectrometry and found decreased accumulation in the tissue of the sms-5 null mutant relative to wild-type animals (Fig. 7f). signiﬁcantly less soluble in water, and have more hydrogen bond acceptors, than the molecules that kill both the wild type and the sms-5 mutant (Fig. 7g). The hypersensitive molecules have nearly the opposite set of properties: they are of lower molecular weight, have a smaller PSA, and have fewer hydrogen bond acceptors and donors than control molecules that fail to kill either the wild type or the sms-5 mutant (Fig. 7g). We infer that SMS-5 contributes to a marginal cell barrier that normally allows the accumulation of large hydrophobic molecules but limits the accumulation of relatively smaller and less hydrophobic molecules. signiﬁcantly less soluble in water, and have more hydrogen bond acceptors, than the molecules that kill both the wild type and the sms-5 mutant (Fig. 7g). The hypersensitive molecules have nearly the opposite set of properties: they are of lower molecular weight, have a smaller PSA, and have fewer hydrogen bond acceptors and donors than control molecules that fail to kill either the wild type or the sms-5 mutant (Fig. 7g). We infer that SMS-5 contributes to a marginal cell barrier that normally allows the accumulation of large hydrophobic molecules but limits the accumulation of relatively smaller and less hydrophobic molecules. We were surprised to ﬁnd that 169 (33%) of the wactives killed the mutants at a concentration that had little-to-no effect on wild type (Fig. 7a). Because the sms-5 mutants are hypersensitive to these 169 molecules, we refer to these as hypersensitive molecules. Detailed analyses with two hypersensitive molecules show that SMS-5 expression speciﬁcally in the anterior marginal cells (SMS- 5(MC)) can rescue hypersensitivity (Fig. 7d, e). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 4 Mutant genes that resist the lethality induced by wact-190. Mutants listed in rows 1 17 were isolated in our forward genetic screen for those that a No. of animals ( ) or % crystals ( ) wt wact-190 sms-5(ok2498) wact-190 Ketosphingonine reductase Ceramide synthase Serine palmityol transferase Dihydroceramide desaturase Sphingomyelin synthase Sphingomyelin sms-1 sms-2 sms-3 sms-4 sms-5 F33D4.4 ttm-5 hyl-1 hyl-2 lagr-1 sptl-1 sptl-2 sptl-3 Many homologues (untested) a b c d Wact-190: – – + + – – + + – – + + – – + + 10 µm >100 80 60 40 20 0 wt sms-5(ok2498) sms-5(ok2498) + sms-5 fos. sms-5(ok2498) + sms-5 (MC) wt wact-190 sms-5(ok2498) wact-190 Sphingomyelin b c 10 µm No. of animals ( ) or % crystals ( ) d Wact-190: – – + + – – + + – – + + – – + + >100 80 60 40 20 0 wt k2498) s-5 fos. 5 (MC) b = Culture overgrown (>>50 animals in the well) = Culture sick (between 11 and 50 animals) = Culture dead (fewer than 11 animals) = Not done Fig. 4 Mutant genes that resist the lethality induced by wact-190. Mutants listed in rows 1–17 were isolated in our forward genetic screen for those that resist the lethal/arrest phenotype of wact-190. In addition to these 17, we identiﬁed 29 other strains with a mutant allele of pgp-14 that will be described elsewhere. Additional mutants beyond row 17 are deletion alleles obtained from either the C. elegans Genetics Centre or from Shohei Mitani. While all of the mutants isolated in our screen resist wact-190 on solid substrate, only some also resist wact-190 effects in liquid culture. F53B1.2 is a paralog of the sms genes, and F33D4.4 is a paralog of ttm-5. Viability assays were done with at least four replicates (see “Methods” for details). Residue numbers for both isoforms of sptl-2 are shown. Protein changes noted in red indicate a presumptive null allele; “X” indicates an early non- sense codon. For ﬁve wact-190-resistant strains (rows 13–17), the mutant gene responsible for wact-190 resistance has not been identiﬁed and are not discussed further. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 g A schematic of the pharynx illustrating the approximate area of the cross-sections imaged by TEM. The marginal cells are indicated in fuchsia. Image adapted from WormAtlas with permission remaining signal is either background staining from the SM probe and/or SM that is produced by other SMSs27. We conclude that sms-5 mutants produce less SM in the anterior pharynx compared to wild-type animals. resistant to wact-190 or whether the mutant exhibits resistance to structurally diverse small molecules. To do this, we examined the viability of the wild type and the sms-5(ok2498) null mutant when incubated in 508 wactives at three different concentrations (7.5, 30, and 60 μM). In addition to wact-190, we found that the sms-5 mutant is resistant to 23 other wactives in at least one of the concentrations tested (Fig. 7a). We refer to these 24 molecules as The anterior pharynx is a ﬁlter for xenobiotic accumulation. We investigated whether sms-5 mutant animals are speciﬁcally NATURE COMMUNICATIONS \| (2019) 10:3938 \| https://doi.org/10.1038/s41467-019-11908-0 \| www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 signiﬁcantly less soluble in water, and have more hydrogen bond acceptors, than the molecules that kill both the wild type and the sms-5 mutant (Fig. 7g). The hypersensitive molecules have nearly the opposite set of properties: they are of lower molecular weight, have a smaller PSA, and have fewer hydrogen bond acceptors and donors than control molecules that fail to kill either the wild type or the sms-5 mutant (Fig. 7g). We infer that SMS-5 contributes to a marginal cell barrier that normally allows the accumulation of large hydrophobic molecules but limits the accumulation of relatively smaller and less hydrophobic No. of animals ( ) or % crystals ( ) wt 1.5 1.0 0.5 0 sms-5(ok2498) sms-5(tr433) wt wact-190 sms-5(ok2498) wact-190 Ketosphingonine reductase Ceramide synthase Serine palmityol transferase Dihydroceramide desaturase Sphingomyelin synthase Sphingomyelin sms-1 sms-2 sms-3 sms-4 sms-5 F33D4.4 ttm-5 hyl-1 hyl-2 lagr-1 sptl-1 sptl-2 sptl-3 Many homologues (untested) a b c f g d Wact-190: – – + + – – + + – – + + – – + + e 10 µm 10 µm * * >100 80 60 40 20 0 wt Relative abundance of wact-190 by mass-spec sms-5(ok2498) sms-5(ok2498) + sms-5 fos. sms-5(ok2498) + sms-5 (MC) tr466 sms-5 sms-1 - - - - sms-2 sms-3 sms-5 sptl-1 sptl-2 sptl-3 ttm-5 ttm-5 ok2095 ok2843 ok1927 ok2753 ok2498 ok2444 tm4022 tm2613 tm2666 gk13075 tm6585 F33D4.4 F53B1.2 N2 Strain Gene Allele Liquid 0 1.9 3.8 7.5 15 30 60 0 30 60 Solid Protein change Viability assays wact-190 concentration (µM) 1 RP2904 RP2788 RP2758 RP2785 RP2799 RP2897 RP3006 RP3009 RP2900 RP2909 RP2907 RP3004 RP2802 Unknown Unknown Unknown Unknown Unknown Deletion Deletion Deletion Deletion Deletion Deletion Deletion Deletion Deletion Deletion Deletion RP2906 RP2942 RP2943 RP3002 RB1892 RB1919 VC20262 VC2358 RB1579 RB2135 RB1685 = Culture overgrown (>>50 animals in the well) = Culture sick (between 11 and 50 animals) = Culture dead (fewer than 11 animals) = Not done 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 wt E122K G355D-G383D G380R-G408R G390E-G418E H262Y , L252P C247S W100X W100X W179X Q371X W112X Y178X sms-5 sms-5 sms-5 sms-5 sptl-2 sptl-2 sptl-2 sptl-2 sptl-2 tr444 tr433 tr441 tr448 tr459 tr490 tr493 tr462 tr471 ttm-5 ttm-5 tr469 tr488 tr451 tr468 tr474 tr475 tr486 Fig. 4 Mutant genes that resist the lethality induced by wact-190. NATURE COMMUNICATIONS \| (2019) 10:3938 \| https://doi.org/10.1038/s41467-019-11908-0 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 Mutants listed in rows 1–17 were isolated in our forward genetic screen for those that resist the lethal/arrest phenotype of wact-190. In addition to these 17, we identiﬁed 29 other strains with a mutant allele of pgp-14 that will be described elsewhere. Additional mutants beyond row 17 are deletion alleles obtained from either the C. elegans Genetics Centre or from Shohei Mitani. While all of the mutants isolated in our screen resist wact-190 on solid substrate, only some also resist wact-190 effects in liquid culture. F53B1.2 is a paralog of the sms genes, and F33D4.4 is a paralog of ttm-5. Viability assays were done with at least four replicates (see “Methods” for details). Residue numbers for both isoforms of sptl-2 are shown. Protein changes noted in red indicate a presumptive null allele; “X” indicates an early non- sense codon. For ﬁve wact-190-resistant strains (rows 13–17), the mutant gene responsible for wact-190 resistance has not been identiﬁed and are not discussed further. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 Source data and homozygous mutation information for the relevant strains are provided in the Source Data ﬁle Ketosphingonine reductase Ceramide synthase Serine palmityol transferase Dihydroceramide desaturase Sphingomyelin synthase sms-1 sms-2 sms-3 sms-4 sms-5 F33D4.4 ttm-5 hyl-1 hyl-2 lagr-1 sptl-1 sptl-2 sptl-3 Many homologues (untested) a tr466 sms-5 sms-1 - - - - sms-2 sms-3 sms-5 sptl-1 sptl-2 sptl-3 ttm-5 ttm-5 ok2095 ok2843 ok1927 ok2753 ok2498 ok2444 tm4022 tm2613 tm2666 gk13075 tm6585 F33D4.4 F53B1.2 N2 Strain Gene Allele Liquid 0 1.9 3.8 7.5 15 30 60 0 30 60 Solid Protein change Viability assays wact-190 concentration (µM) 1 RP2904 RP2788 RP2758 RP2785 RP2799 RP2897 RP3006 RP3009 RP2900 RP2909 RP2907 RP3004 RP2802 Unknown Unknown Unknown Unknown Unknown Deletion Deletion Deletion Deletion Deletion Deletion Deletion Deletion Deletion Deletion Deletion RP2906 RP2942 RP2943 RP3002 RB1892 RB1919 VC20262 VC2358 RB1579 RB2135 RB1685 = Culture overgrown (>>50 animals in the well) = Culture sick (between 11 and 50 animals) = Culture dead (fewer than 11 animals) = Not done 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 wt E122K G355D-G383D G380R-G408R G390E-G418E H262Y , L252P C247S W100X W100X W179X Q371X W112X Y178X sms-5 sms-5 sms-5 sms-5 sptl-2 sptl-2 sptl-2 sptl-2 sptl-2 tr444 tr433 tr441 tr448 tr459 tr490 tr493 tr462 tr471 ttm-5 ttm-5 tr469 tr488 tr451 tr468 tr474 tr475 tr486 tr466 sms-5 sms-1 - - - - sms-2 sms-3 sms-5 sptl-1 sptl-2 sptl-3 ttm-5 ttm-5 ok2095 ok2843 ok1927 ok2753 ok2498 ok2444 tm4022 tm2613 tm2666 gk13075 tm6585 F33D4.4 F53B1.2 N2 Strain Gene Allele Liquid 0 1.9 3.8 7.5 15 30 60 0 30 60 Solid Protein change Viability assays wact-190 concentration (µM) 1 RP2904 RP2788 RP2758 RP2785 RP2799 RP2897 RP3006 RP3009 RP2900 RP2909 RP2907 RP3004 RP2802 Unknown Unknown Unknown Unknown Unknown Deletion Deletion Deletion Deletion Deletion Deletion Deletion Deletion Deletion Deletion Deletion RP2906 RP2942 RP2943 RP3002 RB1892 RB1919 VC20262 VC2358 RB1579 RB2135 RB1685 = Culture overgrown (>>50 animals in the well) = Culture sick (between 11 and 50 animals) = Culture dead (fewer than 11 animals) = Not done 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 wt E122K G355D-G383D G380R-G408R G390E-G418E H262Y , L252P C247S W100X W100X W179X Q371X W112X Y178X sms-5 sms-5 sms-5 sms-5 sptl-2 sptl-2 sptl-2 sptl-2 sptl-2 tr444 tr433 tr441 tr448 tr459 tr490 tr493 tr462 tr471 ttm-5 ttm-5 tr469 tr488 tr451 tr468 tr474 tr475 tr486 Fig. ARTICLE For each pair of images, ﬂuorescent image is on the left and the differential interference contrast image is on the right. The scale is the same for all corresponding panels. Error bars represent standard error of the mean (SEM) for all graphs in this ﬁgure. Source data for d and e are provided in the Source Data ﬁle Given SMS-5’s role in the accumulation of hydrophobic small molecules, we asked whether SMS-5 might function in parallel with CHUP-1 to help absorb cholesterol, which is also a small hydrophobic molecule. Wild-type hatchling parental (P0) animals placed on cholesterol-limited plates are themselves able to grow to adulthood, likely because of maternally contributed cholesterol stores29. However, these P0 animals produce fewer F1 progeny and these F1s are slow to develop to the L4 stage (Fig. 9a, ﬁrst column). We tested how the sms-5 null mutant behaves on cholesterol-limited plates. Like the chup-1 null control, sms-5 null animals had difﬁculty growing on cholesterol-limited conditions (Fig. 9a; Supplementary Fig. 9). The growth deﬁcit of the sms-5 mutant is rescued by the expression of SMS-5 speciﬁcally in the anterior marginal cells. Furthermore, the chup-1; sms-5 double mutant grows worse on cholesterol-limited conditions than either single mutant (Fig. 9a; Supplementary Fig. 9). This double mutant analysis indicates that SMS-5 may function in parallel to CHUP-1 in the absorption of cholesterol. We also investigated whether the sms-5 mutant is deﬁcient in cholesterol absorption by examining the uptake of a ﬂuorescent analog called NBD-cholesterol, which was previously used to characterize chup-1’s role in cholesterol uptake28. Similar to previous results, we observed an obvious decrease in NBD- cholesterol accumulation in the chup-1 mutants relative to wild- type control after synchronized L1s were incubated in NBD- cholesterol for 6 days (Fig. 9b–d; Supplementary Fig. 10). We observed a similar decrease in NBD-cholesterol accumulation in the sms-5 mutant (Fig. 9e), which could be rescued by expressing SMS-5 speciﬁcally in the anterior marginal cells (Fig. 9f). Together, the data indicate that SMS-5 contributes to cholesterol absorption. disrupted in the same cells in which crystals form (Figs. 1 and 2). Third, sms-5 mutants coincidentally resist the crystal formation and lethality induced by wact-190 (Figs. 5d and 7b, c). Fourth, the restoration of SMS-5 activity speciﬁcally in the cells in which crystals normally form restores both crystal formation and leth- ality to the sms-5 mutant (Figs. 5d and 7b, c). ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 sms-5 mutants are hypersensitive to the loss of cholesterol. C. elegans is a cholesterol auxotroph, requiring as little as 2.5 ng/mL to survive4. CHUP-1 (initially called CUP-1) is a key trans- membrane protein involved in cholesterol absorption in C. ele- gans28. Consequently, chup-1 mutants are hypersensitive to cholesterol-limited conditions28. CHUP-1 is expressed in the posterior pharynx but is more prominently expressed in the intestine28. Despite C. elegans’ reliance on mature sterols for development4, chup-1 null mutants are viable, suggesting that additional mechanisms of cholesterol uptake exist28. Fig. 5 Mutations in a predicted sphingomyelin synthesis pathway confer resistance to wact-190. a A predicted sphingomyelin synthesis pathway. 5) are sufﬁcient to confer resistance to wact 190 (see Fig. 4). Independently derived deletion alleles were tested for each of these genes (in green), together with their respective paralogs (in black), and only the deletions of sptl-2, ttm-5, and sms-5 confer resistance (see Fig. 4). b, c 60 μM wact-190 forms crystals in wild-type worms but not in the sms-5 (ok2498) mutant. Animals were incubated in the small molecule for 24 h as L4-staged animals. Anterior is up; white and green arrows indicate channels and crystals, respectively. d Quantiﬁcation of wact-190’s effects on viability and crystal formation. The sms-5-containing fosmid (WRM0626dC03) is harbored as an extra-chromosomal array. The construct expressing SMS-5:: FLAG::mCherry from an anterior marginal cell-speciﬁc promoter (SMS-5 (MC)) (see Supplementary Fig. 7) is harbored by the transgenic array (trIs104). Viability is quantiﬁed with n = 8 independent experiments; crystal counts were done with n = 3 independent experiments. In samples where viability is indicated as <40, the respective wells have only the original young larvae that are arrested or dead. e Accumulation of the indicated small molecule in sms-5 mutants relative to wild-type animals as measured by mass spectrometry. n = 3 independent experiments. Asterisk () indicates a signiﬁcant difference (p < 0.001) using a Student’s T-test. f, g Two focal planes of an adult mosaic for the extra-chromosomal (Ex) array harboring pPRHM1051 (a construct with the SMS-5 fosmid WRM0626dC03 with yellow ﬂuorescent protein (YFP) coding sequence inserted in frame at the C-terminus of SMS-5). The YFP-fusion protein is localized to the anterior marginal cells and is enriched basally at the borders with adjacent muscle cells (white arrows). Supplementary Fig. 6 shows the non-mosaic expression pattern of SMS-5. ARTICLE We therefore expected that all of the crystal-forming compounds would behave like wact-190. That is, we expected that all crystal-forming compounds would: (i) fail to form crystals in the sms-5 mutant, and coincidentally, (ii) fail to kill the sms-5 mutant. However, we were surprised to ﬁnd that 53% of the crystallizing wactives that kill the wild type remain effective at killing the sms-5 mutant (Fig. 8a). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 In contrast to the decreased accumulation of resistant molecules in the sms-5 mutant, we examined the accumulation of three hypersensitive molecules and found increased accumulation in the sms-5 mutant relative to wild type (Fig. 7f). Some crystallizing wactives kill through multiple mechanisms. Several lines of evidence are consistent with the idea that wact- 190 crystals induced developmental arrest and/or lethality. First, the concentration at which crystals form is coincident with the concentration that kills worms (Fig. 2a). Second, the PM is We investigated the physical–chemical properties of the resistant and hypersensitive wactives. We found that the resistant molecules have a larger mass, have a greater topological PSA, are TURE COMMUNICATIONS \| (2019) 10:3938 \| https://doi.org/10.1038/s41467-019-11908-0 \| www.nature.com/naturecommunications 7 7 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 In both cases, differences are signiﬁcant (p < 0.05) at a detergent concentration of 0.8% (w/v) using a Student’s T test. SMS-5(MC) represents marginal-cell-speciﬁc expression of SMS-5::FLAG::mCherry from the trIs104 integrated transgenic array. Three biological repeats were done with four technical repeats done during each biological repeat. c, d Wild-type and sms-5 (ok2498) mutant animals are ﬁxed and stained with the GFP-NT-Lysenin protein probe that recognizes clustered sphingomyelin. White arrows indicate buccal cavity and white arrowheads indicate anterior channels. For each pair of images, the differential interference contrast image is on the left, and the ﬂuorescent image is on the right. The scale is the same for all panels. e Quantiﬁcation of the relative green ﬂuorescent protein (GFP) signal from more than ﬁve biological replicates of GFP-NT-Lysenin staining (see “Methods” for details). Asterisk () indicates a signiﬁcant difference (p = 0.024) using a Student’s T test. n = 7 independent trials with at least 10 worms analyzed during each trial. Error bars represent standard error of the mean (SEM) for all graphs in this ﬁgure. Source data for a, b, and e are provided in the Source Data ﬁle associated with cholesterol within the PM and creates sub- domains of densely packed regions within the lipid bilayer32. In addition, sphingolipids and cholesterol have been shown to act as molecular traps for one another33,34, which is consistent with the model that the SM-rich marginal cells of C. elegans can act as a sink for cholesterol and other hydrophobic molecules. Notably, a reduction in SM abundance decreases the packing density of lipids23,24 and likely makes it easier for lower-molecular-weight molecules to pass through the PM barrier (Fig. 10). Hence, the biological properties of SM are consistent with our ﬁndings that SMS-5 activity leads to the accumulation of larger hydrophobic wactives while creating a barrier to smaller wactives. but be of little evolutionary consequence because crystal-forming concentrations of xenobiotics may rarely be encountered in the wild. Our discovery that small molecules can kill worms through crystallization has important implications for drug screens using C. elegans as a model system. Crystallizing wactives are varied in structure and potency, but all have the potential to kill or arrest young larvae through perforation of the PM. For many of these crystallizing wactives, both crystallization and its associated lethality is dependent on an intact SMS-5 SM synthesis pathway (Fig. 10). Discussion H h Here we show that the marginal cells within the anterior pharynx have distinct properties that facilitate unique interactions with exogenous small molecules. We can speculate why these marginal cells play a special role in the animal by ﬁrst considering that nematodes are cholesterol auxotrophs and that free cholesterol is relatively rare within the environment1,4. The marginal cells form the channels through which excess ingested ﬂuids are expelled from the animal7,9. Hence, the marginal cells may function as a salvage system to ensure that scarce nutrients are not again lost to the environment. This model is consistent with several observa- tions: First, exogenous small molecules visibly accumulate almost exclusively in the cells that line the anterior channels. Second, the pharynx is the ﬁrst tissue to accumulate a ﬂuorescent analog of cholesterol in time-course analyses30. Third, the anterior pharynx accumulates obviously higher levels of cholesterol compared to the posterior pharynx10. Finally, absorption of these small molecules is SMS-5 dependent and SM-rich membrane has been repeatedly shown to act as a sink for cholesterol24. To investigate the discordance of the ability to kill the sms-5 mutant among crystal-forming wactives, we investigated a handful of crystal-forming compounds in detail for their ability to kill and form crystals in the sms-5 mutant. In all cases, we found that the sms-5 mutant resists crystal formation, but half of these wactives are still able to kill the mutant (Fig. 8b). Given that several lines of evidence indicate that crystals can kill larvae, these data suggest that some crystal-forming wactives may kill through both crystal formation and through crystal-independent mechanisms. We also performed similar dose–response analyses for a handful of sphere-forming compounds. In all cases examined, sms-5 mutants remained sensitive to these wactives despite a lack of sphere formation (Fig. 8b). This suggests that the sphere formation itself is simply a visible marker of compound accumulation and is not directly related to how the sphere- forming wactives kill the animals. How SMS-5 facilitates the absorption of hydrophobic mole- cules is not clear. We do know that sphingomyelin incorporation into a lipid bilayer alters the physicochemical properties of that bilayer (reviewed in refs. 31,32). In other animals, SM is tightly NATURE COMMUNICATIONS \| (2019) 10:3938 \| https://doi.org/10.1038/s41467-019-11908-0 \| www.nature.com/naturecommunications 8 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 –1 Wild type sms-5 (ok2498) 0 1 2 3 4 c WT d sms-5 e 0 20 40 60 80 0 0.1 0.2 0.4 0.8 1.6 wt sms-5(ok2498) sms-5(ok2498) + SMS-5(MC) 0 20 40 60 80 0 0.1 0.2 0.4 0.8 1.6 a b Triton-X100 (%) Worms alive (#) >100 Worms alive (#) IGEPAL CA-630 (%) >100 NT-lysenin::GFP signal (relative) 10 µm * Fig. 6 sms-5 mutants have less sphingomyelin in the anterior pharynx. a, b The sms-5 deletion allele is hypersensitive to the lethality induced by detergents triton-X 100 and IGEPAL CA-630, shown as a v/v percentage of the liquid media. In both cases, differences are signiﬁcant (p < 0.05) at a detergent concentration of 0.8% (w/v) using a Student’s T test. SMS-5(MC) represents marginal-cell-speciﬁc expression of SMS-5::FLAG::mCherry from the trIs104 integrated transgenic array. Three biological repeats were done with four technical repeats done during each biological repeat. c, d Wild-type and sms-5 (ok2498) mutant animals are ﬁxed and stained with the GFP-NT-Lysenin protein probe that recognizes clustered sphingomyelin. White arrows indicate buccal cavity and white arrowheads indicate anterior channels. For each pair of images, the differential interference contrast image is on the left, and the ﬂuorescent image is on the right. The scale is the same for all panels. e Quantiﬁcation of the relative green ﬂuorescent protein (GFP) signal from more than ﬁve biological replicates of GFP-NT-Lysenin staining (see “Methods” for details). Asterisk () indicates a signiﬁcant difference (p = 0.024) using a Student’s T test. n = 7 independent trials with at least 10 worms analyzed during each trial. Error bars represent standard error of the mean (SEM) for all graphs in this ﬁgure. Source data for a, b, and e are provided in the Source Data ﬁle 0 20 40 60 80 0 0.1 0.2 0.4 0.8 1.6 wt sms-5(ok2498) sms-5(ok2498) + SMS-5(MC) a Triton-X100 (%) Worms alive (#) >100 c WT 10 µm Triton-X100 (%) 0 20 40 60 80 0 0.1 0.2 0.4 0.8 1.6 b Worms alive (#) IGEPAL CA-630 (%) >100 b d sms-5 Fig. 6 sms-5 mutants have less sphingomyelin in the anterior pharynx. a, b The sms-5 deletion allele is hypersensitive to the lethality induced by detergents triton-X 100 and IGEPAL CA-630, shown as a v/v percentage of the liquid media. NATURE COMMUNICATIONS \| (2019) 10:3938 \| https://doi.org/10.1038/s41467-019-11908-0 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 ARTICLE wt sms-5(0) sms-5(0) + SMS-5(mc) Complex Hypersensitive (169) 0 20 40 60 80 100 0 0.12 0.23 0.47 0.94 1.88 3.75 7.5 15 30 60 120 0 20 40 60 80 100 0 0.12 0.23 0.47 0.94 1.88 3.75 7.5 15 30 60 120 200 250 300 350 400 450 500 20 40 60 80 100 −8 −6 −4 −2 0 −1 0 1 2 3 4 0 2 4 6 8 0 2 4 6 8 7.5 µM 30 µM 60 µM Wactive library (508 molecules) a d Resistant (24) b c [wact-190] (µM) [wact-455] (µM) [wact-416] (µM) [wact-572] (µM) Animals (#/well) Animals (#/well) Animals (#/well) e LogSw (solubility in water) Mass (Da) Hydrogen bond donors Hydrogen bond acceptors Topological polar surface area Rotatable bonds * * * * * g Thriving ( ) Sick ( ) Dead ( ) No data ( ) * 0 20 40 60 80 100 7.5 15 30 60 120 0 20 40 60 80 100 7.5 15 30 60 120 Accumulation in sms-5 mutant relative to wild type (log2 scale) Resistant Hypersensitive Dead Wact-190 Wact-171 Wact-498 Wact-406 Wact-455 Wact-519 Resistant Hypersensitive Alive Dead Resistant Hypersensitive Alive Dead Resistant Hypersensitive Alive Dead Resistant Hypersensitive Alive Dead Resistant Hypersensitive Alive Dead Resistant Hypersensitive Alive f Animals (#/well) 8 4 2 1 0.5 0.25 0.13 0.06 * * * * * * Wild type Wild type Wild type sms-5 () sms-5 () sms-5 () Fig. 7 sms-5 mutants have altered sensitivity to small molecules. a L1 viability assays of animals of the indicated genotype (top) grown in liquid cultu quadruplicate in 508 different wactive small molecules at 7.5, 30, or 60 μM. Wells were inspected after 6 days of growth; wells that had ≥50 animals shown in green, wells that have between 11 and 50 animals are shown in yellow, and wells with ≤11 animals are shown in red. The resulting popula growth of each of the four replicates is shown in each column. Each row corresponds to a distinct wactive molecule. The data are clustered along the y The 24 wactives with reduced potency in the mutants are referred to as resistant wactives, and the 169 wactives with increased potency in the mutants referred to as hypersensitive wactives. b–e Dose–response analyses of C. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 elegans population growth with two resistant molecules (wact-190 and wact-4 and two hypersensitive molecules (wact-455 and wact-572). The sms-5(ok2498) deletion mutant (“sms-5(0)”) was used in this analysis, along with rescuing sms-5 transgene whereby SMS-5::FLAG::mCherry (SMS-5(MC)) is expressed exclusively in the anterior marginal cells. Resulting wells with animals in the wact-190 and wact-416 conditions were invariably arrested or dead L1s. Viability counts were done with n = 8 independent experime crystal counts were done with n = 3 independent experiments. f Accumulation of the indicated small molecule in the sms-5(ok2498) mutant relativ wild-type L1 animals as measured by mass spectrometry. Standard error of the mean is shown for graphs b–f. n = 6 independent experiments for wact- wact-171, wact-498, wact-406, and wact-519; n = 4 for wact-455. g Violin plots comparing the physical–chemical properties of molecules that are classiﬁed as lethal in all conditions (dead), resistant, hypersensitive, or not obviously bioactive (alive) at 30 μM. See the legend of Fig. 1 for the descrip of the violin plots. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 This class of wactives likely has no other mechanism of killing aside from crystal formation. Our unpublished observa- tions suggest that wactives that kill C. elegans through crystal formation alone do not have wide-ranging utility against diverse parasitic nematodes. Hence, our future screens for nematicides using C. elegans as a screening platform will include a step to determine whether the wactive’s bioactivity is SMS-5 dependent. Our observations suggest a potential paradox: C. elegans has evolved a sterol-salvaging system as part of its ﬁlter-feeding strategy despite simultaneously creating a vulnerability to xeno- biotic crystal formation. However, it is unlikely that high con- centrations of hydrophobic xenobiotics are commonplace in nature. Death by crystal formation may therefore be rare beyond the laboratory. Hence, a cholesterol-salvaging system may inad- vertently facilitate the accumulation of hydrophobic xenobiotics A second class of crystallizing wactives are those whose leth- ality is not dependent on SMS-5. In an sms-5 mutant background, the crystallization of this class of wactives is abolished, but their 9 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 a L1 viability assays of animals of the indicated genotype (top) grown in liquid culture in quadruplicate in 508 different wactive small molecules at 7.5, 30, or 60 μM. Wells were inspected after 6 days of growth; wells that had ≥50 animals are shown in green, wells that have between 11 and 50 animals are shown in yellow, and wells with ≤11 animals are shown in red. The resulting population growth of each of the four replicates is shown in each column. Each row corresponds to a distinct wactive molecule. The data are clustered along the y axis. The 24 wactives with reduced potency in the mutants are referred to as resistant wactives, and the 169 wactives with increased potency in the mutants are referred to as hypersensitive wactives. b–e Dose–response analyses of C. elegans population growth with two resistant molecules (wact-190 and wact-416) and two hypersensitive molecules (wact-455 and wact-572). The sms-5(ok2498) deletion mutant (“sms-5(0)”) was used in this analysis, along with the rescuing sms-5 transgene whereby SMS-5::FLAG::mCherry (SMS-5(MC)) is expressed exclusively in the anterior marginal cells. Resulting wells with <50 animals in the wact-190 and wact-416 conditions were invariably arrested or dead L1s. Viability counts were done with n = 8 independent experiments; crystal counts were done with n = 3 independent experiments. f Accumulation of the indicated small molecule in the sms-5(ok2498) mutant relative to wild-type L1 animals as measured by mass spectrometry. Standard error of the mean is shown for graphs b–f. n = 6 independent experiments for wact-190, wact-171, wact-498, wact-406, and wact-519; n = 4 for wact-455. g Violin plots comparing the physical–chemical properties of molecules that are classiﬁed as lethal in all conditions (dead), resistant, hypersensitive, or not obviously bioactive (alive) at 30 μM. See the legend of Fig. 1 for the description of the violin plots. One, two, and three asterisks indicate a signiﬁcant difference (p < 0.05, p < 0.01, and p < 0.001, respectively) between the indicated datasets using Student’s T test. Source data are provided in the Source Data ﬁle Fig. 7 sms-5 mutants have altered sensitivity to small molecules. a L1 viability assays of animals of the indicated genotype (top) grown in liquid culture in quadruplicate in 508 different wactive small molecules at 7.5, 30, or 60 μM. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 One, two, and three asterisks indicate a signiﬁcant difference (p < 0.05, p < 0.01, and p < 0.001, respectively) between the indica ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-1190 Complex Hypersensitive (169) 7.5 µM 30 µM 60 µM Wactive library (508 molecules) a Resistant (24) Thriving ( ) Sick ( ) Dead ( ) No data ( ) Wild type Wild type Wild type sms-5 () sms-5 () sms-5 () wt sms-5(0) 0 20 40 60 80 100 0 0.12 0.23 0.47 0.94 1.88 3.75 7.5 15 30 60 120 b [wact-190] (µM) Animals (#/well) b sms-5(0) + SMS-5(mc) d Animals (#/well) 0 20 40 60 80 100 7.5 15 30 60 120 Animals (#/well) Resistant (24) Resistant (24) Hypersensitive (169) Resistant (24) Hypersensitive (169) Resistan ex Hypersensitive (169) Res [wact-455] (µM) [wact-572] (µM) e 0 20 40 60 80 100 7.5 15 30 60 120 Animals (#/well) Hypersensitive (169 0 20 40 60 80 100 0 0.12 0.23 0.47 0.94 1.88 3.75 7.5 15 30 60 120 c [wact-416] (µM) Animals (#/well) c e Animals (#/well) Animals (#/well) 0.12 0.23 0.47 0.94 1.88 3.75 7.5 15 30 60 120 [wact-416] (µM) [wact-416] (µM) −8 −6 −4 −2 0 * LogSw (solubility in water) g ( ) ( ) Dead Resistant Hypersensitive Alive 200 250 300 350 400 450 500 20 40 60 80 100 −8 −6 −4 −2 0 −1 0 1 2 3 4 0 2 4 6 8 0 2 4 6 8 [wact 416] (µM) [wact 572] (µM) * LogSw (solubility in water) Mass (Da) Hydrogen bond donors Hydrogen bond acceptors Topological polar surface area Rotatable bonds * * * * * g g ( ) ( ) ( ) ( ) * Accumulation in sms-5 mutant relative to wild type (log2 scale) Resistant Hypersensitive Dead Wact-190 Wact-171 Wact-498 Wact-406 Wact-455 Wact-519 Resistant Hypersensitive Alive Dead Resistant Hypersensitive Alive Dead Resistant Hypersensitive Alive Dead Resistant Hypersensitive Alive Dead Resistant Hypersensitive Alive Dead Resistant Hypersensitive Alive f 8 4 2 1 0.5 0.25 0.13 0.06 * * * * * * Accumulation in sms-5 mutant relative to wild type (log2 scale) Resistant Hypersensitive Wact-190 Wact-171 Wact-498 Wact-406 Wact-455 Wact-519 f 8 4 2 1 0.5 0.25 0.13 0.06 * * * * * * f g LogSw (solubility in water) Hydrogen bond donors Fig. 7 sms-5 mutants have altered sensitivity to small molecules. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 The standard error are provided in the Source Data ﬁle wact-34 b wt sms-5(0) wact-190 wact-390 wact-469 wact-2 wact-220 wact-128 wact-124 Sphere-forming wactives Crystal-forming wactives Mutants decrease lethal potency of wactive Mutants do not alter lethal potency of wactive 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 100 >100 50 0 >100 50 0 >100 50 0 >100 50 0 >100 50 0 >100 50 0 >100 50 0 >100 50 0 >100 wact-34 b wt sms-5(0) Animals with objects (%) Animals alive (#) wact-190 wact-390 wact-469 wact-2 wact-220 wact-128 wact-124 wact-140 0 0.5 3.75 15 120 0 0.5 3.75 15 120 Sphere-forming wactives Crystal-forming wactives Mutants decrease lethal potency of wactive Mutants do not alter lethal potency of wactive Wactive concentration (µM) 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 >100 50 0 >100 50 0 >100 50 0 >100 50 0 >100 50 0 >100 50 0 >100 50 0 >100 50 0 >100 50 0 b a 38 crystallizing wactives (30 µM) 33 sphere-forming wactives (30 µM) Viability Object wt sms-5(0) Wactive wt Crystal-forming Mix; crystals predominate Sphere-forming Mix-spheres predominate Culture thriving Culture struggling Culture dead/arrested 50 153 158 174 190 381 390 416 498 24 81 105 391 415 469 106 154 2 15 49 58 113 120 128 165 169 184 220 393 558 425 570 134 102 156 86 135 136 51 3 16 18 20 28 34 43 53 59 62 63 71 83 92 116 123 124 126 140 142 145 203 378 588 17 38 39 213 513 133 82 70 b a wact-34 38 crystallizing wactives (30 µM) 33 sphere-forming wactives (30 µM) Animals with objects (%) Animals alive (#) wac wact-390 wact-469 wact-2 wact-220 wact-128 wact-124 wact-140 0 0.5 3.75 15 120 0 0.5 3.75 15 120 Sphere-forming wactives Crystal-forming wactives Mutants decrease lethal potency of wa Mutants do not alter lethal potency of wactive Crystal-forming Mix; crystals predominate Sphere-forming Mix-spheres predominate Culture thriving Culture struggling Culture dead/arrested Wactive concentration (µM) 0 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 0 >100 50 0 >100 50 0 >100 50 0 >100 50 0 >100 50 0 >100 50 0 >100 50 0 >100 50 0 153 158 174 190 381 390 416 498 24 81 105 391 415 469 106 154 2 15 49 58 113 120 128 165 169 184 220 393 558 425 570 134 102 156 86 135 136 51 3 16 18 20 28 34 43 53 59 62 63 71 83 92 116 123 124 126 140 142 145 203 378 588 17 38 39 213 513 133 82 70 Fig. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 Wells were inspected after 6 days of growth; wells that had ≥50 animals are shown in green, wells that have between 11 and 50 animals are shown in yellow, and wells with ≤11 animals are shown in red. The resulting population growth of each of the four replicates is shown in each column. Each row corresponds to a distinct wactive molecule. The data are clustered along the y axis. The 24 wactives with reduced potency in the mutants are referred to as resistant wactives, and the 169 wactives with increased potency in the mutants are referred to as hypersensitive wactives. b–e Dose–response analyses of C. elegans population growth with two resistant molecules (wact-190 and wact-416) and two hypersensitive molecules (wact-455 and wact-572). The sms-5(ok2498) deletion mutant (“sms-5(0)”) was used in this analysis, along with the rescuing sms-5 transgene whereby SMS-5::FLAG::mCherry (SMS-5(MC)) is expressed exclusively in the anterior marginal cells. Resulting wells with <50 animals in the wact-190 and wact-416 conditions were invariably arrested or dead L1s. Viability counts were done with n = 8 independent experiments; crystal counts were done with n = 3 independent experiments. f Accumulation of the indicated small molecule in the sms-5(ok2498) mutant relative to wild-type L1 animals as measured by mass spectrometry. Standard error of the mean is shown for graphs b–f. n = 6 independent experiments for wact-190, wact-171, wact-498, wact-406, and wact-519; n = 4 for wact-455. g Violin plots comparing the physical–chemical properties of molecules that are classiﬁed as lethal in all conditions (dead), resistant, hypersensitive, or not obviously bioactive (alive) at 30 μM. See the legend of Fig. 1 for the description of the violin plots. One, two, and three asterisks indicate a signiﬁcant difference (p < 0.05, p < 0.01, and p < 0.001, respectively) between the indicated datasets using Student’s T test. Source data are provided in the Source Data ﬁle NATURE COMMUNICATIONS \| (2019) 10:3938 \| https://doi.org/10.1038/s41467-019-11908-0 \| www.nature.com/naturecommunications 10 ARTICLE NATURE COMMUNICATIONS \| (2019) 10:3938 \| https://doi.org/10.1038/s41467-019-11908-0 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 This suggests that this class of crystallizing east two mechanisms of killing— one that is and one that is crystal independent. Knowing lass of wactives exist has important ramiﬁca- molecules. Typically, our ﬁrst approach toward cha wactive’s mechanism of action is to screen for C. ele that resist the molecule’s lethal effects. When su mutant gene that confers resistance provides insi wact-34 a 38 crystallizing wactives (30 µM) 33 sphere-forming wactives (30 µM) b Viability Object wt sms-5(0) Wactive wt wt sms-5(0) Animals with objects (%) Animals alive (#) wact-190 wact-390 wact-469 wact-2 wact-220 wact-128 wact-124 wact-140 0 0.5 3.75 15 120 0 0.5 3.75 15 120 Sphere-forming wactives Crystal-forming wactives Mutants decrease lethal potency of wactive Mutants do not alter lethal potency of wactive Crystal-forming Mix; crystals predominate Sphere-forming Mix-spheres predominate Culture thriving Culture struggling Culture dead/arrested Wactive concentration (µM) 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 >100 50 0 >100 50 0 >100 50 0 >100 50 0 >100 50 0 >100 50 0 >100 50 0 >100 50 0 >100 50 0 50 153 158 174 190 381 390 416 498 24 81 105 391 415 469 106 154 2 15 49 58 113 120 128 165 169 184 220 393 558 425 570 134 102 156 86 135 136 51 3 16 18 20 28 34 43 53 59 62 63 71 83 92 116 123 124 126 140 142 145 203 378 588 17 38 39 213 513 133 82 70 of the lethality and object-forming capability of wactives in animals lacking SMS-5. a The behavior of molecules that fo ulation are analyzed with respect to their lethal potency in wild-type and the sms-5(ok2498) deletion mutant. The wa nd black arrowheads indicate the wactives analyzed in b. b. Dose–response analyses for select crystal and sphere-form background (indicated at the top of the two columns of graphs). Each of the wactives is analyzed over a twofold di ated at the bottom of the three columns of graphs). Wactive molecules are indicated on the left. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 However, with a 11 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 a b NBD-cholesterol accumulation in mutant relative to wt control c Wild type d chup-1(gk245) e sms-5(ok2498) f sms-5 + SMS-5(MC) 250 µm 0.0 wt chup-1(gk245) chup-1(gk245) sms-5(ok298) sms-5(ok298) sms-5(ok2498;) chup-1(gk245) sms-5(ok2498) + SMS-5(MC) sms-5(ok2498) + SMS-5(MC) 0.2 0.4 0.6 0.8 1.0 * * * * Population growth rate on XOL-limited plates 0.0 0.2 0.4 0.6 0.8 * Fig. 9 SMS-5 is required for development in cholesterol-limited conditions a The growth rate of animals of the indicated genotype grown on cholestero (xol)-limited plates (containing 50 ng/mL xol) is reported relative to the same strains grown on plates with standard concentrations of cholesterol (5000 ng/mL xol) (y axis). See “Methods” for additional details. Asterisks indicate signiﬁcance (p < 0.001) relative to the wild-type (wt) control; the red asterisks indicate signiﬁcance (p < 0.05) relative to the sms-5(ok2498) deletion mutant. The double mutant has a more severe growth defect relative to either single mutant (p = 0.06). n = 10 independent biological trials for the control (wt); n = 3 independent biological trials for all experimental genotypes. b Accumulation of NBD-cholesterol signal of the indicated mutants relative to wild-type controls. Black asterisks indicate signiﬁcance (p < 0.01) relative to the wild-type control; red asterisk indicates signiﬁcance (p < 0.05) relative to the sms-5(ok2498) deletion mutant. n = 6 a b NBD-cholesterol accumulation in mutant relative to wt control 0.0 wt chup-1(gk245) chup-1(gk245) sms-5(ok298) sms-5(ok298) sms-5(ok2498;) chup-1(gk245) sms-5(ok2498) + SMS-5(MC) sms-5(ok2498) + SMS-5(MC) 0.2 0.4 0.6 0.8 1.0 * * * * Population growth rate on XOL-limited plates 0.0 0.2 0.4 0.6 0.8 * a 0.0 0.2 0.4 0.6 0.8 1.0 * * * * Population growth rate on XOL-limited plates b NBD-cholesterol accumulation in mutant relative to wt control 0.0 0.2 0.4 0.6 0.8 * molecule that kills via two distinct mechanisms, any worm with a mutation that confers resistance to one mechanism will likely be killed by the second mechanism. Isolating a mutant that resists both mechanisms of action would be rare, occurring roughly once in every four million mutant genomes if loss-of-function muta- tions were sufﬁcient to confer resistance to each lethal mechanism (and even rarer if resistance is conferred by gain of function only). Methods D l Developmental synchronization of C. elegans population. To obtain synchro- nized populations of L1s, gravid adults were washed off the plates with M9 buffer, centrifuged at 800 × g to remove supernatant, followed by additional washes in M9 buffer until all bacteria were removed after which worms were collected as a 1.5-mL pellet in a 15-mL conical tube36. Next, 1 mL of 10% hypochlorite solution (Sigma) was added to the tube followed by 2.5 mL of 1 M sodium hydroxide solution and 1 mL double-distilled water, and the mixture was incubated on a nutator for 5 min. With 1.5 min remaining for incubation, the tube was vortexed for 10 s with two 5-s pulses after which the harvested eggs were washed 4 times with 12 mL M9 buffer, with vortexing after every addition of M9 buffer. After the ﬁnal wash, the tube was incubated overnight on a nutator at 20 °C to allow egg-hatching and checked the next day for synchronized L1s. To obtain other synchronized stages, the syn- chronize L1s are plated on solid agar substrate with E. coli food and allowed to progress to the desired stage before processing. Fig. 9 SMS-5 is required for development in cholesterol-limited conditions. a The growth rate of animals of the indicated genotype grown on cholesterol (xol)-limited plates (containing 50 ng/mL xol) is reported relative to the same strains grown on plates with standard concentrations of cholesterol (5000 ng/mL xol) (y axis). See “Methods” for additional details. Asterisks indicate signiﬁcance (p < 0.001) relative to the wild-type (wt) control; the red asterisks indicate signiﬁcance (p < 0.05) relative to the sms-5(ok2498) deletion mutant. The double mutant has a more severe growth defect relative to either single mutant (p = 0.06). n = 10 independent biological trials for the control (wt); n = 3 independent biological trials for all experimental genotypes. b Accumulation of NBD-cholesterol signal of the indicated mutants relative to wild-type controls. Black asterisks indicate signiﬁcance (p < 0.01) relative to the wild-type control; red asterisk indicates signiﬁcance (p < 0.05) relative to the sms-5(ok2498) deletion mutant. n = 6 independent biological trials for wt and the sms-5 mutant; n = 3 independent biological trials for the other two strains. In both a and b, standard error of the mean is shown and signiﬁcance is measured using Student’s T test. Source data are provided in the Source Data ﬁle. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 8 A comparison of the lethality and object-forming capability of wactives in animals lacking SMS-5. a The behavior of molecules that form objects in at least 25% of the population are analyzed with respect to their lethal potency in wild-type and the sms-5(ok2498) deletion mutant. The wactive number is shown on the right and black arrowheads indicate the wactives analyzed in b. b. Dose–response analyses for select crystal and sphere-forming wactives in the indicated genetic background (indicated at the top of the two columns of graphs). Each of the wactives is analyzed over a twofold dilution series of concentrations (indicated at the bottom of the three columns of graphs). Wactive molecules are indicated on the left. The standard error of the mean is shown. Source data are provided in the Source Data ﬁle Crystal-forming wactives 3 3 Wactive concentration (µM) Animals with objects (%) Animals alive (#) Fig. 8 A comparison of the lethality and object-forming capability of wactives in animals lacking SMS-5. a The behavior of molecules that form objects in at least 25% of the population are analyzed with respect to their lethal potency in wild-type and the sms-5(ok2498) deletion mutant. The wactive number is shown on the right and black arrowheads indicate the wactives analyzed in b. b. Dose–response analyses for select crystal and sphere-forming wactives in the indicated genetic background (indicated at the top of the two columns of graphs). Each of the wactives is analyzed over a twofold dilution series of concentrations (indicated at the bottom of the three columns of graphs). Wactive molecules are indicated on the left. The standard error of the mean is shown. Source data are provided in the Source Data ﬁle lethality persists. This suggests that this class of crystallizing wactives has at least two mechanisms of killing— one that is crystal dependent, and one that is crystal independent. Knowing that this second class of wactives exist has important ramiﬁca- tions for how we investigate the mechanism of action of bioactive molecules. Typically, our ﬁrst approach toward characterizing a wactive’s mechanism of action is to screen for C. elegans mutants that resist the molecule’s lethal effects. When successful, the mutant gene that confers resistance provides insight into the molecule’s mechanism of action12–14,35. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 Knowing that a bioactive molecule behaves in this way may facilitate the characterization of its mechanism of action by screening for resistant mutants in an sms-5 null background wherein crystal formation is abolished. a b NBD-cholesterol mutant relative c Wild type d chup-1(gk245) e sms-5(ok2498) f sms-5 + SMS-5(MC) 250 µm 0.0 wt chup-1(gk245) chup-1(gk245) sms-5(ok298) sms-5(ok298) sms-5(ok2498;) chup-1(gk245) sms-5(ok2498) + SMS-5(MC) sms-5(ok2498) + SMS-5(MC) 0.2 0.4 * * * Population on XOL-lim 0.0 0.2 0.4 y Investigating the sensitivity of the sms-5 mutant to the entire wactive library revealed that this mutant is at least twofold more sensitive than wild type to more than a third of the small mole- cules surveyed. Detailed analysis with a small subset of these hypersensitive molecules indicates that restoring SMS-5 function to only the marginal cells is sufﬁcient to confer normal sensitivity to these wactives. These observations raise several important points. First, it raises the possibility that the tissue of the anterior pharynx is either the target of many bioactive molecules and/or serves as the conduit through which many bioactive molecules traverse en route to their targets in other tissues. Intriguingly, there are several sphere-generating wactives that fail to generate spheres in the mutant background but simultaneously increase in potency in the mutant background. This suggests that, without SMS-5 in the pharynx, the sphere-generating wactives cannot be contained within the pharynx and are better able to disrupt tar- gets throughout the animal. Second, the sms-5 mutant becomes a tool with which we can increase the potency of many small molecules. Given that the sms-5 deletion mutant is otherwise healthy, it is a useful sensitized background for future small molecule screens and other chemical genetic experiments. 0.0 wt chup-1(gk245) sms-5(ok298) sms-5(ok2498;) chup-1(gk245) sms-5(ok2498) + SMS-5(MC) c Wild type d chup-1(gk245) 250 µm c Wild type 250 µm d chup-1(gk245) c Wild type d chup-1(gk245) e sms-5(ok2498) f sms-5 + SMS-5(MC) 250 µm d e sms-5(ok2498) f f sms-5 + SMS-5(MC) e Methods D l c–f Images of multiple animals after the indicated strains were incubated in NBD-cholesterol for 6 days. The scale is the same for all images. Green lines exemplify the area used in each animal to calculate signal in b. Red arrow indicates the expression of yellow ﬂuorescent protein that is restricted to the anterior pharynx that is used as a marker to follow the rescuing transgene trIs104. In a, b, and f, SMS-5(MC) represents marginal-cell-speciﬁc expression of SMS-5 from the trIs104 integrated transgenic array. Supplementary Fig. 9 shows that, in the absence of NBD-cholesterol, each strain auto-ﬂuoresces in the green ﬂuorescent protein channel to the same extent Fig. 9 SMS-5 is required for development in cholesterol-limited conditions. a The growth rate of animals of the indicated genotype grown on cholesterol (xol)-limited plates (containing 50 ng/mL xol) is reported relative to the same strains grown on plates with standard concentrations of cholesterol (5000 ng/mL xol) (y axis). See “Methods” for additional details. Asterisks indicate signiﬁcance (p < 0.001) relative to the wild-type (wt) control; the red asterisks indicate signiﬁcance (p < 0.05) relative to the sms-5(ok2498) deletion mutant. The double mutant has a more severe growth defect relative to either single mutant (p = 0.06). n = 10 independent biological trials for the control (wt); n = 3 independent biological trials for all experimental genotypes. b Accumulation of NBD-cholesterol signal of the indicated mutants relative to wild-type controls. Black asterisks indicate signiﬁcance (p < 0.01) relative to the wild-type control; red asterisk indicates signiﬁcance (p < 0.05) relative to the sms-5(ok2498) deletion mutant. n = 6 independent biological trials for wt and the sms-5 mutant; n = 3 independent biological trials for the other two strains. In both a and b, standard error of the mean is shown and signiﬁcance is measured using Student’s T test. Source data are provided in the Source Data ﬁle. c–f Images of multiple animals after the indicated strains were incubated in NBD-cholesterol for 6 days. The scale is the same for all images. Green lines exemplify the area used in each animal to calculate signal in b. Red arrow indicates the expression of yellow ﬂuorescent protein that is restricted to the anterior pharynx that is used as a marker to follow the rescuing transgene trIs104. In a, b, and f, SMS-5(MC) represents marginal-cell-speciﬁc expression of SMS-5 from the trIs104 integrated transgenic array. Supplementary Fig. ARTICLE Because of decreased molecula density of a SM-poor PM, relatively smaller molecules can now penetr the PM barrier Phosphatidylcholine Sphingomyelin Crystalizing wactive ‘Hypersensitive’ wactive Extracellular Intracellular SMS-5 a Wild type Extracellular Intracellular SMS-5 b Wild type (+ time) Phosphatidylcholine Sphingomyelin Crystalizing wactive ‘Hypersensitive’ wactive Extracellular Intracellular SMS-5 a Wild type To analyze the ability of animals to recover from exposure to crystallizing wactives, synchronized L1s were incubated in 30 µM each wactive (or 1% DMSO control) using 50 L1s/well of a 24-well plate (Sarstedt) containing 100 µL of NGM plus HB101 bacteria (t = 0). The experimental samples were prepared with four technical replicates and the DMSO control with two technical replicates. At the indicated time points (t = 0.5, 1, 2, 6, 24, and 48 h), the entire liquid volume was withdrawn from the well and pipetted onto 6 cm MYOB plates seeded with OP50 and left to dry. The next day, the total number of L1s were counted on plate. Two days after that, the number of L4s and adults on each plate was recorded and expressed as a fraction of total number of L1s. Results are from two biological trials. All experiments were done at 20 °C. Phosphatidylcholine Sphingomyelin Crystalizing wactive ‘Hypersensitive’ wactive Extracellular Intracellular SMS-5 b Wild type (+ time) To analyze the response of various strains to detergents, 25 synchronized L1 larvae were added to each well of a 24-well plate seeded with 25 μL OP50 and containing a ﬁnal concentration of 0–1.6% detergent (Triton-X-100 or IGEPAL- 630) dissolved in water (day 0). On day 6, the number of worms alive was counted and recorded. Results are from three independent trials with four technical replicates. Analysis of Evans Blue dye penetration into the pharynx. L1-stage worms were incubated with 1% DMSO (control) or 60 µM wact-190 (in 1% DMSO) in liquid for 24 h. Worms were washed once with M9 and incubated with a ﬁnal con- centration of 0.1% Evans Blue dye in 500 µL of M9 in siliconized Eppendorf tubes for 4 h. Worms were then washed three times with M9 solution, suspended in 10 µL M9, and paralyzed for microscopy by adding 4 µL of 50 mM levamisole. Live worms were mounted on 3% agarose pad. ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 the 96-well plates using a 96-pin replicator with a 300-nL slot volume (V&P Sci- entiﬁc). The ﬁnal concentration of DMSO in each well was 0.6% v/v. Approximately 20 synchronized L1 larval stage worms were added to each well of the 96-well plates in 10 μL of M9 buffer36. For some experiments, approximately 50 synchronized L1-stage worms were added to 80 μL of bacterial suspension in 20 μL of M9 buffer. Worms were synchronized to L1 stage through hypochlorite bleaching of gravid adults performed the previous day36. Assay plates were sealed with Paraﬁlm and incubated for 6 days at 20 °C with shaking at 200 in a Newbrunswick scientiﬁc I26 incubator shaker. After 6 days, the plates were observed using a dissection micro- scope and the wells were categorized according to the number of viable adult and larval stage worms in each well. Wells with >50 animals after 6 days were categorized as over-grown. The number of worms in wells with approximately ≤50 worms were counted. All 6-day viability assays were completed in quadruplicate. diverse properties within the limits of our search parameters. We conducted Phosphatidylcholine Sphingomyelin Crystalizing wactive ‘Hypersensitive’ wactive Extracellular Intracellular SMS-5 a Wild type c sms-5 mutant Intracellular Extracellular Intracellular SMS-5 b Wild type (+ time) Fig. 10 A model of SMS-5’s role in small molecule accumulation. a SMS- key in synthesizing sphingomyelin (SM) on the outer leaﬂet of the plas membrane (PM) of the marginal cells of the anterior pharynx. The SM- membrane acts as a sink for relatively larger hydrophobic crystallizing wactives but a barrier to comparatively smaller molecules. b Over time, hydrophobic wactives precipitate out of solution within the SM-rich PM the wild-type marginal cells of the anterior pharynx. c The absence of SM 5 results in less SM in the PM, which in turn reduces the accumulatio relatively large hydrophobic molecules. ARTICLE Dye ﬂuorescence was observed in TX2 channel of a Leica DMRA microscope at ×630 total magniﬁcation and quantiﬁed using the Fiji (ImageJ) software by calculating the mean gray value (MGV) in the anterior pharynx relative to the MGV in a nearby space in the frame devoid of worms. Experiments were done measuring at least six worms for each trial with a total of three biological trials. c sms-5 mutant Intracellular c Molecular biology. pPRHM1051 is the wrm0626dC03 fosmid, which contains the sms-5 locus (W07E6.3), wherein the SMS-5 coding sequence is C-terminally tagged with YFP. This construct rescues mutant sms-5’s resistance to wact-190. pPRHM1051 was created using Oliver Hobert’s fosmid tagging methodology37. Brieﬂy, we PCR ampliﬁed the YFPint-FRT-galK-FRT cassette from the pBALU2 plasmid (a gift from Oliver Hobert) using the cassette-sms-5 fusion primers, Fw (5’-cgagttccaaaaacgtgtcgacattgaaaaaatcacgaagatctttcgaaatgagtaaaggagaagaacttttcac- 3’) and Rev (5’-gagattttttattcaatttttgttagcaaaaataaattgttcagcctaatTTAtttgta- tagttcatccatgccatg-3’). We transformed the bacterial strain carrying the wrm0626dC03 fosmid (a gift from Don Moerman) with the ampliﬁed PCR product and followed the detailed recombineering protocol described in Tursun et al. (2009) to integrate the YFP tag directly into the sms-5-containing fosmid37. pPRHZ1138 (pgp-14p::SMS-5B(genomic)::FLAG::mCherry) (aka SMS-5(MC)) construct is based upon pPRHM1065 (myo-2p::SMS-5::FLAG::mCherry). Brieﬂy, we used primers HZ4pgp14hind.for (5’- aaattaagcttcaacagagagcaaggtg-3’) and HZ3pgp14prokpn.rev (5’-aaattggtaccgtttaattatcgtacatcg-3’) to amplify by PCR a 1.6-kb pgp-14 promoter fragment from the pPRZH1160 (the pgp-14 fosmid (WRM065dH09) 7.748 kb NaeI/SacI fragment in pKS) construct. We then cut the 1.6-kb PCR fragment with HindIII and KpnI. We cut out the myo-2 promoter in pPRHM1065 by using HindIII and KpnI and purifying the 7.5-kb fragment and ligating it to the 1.6-kb pgp-14 promoter. We veriﬁed the construct through restriction digest analyses. Fig. 10 A model of SMS-5’s role in small molecule accumulation. a SMS-5 is key in synthesizing sphingomyelin (SM) on the outer leaﬂet of the plasma membrane (PM) of the marginal cells of the anterior pharynx. The SM-rich membrane acts as a sink for relatively larger hydrophobic crystallizing wactives but a barrier to comparatively smaller molecules. b Over time, the hydrophobic wactives precipitate out of solution within the SM-rich PM of the wild-type marginal cells of the anterior pharynx. c The absence of SMS- 5 results in less SM in the PM, which in turn reduces the accumulation of relatively large hydrophobic molecules. Because of decreased molecular density of a SM-poor PM, relatively smaller molecules can now penetrate the PM barrier Fig. 10 A model of SMS-5’s role in small molecule accumulation. Methods D l 9 shows that, in the absence of NBD-cholesterol, each strain auto-ﬂuoresces in the green ﬂuorescent protein channel to the same extent Crystal and sphere analyses. Synchronized L1s were added to wells of a 96-well plate (50 L1s/well) containing liquid Nematode Growth Medium (NGM), E. coli (HB101) as food source (OD600 = 1.2), and 30 μM of 240 wactives in duplicate wells. Forty-eight hours later, worms were transferred to Eppendorf tubes and washed once with fresh M9 and spun down at 1800 × g for 1 min to form a tight worm pellet of ∼10 μL. Then 5 μL of 50 mM levamisole (to a ﬁnal concentration of ∼16.7 mM) was added to paralyze worms. Worms were then mounted on a 2% agarose pad on a glass slide. Live worms were then observed for the presence of birefringent crystals or non-birefringent spheres in the pharynx using ×40 objective of a Leica DMRA microscope. For both the survey of 238 molecules and the dose–response analyses, a minimum of three biological replicates were processed, and at least 20 worms per replicate were counted for the presence or absence of spheres and/or crystals. To investigate the physicochemical property trends that are consistent with crystal-forming molecules and non-object forming molecules, we started with Chembridge’s set of 500,000 “express-pick” molecules and applied the desired physicochemical ﬁlters. The selected range of crystal-like properties are: log Sw (approximately from −5 to −6); rotatable bonds (1 or 2); tPSA (≥50); H bond acceptors (≥4); H bond donors (0); mass (between 305 and 315). The selected range of no-object-like properties are: log Sw (approximately from −4.0 to −3.5); rotatable bonds (4–6); tPSA (≤40); H bond acceptors (1 or 2); H bond donors (1); mass (between 305 and 315). This left 122 molecules for the “crystal properties” list and 142 molecules for the “no-object” list. From these, we selected molecules with 12 NATURE COMMUNICATIONS \| (2019) 10:3938 \| https://doi.org/10.1038/s41467-019-11908-0 \| www.nature.com/naturecommunications ARTICLE The plates incorporate either standard (5000 ng/mL, “+xol”) or low (50 ng/mL, “−xol”) concentrations of cholesterol. We did not prepare plates without choles- terol because resulting F1s arrest as L1s, regardless of the genotype we tested. All plates are then seeded with 50 µL of an overnight OP50 LB culture. GFP-NT-Lysenin protein production and testing. GFP-NT-Lysenin protein38 was produced by ﬁrst transforming BL21 (DE3) Rosetta cells with pMAL-C2-GFP-NT- Lysenin vector (a gift from Dr. Gregory Fairn, University of Toronto) and cultured in 1 L of LB broth at 37 °C until OD600 reached 0.639. Expression was induced with 1 mM IPTG for 6 h at 25 °C. Cells were frozen overnight at −80 °C. Cells were lysed using B-PER reagent (Thermo Fisher Scientiﬁc) according to the manufacturer’s instructions in the presence of excess MgCl2, DNase I, lysozyme, and protease inhibitor cocktail. Crude cell lysate was passed through a Sepharose column con- taining 5 mL amylose resin (New England Biolabs) and the MBP-tagged GFP-NT- Lysenin fusion protein was eluted with fresh 10 mM maltose in 1× phosphate-buffer saline (PBS; pH 7.4). All puriﬁcation steps were carried out at 4 °C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis conﬁrmed the presence of the 90 kDa fusion protein, which was stored at 4 °C until use. Three days after seeding plates with L1 P0s, 20 gravid adults are transferred to fresh plates in triplicate and allowed to lay eggs. P0s raised on +xol plates are transferred to new +xol plates; P0s raised on −xol plates are transferred to new −xol plates. After 6 h of egg-laying, the P0s are removed. We deﬁne this as “day 0”. On day 4, the number of F1 L4s and/or adults on the plates are counted. The experiment was repeated at least three independent times (each with three technical replicates) at 20 °C. For each strain, we measure the impact of xol limitation by calculating the ratio of F1 L4s/adults on the −xol plates relative to the F1 L4s/adults on the +xol plates (to account for fecundity/growth issues unrelated to xol). Note that we store our ﬁlter-sterilized cholesterol stock (in 100% ethanol) in a 50-mL falcon tube sealed with paraﬁlm at 4 °C to prevent evaporation. ARTICLE p , To test the efﬁcacy of the GFP-NT-Lysenin protein, we created an artiﬁcial lipid bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine either with or without brain SM (1:1) prepared in 10 mM HEPES buffer and 150 mM NaCl, pH 7.4. DiI- C18 (Invitrogen, Oakville, ON) was added to the lipid mixtures from a stock solution to a ﬁnal concentration of 1 mol%40. To create the bilayers, the lipid mixture was dried by rotary evaporation for 1.5 h. The resulting ﬁlm was rehydrated in the HEPES buffer to a concentration of 1 mM and sonicated at 65 °C for 20 min to produce small unilamellar vesicles (SUVs). The lipid bilayer was formed by vesicle fusion onto freshly cleaved V1 grade muscovite mica afﬁxed using UV-curable adhesive (Norland Optical Adhesive 63, Norland Products, Cranbury, NJ) to a glass-bottom Willco dish. The ﬂuid cell was incubated for 5 min at 50 °C with 400 μL HEPES buffer and 5 mM CaCl2 to make the surface more hydrophilic prior to adding 100 μL of the SUV suspension. To facilitate liposome fusion and bilayer formation, the freshly cleaved mica sealed in the ﬂuid cell was incubated for ∼10 min with HEPES buffer (10 mM HEPES, 150 mM NaCl, pH 7.4, 1 M CaCl2) prior to the introduction of ∼500 µL of hydrated liposomes heated to ∼70 °C. After ∼30 min of incubation at RT to allow for bilayer formation, the ﬂuid cell was ﬂushed with liposome-free buffer. If an excessive number of vesicles were observed under TIRF illumination, 1 mL aliquots of buffer were exchanged from the dish as needed. If a continuous bilayer was not present, 100 μL aliquots of 1 mM lipid stock were exchanged for 100 μL buffer from the dish as needed. A home-built polarized TIRF microscope built around an Olympus IX70 inverted microscope that accommodates multiple excitation laser lines was utilized for pTIRF microscopy. The bottom of the dish was brought into focus under ambient lighting under oil immersion using a ×60 1.45 NA TIRF objective. Appropriate ﬁlters were then inserted and a region of the supported bilayer was brought into focus under pTIRF illumination. Images were captured with an Evolve 512 EMCCD camera (Photometrics, Tucson, AZ) controlled by Micro-Manager (http://micro-manager.org). Fluorescent probes were excited by parallel (s) or perpendicular (p) polarized light, relative to the substrate surface, through the rotation of a half-wave plate in the excitation path. ARTICLE Images were acquired before and after the addition of 20 µg/mL GFP-NT-Lysenin protein to the imaging chamber. NBD-cholesterol accumulation assay. NBD-cholesterol accumulation assays were performed by modifying a previously published protocol28 as follows. An overnight OP50 culture was pelleted and diluted with autoclaved water to which was added 22-NBD-cholesterol dissolved in ethanol (Thermoﬁsher Inc.) at a concentration of 200 µM and vortexed vigorously. One hundred and seventy-ﬁve microliters of the OP50 + 22-NBD-cholesterol mixture was added to 6 cm NGM Petri plates containing 5 µg/mL (∼13 µM) cholesterol made on the same day and then dried in a laminar ﬂow hood for 1 h. The next day (deﬁned as “day 0”), 25 synchronized L1-stage larvae were plated onto each of three 6 cm NGM Petri plates for each strain. On day 6, worms were removed from plates and washed three times with M9. After the ﬁnal wash, 200 µL of worm pellet was added to an Eppendorf tube, to which was added 75 µL of 20% of freshly prepared PFA diluted to a ﬁnal volume of 500 µL with M9 (to ﬁnal concentration of 3%). Worms were ﬁxed for 30 min at RT. Next, the worm pellet was washed twice with 1 mL of a 100 mM glycine solution to quench the PFA. A minimal volume of concentrated worm pellet (∼5 µL) was then deposited onto a 3% agarose pad on a glass slide. A small drop of a ﬂuorescent mounting medium (Thermo Scientiﬁc™Richard-Allan Sci- entiﬁc™Cytoseal 280) was added to the sample before adding a coverslip. For each sample, 30–70 worms were imaged using identical exposure times and ×10–×20 objectives. ImageJ was used to quantify ﬂuorescence intensity in the anterior and posterior halves of the worm gut, along with background areas next to the anterior and posterior halves to control for differences in background signals. The ﬂuor- escence intensity is expressed as MGV. The experiment was performed at least three times. Except where noted, worms were incubated at 20 °C. Emission spectra of wactives. Emission spectra for select wactives were measured by making a 50-µL solution of each compound at a concentration of 250 µM in double-distilled water. A ﬂuorescence intensity scan was performed in 96-well plate format using an Inﬁnite M200 Pro microplate reader (Tecan Life Sciences) with an excitation wavelength set at 390 nm and the range of emission wavelength mea- sured at 425–700 nm with 25-nm steps. ARTICLE Worms were grown on MYOB plates seeded with E. coli (OP50). Gravid adults were bleached and the resulting embryos were incubated overnight at 20 °C to yield synchronized L1 parents (P0s). The next day, ∼80 synchronized L1 P0s were plated onto modiﬁed NGM plates (35 mm) in triplicate. The modiﬁed NGM recipe is used to deplete its contents of sterols, and is described in ref. 10. Brieﬂy, the agar is replaced with agarose (Froggarose) and the peptone is extracted with ether three times and dried in a fume hood overnight. The plates incorporate either standard (5000 ng/mL, “+xol”) or low (50 ng/mL, “−xol”) concentrations of cholesterol. We did not prepare plates without choles- terol because resulting F1s arrest as L1s, regardless of the genotype we tested. All plates are then seeded with 50 µL of an overnight OP50 LB culture. Three days after seeding plates with L1 P0s, 20 gravid adults are transferred to fresh plates in triplicate and allowed to lay eggs. P0s raised on +xol plates are transferred to new +xol plates; P0s raised on −xol plates are transferred to new −xol plates. After 6 h of egg-laying, the P0s are removed. We deﬁne this as “day 0”. On day 4, the number of F1 L4s and/or adults on the plates are counted. The experiment was repeated at least three independent times (each with three technical replicates) at 20 °C. For each strain, we measure the impact of xol limitation by calculating the ratio of F1 L4s/adults on the −xol plates relative to the F1 L4s/adults on the +xol plates (to account for fecundity/growth issues unrelated to xol). Note that we store our ﬁlter-sterilized cholesterol stock (in 100% ethanol) in a 50-mL falcon tube sealed with paraﬁlm at 4 °C to prevent evaporation. Cholesterol-limitation assay. Worms were grown on MYOB plates seeded with E. coli (OP50). Gravid adults were bleached and the resulting embryos were incubated overnight at 20 °C to yield synchronized L1 parents (P0s). The next day, ∼80 synchronized L1 P0s were plated onto modiﬁed NGM plates (35 mm) in triplicate. The modiﬁed NGM recipe is used to deplete its contents of sterols, and is described in ref. 10. Brieﬂy, the agar is replaced with agarose (Froggarose) and the peptone is extracted with ether three times and dried in a fume hood overnight. ARTICLE a SMS-5 is key in synthesizing sphingomyelin (SM) on the outer leaﬂet of the plasma membrane (PM) of the marginal cells of the anterior pharynx. The SM-rich membrane acts as a sink for relatively larger hydrophobic crystallizing wactives but a barrier to comparatively smaller molecules. b Over time, the hydrophobic wactives precipitate out of solution within the SM-rich PM of the wild-type marginal cells of the anterior pharynx. c The absence of SMS- 5 results in less SM in the PM, which in turn reduces the accumulation of relatively large hydrophobic molecules. Because of decreased molecular density of a SM-poor PM, relatively smaller molecules can now penetrate the PM barrier Sample preparation for mass spectrometric (MS) analyses. We grew a mixed stage population of wild-type (N2) C. elegans and collected embryos through hypochlorite bleaching of gravid adults performed the previous day36. Embryos were allowed to hatch overnight in M9 buffer. The resulting synchronized L1s were harvested the next morning. L1s were then grown on 10 cm plates, with 10,000 L1s per plate, and incubated at room temperature (RT) for 48 h. Worms were then washed off the two plates with M9 buffer, collected into a 15-mL conical tube, and washed three times. In parallel to this, we prepared a “drug”-incubation buffer by ﬁrst inoculating 400 mL of LB with 50 μL of an overnight culture of E. coli (HB101), grew it overnight, and centrifuged 50 mL of this fresh culture for 10 min at 2100 × g. We decanted the LB and rinsed the bacteria in ∼50 mL of NGM buffer once and then resuspended the bacteria in 25 mL of NGM. We resuspended 20,000 diverse properties within the limits of our search parameters. We conducted the tests in the same way as our initial survey of 238 molecules. C. elegans viability assays. Six-day viability assays were conducted by ﬁrst gen- erating a saturated culture of HB101. E. coli was concentrated twofold in liquid NGM and 40 μL of bacterial suspension was dispensed into each well of a ﬂat-bottom 96- well plate. Compounds and dimethyl sulfoxide (DMSO) controls were pinned into 13 NATURE COMMUNICATIONS \| (2019) 10:3938 \| https://doi.org/10.1038/s41467-019-11908-0 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 worms in 1 mL of the NGM+HB101 solution in either 1% DMSO or 30 µM of small molecule (to a ﬁnal DMSO concentration of 1%) in 1.5-mL siliconized microcentrifuge tubes. We then incubated the tubes for 4 h at 20 °C on a nutator. Thereafter, we washed the worms ﬁve times with ice-cold M9 buffer, keeping the samples on ice as much as possible. We removed the M9 and ﬂash froze the samples using liquid nitrogen and stored the worms at −80 °C until the samples were ready to process by MS. Staining worms with GFP-NT-Lysenin protein. Synchronized adults were ﬁxed in 1.5-mL Eppendorf tubes using the Modiﬁed Finney-Ruvkun protocol41. Brieﬂy, worms were washed with chilled M9 buffer three times, removing as much bacteria as possible. The worm pellet was incubated in ice for 30 min and then mixed with 2× MRWB (5% methanol) and freshly prepared paraformaldehyde (PFA; ﬁnal con- centration—4%), freeze–thawed in an ice-ethanol bath four times, and then incubated on ice for 2 h. The worm suspension was then reduced and oxidized with 1% beta- mercaptoethanol and 10 mM dithiothreitol and 0.3% hydrogen peroxide, respectively. The following changes were made to the protocol: All washes were done with tris- triton buffer (TTB) lacking triton-X-100; triton-X-100 was also replaced with 1× PBS in Buffers A and B in the ﬁnal steps. The quality of ﬁxation was tested by actin staining of ﬁxed worms following 2 h incubation at RT with 0.328 nM phalloidin. y p y To perform an methyl tert-butyl ether (MTBE) extraction of small molecules from the worm lysate, we ﬁrst lyophilized the worm pellet and resuspended it in 200 μL of 0.1 M NaCl, followed by sonication for 5 min at 100 W in a Misonix cup sonicator. Samples were then spiked with 43 ng of internal standard (triamcinolone acetonide) and extracted twice with MTBE (sample: MTBE, 1:5, v/v). Organic layers were pooled and evaporated to dryness under N2. Extracts were re- suspended in 200 μL of high-performance liquid chromatography (HPLC)-grade methanol and analyzed as described below. y p y To perform an methyl tert-butyl ether (MTBE) extraction of small molecules from the worm lysate, we ﬁrst lyophilized the worm pellet and resuspended it in 200 μL of 0.1 M NaCl, followed by sonication for 5 min at 100 W in a Misonix cup sonicator. ARTICLE Samples were then spiked with 43 ng of internal standard (triamcinolone acetonide) and extracted twice with MTBE (sample: MTBE, 1:5, v/v). Organic layers were pooled and evaporated to dryness under N2. Extracts were re- To stain worms with GFP-NT-Lysenin, 20 µL of ﬁxed worms were suspended in 0.5 mg/mL of GFP-NT-Lysenin in the ﬁnal volume of 100 µL of 1× PBS in a 0.6-mL tube. Fixed worms were incubated at 4 °C for 4 h in the dark with constant mixing. Worms were washed two times with 5.5 mL ice-cold 1× PBS. Ten microliters of worms were mounted on a glass slide containing 3% agarose and a cover-slip was applied. Animals were imaged in the GFP channel using ×20 magniﬁcation and identical exposure times for all strains. The experiment was repeated at least three times. Fluorescence intensity in the anterior pharynx was quantiﬁed using the ImageJ software. For each worm, ﬂuorescence intensity was determined by subtracting the background MGV from MGV of the procorpus. Student’s T test was performed on mean values of the different trials using GraphPad 6.0. y p p y 2 suspended in 200 μL of high-performance liquid chromatography (HPLC)-grade methanol and analyzed as described below. MS analyses of wactive accumulation. The samples were analyzed by LC/MS/MS using a 6410 LC/MS/MS instrument (Agilent Technologies) with an ESI source in positive ion mode. Samples were separated on a Zorbax XDB-C18 column (4.6 × 50 mm, 3.5 μm) at 0.4 mL/min. The mobile phase consisted of HPLC-grade water (A) and methanol (B) both containing 5 mM NH4Ac. The following gradient was run: 0–1 min, 60% (B); 1–3.3 min, 60–100% (B); 3.3–7 min, 100% (B); stop time, 10 min; post-time, 5.5 min. MS parameters were as follows: nebulizer pressure 35 psi, drying gas (nitrogen) 10 L/min, VCap 6000 V, Delta EMV 800 V, column temperature 40 °C, and drying gas temperature 350 °C for all compounds. For each of the following molecules, the following transitions were measured using multiple reaction monitoring (following the molecule’s name, the run-time (min), MRM, Fragmentor (voltage), and Collision Energy (voltage) are provided): trimacinolone acetonide, 5.7, 435 →415, 108, 5; wact-190, 7.7, 330 →107, 155, 22; wact-171, 7.3, 386 →131, 45, 12; wact-498, 7.4, 346 →238, 45, 40; wact-406, 5.9, 240 →119, 45, 16; wact-455, 6.2, 254 →105.1, 45, 40; wact-519, 6.8, 324 →240.2, 45, 16. Cholesterol-limitation assay. References 1. Samuel, B. S., Rowedder, H., Braendle, C., Felix, M. A. & Ruvkun, G. Caenorhabditis elegans responses to bacteria from its natural habitats. Proc. Natl Acad. Sci. USA 113, E3941–E3949 (2016). 33. Eggeling, C. et al. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159–1162 (2009). Caenorhabditis elegans responses to bacteria from its natural habitats. Proc. Natl Acad. Sci. USA 113, E3941–E3949 (2016). 34. 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IUBMB Life 63, 736–746 (2011). Transmission electron microscopic (TEM) analyses. Approximately 1000 syn- chronized L1-stage worms were added to 60-mm plates containing 60 µM of wact- 190 or 1% DMSO vehicle control seeded with OP50 bacteria. Twenty-four hours later, worms were collected and prepared for TEM42. Live animals were loaded into a metal planchette in a slurry of E. coli and fast frozen under high pressure using a Balt-tec HM 010 freezing device, placed into 2% osmium tetroxide, 0.1% UAc, 2% dH2O in acetone at −90 °C for 96 h using a Boeckler freeze substitution unit, slowly warmed (5 °C/h) to −30 °C, held 16 h, then slowly warmed (5 °C/h) to 0 °C, then rinsed in cold acetone at 0 °C, and again rinsed several more times at RT before inﬁltration into plastic resin over 3 days (HardPlus Embed812). Once fully inﬁltrated, the samples were cured at 60 °C for 2 days and thin sectioned for views at high resolution using a Philips CM10 electron microscope. Some samples were viewed in cross-section, and others in lengthwise section for comparison. Control animals were exposed to DMSO without drug for 24 h. Transmission electron microscopic (TEM) analyses. Approximately 1000 syn- chronized L1-stage worms were added to 60-mm plates containing 60 µM of wact- 190 or 1% DMSO vehicle control seeded with OP50 bacteria. Twenty-four hours later, worms were collected and prepared for TEM42. Live animals were loaded into a metal planchette in a slurry of E. coli and fast frozen under high pressure using a Balt-tec HM 010 freezing device, placed into 2% osmium tetroxide, 0.1% UAc, 2% dH2O in acetone at −90 °C for 96 h using a Boeckler freeze substitution unit, 20. Huitema, K., van den Dikkenberg, J., Brouwers, J. F. & Holthuis, J. C. Identiﬁcation of a family of animal sphingomyelin synthases. EMBO J. 23, 33–44 (2004). 21. Tafesse, F. G., Ternes, P. & Holthuis, J. C. The multigenic sphingomyelin synthase family. J. Biol. Chem. 281, 29421–29425 (2006). 22. Goni, F. M. & Alonso, A. Biophysics of sphingolipids I. Membrane properties of sphingosine, ceramides and other simple sphingolipids. Biochim. Biophys. Acta 1758, 1902–1921 (2006). y y then rinsed in cold acetone at 0 °C, and again rinsed several more times at RT before inﬁltration into plastic resin over 3 days (HardPlus Embed812). ARTICLE Once fully inﬁltrated, the samples were cured at 60 °C for 2 days and thin sectioned for views at high resolution using a Philips CM10 electron microscope. Some samples were viewed in cross-section, and others in lengthwise section for comparison. Control animals were exposed to DMSO without drug for 24 h. 23. Mattei, B., Lira, R. B., Perez, K. R. & Riske, K. A. Membrane permeabilization induced by Triton X-100: the role of membrane phase state and edge tension. Chem. Phys. Lipids 202, 28–37 (2017). y p vo-Rekila, H., Ramstedt, B., Leppimaki, P. & Slotte, J. P. Cholestero y p 24. Ohvo-Rekila, H., Ramstedt, B., Leppimaki, P. & Slotte, J. P. Cholesterol interactions with phospholipids in membranes. Prog. Lipid Res. 41, 66–97 (2002). Statistics and graphs. Except where indicated, statistical differences were mea- sured using a two-tailed Student’s T test. Violin plots were generated using the online tool BoxPlotR (http://shiny.chemgrid.org/boxplotr/). 25. Ishitsuka, R., Yamaji-Hasegawa, A., Makino, A., Hirabayashi, Y. & Kobayashi, T A lipid-speciﬁc toxin reveals heterogeneity of sphingomyelin-containing membranes. Biophys. J. 86, 296–307 (2004). p y 26. Yilmaz, N., Yamaji-Hasegawa, A., Hullin-Matsuda, F. & Kobayashi, T. Molecular mechanisms of action of sphingomyelin-speciﬁc pore-forming toxin, lysenin. Semin. Cell Dev. Biol. 73, 188–198 (2018). Animal ethics statement. We (the authors) afﬁrm that we have complied with all relevant ethical regulations for animal testing and research. Given that our Animal ethics statement. We (the authors) afﬁrm that we have complied with all relevant ethical regulations for animal testing and research. Given that our experiments focused exclusively on the invertebrate nematode worm C. elegans, no ethical approval was required for any of the presented work. Animal ethics statement. We (the authors) afﬁrm that we have complied with all relevant ethical regulations for animal testing and research. Given that our Animal ethics statement. We (the authors) afﬁrm that we have complied with all relevant ethical regulations for animal testing and research. Given that our experiments focused exclusively on the invertebrate nematode worm C. elegans, no ethical approval was required for any of the presented work. y 27. Hao, L. et al. Clozapine modulates glucosylceramide, clears aggregated proteins, and enhances ATG8/LC3 in Caenorhabditis elegans. Neuropsychopharmacology 42, 951–962 (2017). g g experiments focused exclusively on the invertebrate nematode worm C. elegans, no ethical approval was required for any of the presented work. g g experiments focused exclusively on the invertebrate nematode worm C. NATURE COMMUNICATIONS \| (2019) 10:3938 \| https://doi.org/10.1038/s41467-019-11908-0 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11908-0 Author contributions Peer review information: Nature Communications would like to thank the anonymous reviewers for their contributions to the peer review of this work. Peer review reports are available M.K. discovered crystal and sphere formation and generated and analyzed all primary data herein that is not included in the contributions by other authors listed below. H.M. performed the initial screens and characterization of wact-190-resistant mutants. L.M. performed the mass spectrometric analyses. D.H. performed the GFP-NT-Lysenin stains and analyses. K.C.Q.N. performed the TEM analyses. M.Y., J.K., and R.B. conducted detailed viability analyses on the wact-190-resistant mutants in the background of wact- 190 and the entire wactive library. A.M.W. conducted the artiﬁcial lipid bilayer analysis of GFP-NT-Lysenin staining. K.S. conducted some crystal formation counts. C.M.Y. supervised and secured funding for the artiﬁcial bilayer work. C.L.C. supervised and secured funding for the mass spectrometric work. D.H.H. supervised and secured funding for the TEM work. P.J.R. supervised and secured funding for the project, and together with M.K., wrote the manuscript and composed the ﬁgures. 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. Acknowledgements g y g 14. Burns, A. R. et al. Caenorhabditis elegans is a useful model for anthelmintic discovery. Nat. Commun. 6, 7485 (2015). We thank Greg Fairn and Masashi Maekawa for reagents and guidance in characterizing sphingomyelin abundance and localization in the worm; Hong Zheng for building the p1138 construct and Kevin Chan for microinjection and integration work; Don Moerman and Oliver Hobert for fosmids and fosmid recombineering tools; David Hall, Zeynep Altun, and Chris Crocker for permission to modify Worm Atlas schematics43; Andrew Burns for helpful comments on the work and mining Chembridge libraries; and Andy Fraser and Michael Schertzberg for whole-genome sequence analysis; Lindy Holden-Dye and Fernando Calahorro Nunez for preliminary analyses; and the C. elegans Genetics Centre and Shohei Mitani for mutant strains. This work was supported by CIHR grants (376634 and 313296) and a CRC to P.J.R., an NSERC (RGPIN 03666-14) to C.L.C., and an NIH grant (OD 010943) to D.H.H. 15. Shaham, S. in WormBook (ed. Ambros, V.) (The C. elegans Research Community, 2006). 16. Ostermeier, C. in Methods and Results in Crystallization of Membrane Proteins (ed. Iwata, S.) Ch. 5 (International University Line, 2003). 17. Matsuda, R., Nishikawa, A. & Tanaka, H. Visualization of dystrophic muscle ﬁbers in mdx mouse by vital staining with Evans blue: evidence of apoptosis in dystrophin-deﬁcient muscle. J. Biochem. 118, 959–964 (1995). 18. Eckhardt, E. R. et al. Asymmetric distribution of phosphatidylcholine and sphingomyelin between micellar and vesicular phases. Potential implications for canalicular bile formation. J. Lipid Res. 40, 2022–2033 (1999). 15 NATURE COMMUNICATIONS \| (2019) 10:3938 \| https://doi.org/10.1038/s41467-019-11908-0 \| www.nature.com/naturecommunicatio Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Additional information Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 019-11908-0. Competing interests: The authors declare no competing interests. Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ © The Author(s) 2019 NATURE COMMUNICATIONS \| (2019) 10:3938 \| https://doi.org/10.1038/s41467-019-11908-0 \| www.nature.com/naturecommunications 16 NATURE COMMUNICATIONS \| (2019) 10:3938 \| https://doi.org/10.1038/s41467-019-11908-0 \| www.nature.com/naturecommunicatio
https://openalex.org/W4206145444	https://openjournals.ugent.be/vdt/article/id/75425/download/pdf/	Dutch	null	Aderlaten: bloed evacueren	Vlaams dierengeneeskundig tijdschrift	2,021	cc-by	3,373	Vlaams Diergeneeskundig Tijdschrift, 2021, 90 Vlaams Diergeneeskundig Tijdschrift, 2021, 90 Vlaams Diergeneeskundig Tijdschrift, 2021, 90 Vlaams Diergeneeskundig Tijdschrift, 2021, 90 313 Uit het verleden 313 AMENVATTING S Bloed ‘laten’ is een van de oudste en meest toegepaste medische en veterinaire praktijken. Het ontstaan ervan is vermoedelijk te vinden in het geloof in magie. Het gewone volk was er diep van overtuigd dat geheimzinnige magische krachten in het lichaam oorzaak waren van ziekte. Het ‘kwaad’ (ziek) bloed moest zoveel mogelijk uit het lichaam verwijderd worden. Om de praktijk te rechtvaardigen, hielden universitair geschoolde artsen het bij de Grieks-Latijnse leer van on evenwicht (dyscrasie) in de lichaamsvochten. In de negentiende eeuw werd de vermeende dyscra sie vervangen door theorieën die de rol van ontsteking beklemtoonden. Bloed met ‘ontstekings stoffen’ moest geëlimineerd worden. Tegen het einde van die eeuw had ook deze interpretatie krediet verloren en aderlaten verdween stilaan uit de praktijk, eerst uit de humane, pas later en schoorvoetend uit de diergeneeskunde, in die tijd nog vooral uitgeoefend door hoefsmeden – of slachters-veeartsen (maréchaux-vétérinaires). Aan de hand van een dagboek bijgehouden van 1853 tot 1887 door een hoefsmid-veearts wordt aangetoond hoe aderlating tot in het laatste kwart van de negentiende eeuw frequent toegepast werd bij allerhande inwendige aandoeningen van runderen. Aansluitend bij dit verhaal over de achtergronden van aderlaten worden de toe gepaste technieken kort beschreven. ABSTRACT For centuries, evacuation of blood, bloodletting, along with purgation, has been the most employed means to attempt healing in diseased domestic animals, as well as in humans. It was a common belief that unknown and much-feared evil forces causing disease (in French: “le mal”, hence “la maladie”) could be evacuated from the body in this way. Among university-trained medicals, however, blood letting was justified by antique Greek theories on disbalance (dyscrasy) of the different body humors (liquids), as the main cause of illness. Through Roman, Byzantine and Arab medicine, this doctrine was introduced in European medical schools. In the nineteenth century, this was superseded by theories on inflammation as a major cause. Again, evacuation of blood was advocated as a powerful means to cure. In domestic animals, bloodletting was frequently performed by farrier- or butcher-veterinarians, officially licensed in the nineteenth century to attempt most veterinary diagnoses and therapeutic acts. A diary kept by a farrier-veterinarian with a mainly dairy practice in a Flemish rural region from 1853 to 1887, shows that bloodletting was done in nearly half (44.5%, first period) to over one third (38%, second period) of all patients suffering from internal disorders. This was nearly always accompanied by oral application of minerals and plant extracts in drenches. This background information is com pleted and illustrated by a short description of the techniques used. Aderlaten: bloed evacueren Achtergrond en technieken gebruikt in de diergeneeskunde Bloodletting: background and techniques used in veterinary medicine 1L. Devriese, 2C. Van der Meeren, 1J. De Smet 1Museumcollectie Diergeneeskundig Verleden Merelbeke, Faculteit Diergeneeskunde, UGent, Salisburylaan 133, B-9820 Merelbeke 2IJzerbergstraat 5, B-9770 Kruisem 1Museumcollectie Diergeneeskundig Verleden Merelbeke, Faculteit Diergeneeskunde, UGent, Salisburylaan 133, B-9820 Merelbeke 2IJzerbergstraat 5, B-9770 Kruisem INLEIDING chaam, handelde onder meer over het aderlaten, sa men met purgeren, als zijnde de daartoe meest ge bruikte methode (Devriese et al., 2015). Uitvoerders bij dieren waren ‘paarden- en koemeesters’, vage, in Een in dit tijdschrift verschenen artikel over het doen verdwijnen van ‘het kwaad’ uit het zieke li Vlaams Diergeneeskundig Tijdschrift, 2021, 90 314 de marge van de veehouderij opererende figuren. In de negentiende eeuw werden ze opgevolgd door officieel toegelaten hoefsmeden-veeartsen of slachters-veeart sen, in het Franse jargon van die tijd “les maréchaux- vétérinaires”, voorgangers van de dierenartsen (Mam merickx, 1997). De ingreep werd evengoed door min of meer deskundige (of eerder: durvende) veehouders uitgevoerd. ingreep nog populair, in de diergeneeskunde duidelijk langer dan in de humane geneeskunde (d’Houdain – Doniol – Valcrose, 2001). In de Utrechtse collegedic taten van Alexander Numan uit de eerste helft van de 19de eeuw worden enkele tientallen predisponerende factoren en specifieke ziekte-indicaties voor aderlaten behandeld, maar voor onevenwicht wordt geen plaats meer geruimd (Nederbragt, 2018). In de opeenvol gende negentiende-eeuwse uitgaven van zijn ook in Vlaams België wijdverspreide Handboek der Genees- en Verloskunde van het Vee, vooral bestemd voor vee houders, komt aderlaten trouwens uitvoerig aan bod. Het eveneens sterk verspreide handboek De bekwame Veearts van Wagenfeld, oorspronkelijk in het Duits (1841), houdt het erg beknopt, maar is ook positief, getuige volgend citaat. ‘De aderlating is in de meeste ontstekingsziekten, vooral bij goed gevoede dieren, een onschatbaar en bijna het enigste geneesmiddel. Men verzuime het dus niet, want zelfs in twijfelach tige gevallen zal zij meer voor- dan nadeel doen’. g In de diergeneeskundige praktijk liet men meestal bloed vloeien uit de halsvene, de vena jugularis. Men deed dus eigenlijk aan ‘vene laten’, flebotomie, maar de term aderlaten was sterk ingeburgerd. Tot ver in de jaren 1800 was aderlaten een belangrijke taak voor de hoefsmeden-veeartsen, wellicht in iets mindere mate ook voor de geschoolde, gediplomeerde veeartsen. De hieronder besproken gegevens uit het dagboek van de hoefsmid-veearts Christiaens uit Beveren - Leie to nen dat er in de tweede helft van de jaren 1800 bij de meeste inwendige aandoeningen nog bloed gelaten werd. Toen de leer van het onevenwicht (dyscrasie) van de lichaamsvochten stilaan verlaten werd, en het ge loof in magische kwade krachten al eerder aan het ta nen was (Thomas, 1971), zocht men nog verder naar verklaringen voor een mogelijk nut van aderlaten. HET ‘KWAAD’ VERSUS ‘DYSCRASIE’ In de volks(dier)geneeskunde werd sterk geloof gehecht aan het nut van aderlaten als middel om het ‘kwaad’ uit het lichaam van de zieke te laten verdwij nen. Ziekte werd immers beschouwd als (meestal) het resultaat van geheimzinnige, onvatbare ‘kwade’ krachten aanwezig in, of inwerkend op het lichaam. Het is een opvatting die stamt uit het geloof in magi sche krachten die alles beheersen. De ziekteverwek kers werden vaagweg ‘het kwaad’ genoemd. De in de Franse taal algemeen gebruikte termen le mal, le (la) malade en la maladie werden er zelfs van afgeleid. In het Nederlands spreken we van kwalen en kwaaltjes. In een negentiende-eeuws Engels handboek (Per civall, 1855) vinden we de overheersende opinie daaromtrent zeer duidelijk geformuleerd: ‘The blood is the food of inflammation; the more we reduce the one, the more shall we diminish the other; drawing blood therefore, is the most direct means we possess of abating inflammation. Indeed, in practice, it is our most decisive means of cure, and in some cases is the only remedy we have it in our power to employ.’ p j Om daaraan te verhelpen werd vooral beroep ge daan op bloed laten, bloed (af)tappen, aderlaten. Dit wordt aanzien als een van de oudste medische ingre pen, na wondverzorging en castreren. Er bestaan aan wijzingen voor dat dit al gebeurde in prehistorische tijden, lang voor Hippocrates (rond 400 vC), de ver meende ‘vader’ van de geneeskunde. Universitair ge diplomeerde artsen verkondigden de hen onderwezen theorieën - even verkeerd als geleerd - over ‘oneven wicht’, dyscrasie, van de lichaamsvochten, ideeën die teruggingen op de antieke geneeskunde van Hippo crates, in het latere Romeinse rijk gepropageerd door Galenus. Via de Arabische rijken kwam deze visie te recht in het vroege Europese universitaire onderwijs. Bij mensen werd aderlaten voorgeschreven door gedi plomeerde artsen, maar de ingreep zelf werd meestal door chirurgijn-barbiers gedaan. In alle wijdverspreide, zowel de populaire als de universitaire negentiende-eeuwse handboeken, werd ontsteking, of wat men daarvoor aanzag, inderdaad aangegeven als hoofdindicatie voor aderlaten. In Frankrijk en landen onder Franse invloedsfeer groei de die mening onder impuls van de gezaghebbende arts Broussais (1772-1838) zelfs uit tot een doctrine: het Broussaïsme. In de handboeken werd wel ge waarschuwd tegen al te enthousiaste toepassing van de ingreep. Men stelde vaag of expliciet dat aderlaten tegenaangewezen was bij verzwakte en zeker bij ane mische dieren. INLEIDING In de 19de eeuw meende men die vooral te vinden in de effecten op wat zeer vaag ‘ontsteking’ genoemd werd, zoals hierboven in het citaat uit Wagenfeld aangege ven. Men klasseerde er zelfs aandoeningen zoals kalf ziekte onder, ziekten waarin het bekende trio sympto men indicatief voor ontsteking, i.e. “calor”, “rubor” of “tumor” en “dolor”: warmte, roodheid of zwelling en pijn, afwezig was. HET ‘KWAAD’ VERSUS ‘DYSCRASIE’ De gevaren en de mogelijke nadelen werden sterker in de verf gezet. ONTSTEKING g In het laatste kwart van de jaren 1800 zorgde de ontdekking van talrijke infectieuze pathogenen ervoor dat de praktijk samen met de justifiërende theorieën sterk aan populariteit inboette. Het geloof in ‘ont In de negentiende eeuw geraakten de theorieën over ‘onevenwicht’ in onbruik, maar aanvankelijk bleef de Vlaams Diergeneeskundig Tijdschrift, 2021, 90 315 Figuur 1. Fragment uit het dagboek van hoefsmid-veearts Christiaens (in familiebezit). Figuur 1. Fragment uit het dagboek van hoefsmid-veearts Christiaens (in familiebezit). uur 1. Fragment uit het dagboek van hoefsmid-veearts Christiaens (in familiebezit). Figuur 1. Fragment uit het dagboek van hoefsmid-veearts Christiaens (in familiebezit). overheersten ‘ontstekingen’ allerhande. Christiaens vermeldde onder andere maagontsteking, longontste king, ‘ribbevliesontsteking’ en ‘lijfmoederontsteking’ (metritis). Een uitzondering op de ontstekingsindica tie is de tot dusverre onopgehelderde ‘moederhoofd ziekte’ (kalfziekte: hypocalcemie?), die gaandeweg een stijgend aandeel in de indicaties innam van enkele percentages tot 38% in respectievelijk het eerste en laatste jaar van zijn praktijkvoering. Het verwondert ook niet dat hij het ‘bloeyd laten’ toepaste bij ‘voet ontsteking’ (hoefbevangenheid) bij het paard. Bij de meeste aandoeningen werd naast de aderlating een aanvullende therapie ingesteld met kruiden(extracten) en/of scheikundige stoffen uit zijn therapeutisch arse naal. giften’, met ‘Detox!’ als commerciële slogan, bleef echter levendig bij het publiek, ook al voerde Molière reeds enkele eeuwen vroeger in zijn theaterstuk Le Malade Imaginaire (1673) de medicus met de toepas selijke naam Purgon op met de commentaar: ‘Il faut qu’il ait tué bien de gens pour s’être fait si riche’. DIERGENEESKUNDIG GEBRUIK Een zorgvuldig bijgehouden dagboek (Figuur 1) van de allicht niet zo rijke hoefsmid-veearts Christi aens uit Beveren-Leie geeft een vermoedelijk repre sentatief idee van het aandeel van aderlating in de diergeneeskunde in de negentiende eeuw. In de vooral op runderen gerichte praktijk van Christiaens lag het gemiddelde percentage aderlatingen op de uitgevoer de behandelingen in de eerst helft van zijn carrière (steekproef van de jaren 1853+1858+1863+1868) op 44,5%. In een tweede en laatste periode (steekproef van de jaren 1873+1876+1883+1887) was dat 38%. Er bestond dus een neiging om procentueel minder aderlatingen te gaan doen (voortschrijdend inzicht?), maar dan toch niet spectaculair. In 1887, het laatste volledige geregistreerde praktijkjaar, werd dan toch nog altijd bij één patiënt op drie een aderlating toege past. De aard van de indicaties veranderde wel in de loop van de tijd. Enkel in de eerste jaren van zijn prak tijk werd aderlating uitgevoerd bij verlossingen (!): niet minder dan 23% van het totaal indicaties. Later Vlaams Diergeneeskundig Tijdschrift, 2021, 90 316 Figuur 2. Snepper (model Brogniez) en anatomische te kening die de plaats aangeeft waar in de submaxillaire streek bloed kon ‘gelaten’ worden bij aandoeningen van schapen in die zone (uit: Brogniez, 1839-1845). beschrijvingen werden al kort daarna overgenomen, niet enkel in de geleerde traktaten, maar ook in de vroeger wijdverspreide primitieve veeartsenijkundige handboekjes, zoals dat van Jacobus de Smet (1651), ‘Peerde-Meester’ in Borgerhout. Er werden echter geen praktische conclusies aan verbonden. Twee tot drie eeuwen na Harvey bleef men ‘bloed slaan’ op de meest onmogelijke plaatsen. Het leek wel alsof som mige ‘meesters’ daarmee hun kunde (en durf!) wilden bewijzen. Tegen aangepaste tarieven, uiteraard. j g g p , ‘Laten’ of ‘bloed laten’ in de opgestuwde halsader deed men meestal door er bovenop een kleine in snede te maken met een vlijm waarop een stevige tik gegeven werd met een houten ‘klopper’ (Figuur 3). Bekijken we even de daarbij gebruikte techniek aan de hand van het ook bij ons veel gebruikte 19de- eeuwse handboek van Numan. De zevende uitgave (1875) geeft aan dat ‘de ware kundigen’ geen band of riem gebruikten om de ader te laten opzwellen. ‘Men kan namelijk de ader door ze onderaan de hals met de vingers te drukken, en het bloed een weinig naar boven te drijven, zeer goed doen zwellen. Deze wijze is daarom niet alleen beter, maar ook eenvoudi ger. Zij die hun vak goed kennen gebruiken ook geen klopper, maar slaan op de vlijm met de pink van de rechterhand’. Anderen beschikten over een ‘snepper’: een vlijm aangedreven door een ingebouwde veer, een mechanisme analoog aan de snaphaan van geweren en handvuurwapens. Trefzeker maar ook niet zonder ge vaar. Figuur 4 en 5 zijn afbeeldingen van zo’n snepper uit de museumcollectie Diergeneeskundig Verleden in Merelbeke (Faculteit Diergeneeskunde, UGent). Figuur 2. Snepper (model Brogniez) en anatomische te kening die de plaats aangeeft waar in de submaxillaire streek bloed kon ‘gelaten’ worden bij aandoeningen van schapen in die zone (uit: Brogniez, 1839-1845). Figuur 3. Meest voorkomend model van vlijm met kloppers (Museumcollectie Diergeneeskundig Verleden (Faculteit Diergeneeskunde, UGent), Merelbeke). ( g ) Na het ‘bloed slaan’ met de vlijm bleef er een letter lijk vlijmscherp ingesneden wonde over. Het risico op verbloeden was reëel, vooral wanneer de vlijm dwars door de ader heen ging. Om overmatig bloedverlies te vermijden, kon men tijdelijk een metalen klemmetje of meteen een huidhechting aanbrengen. HALSVENE Veruit de gemakkelijkste en meest populaire plaats om bloed te laten was de vena jugularis. Strikt geno men was dit geen (slag)ader-, maar ‘venelating’, flebo tomie. Bij lokale aandoeningen kon de ingreep gebeu ren op of dichtbij de plaats waar men vermoedde dat ‘het kwaad’ huisde. Daarom werden arteriële latingen uitgevoerd op soms moeilijk bereikbare en delicate plaatsen (Figuur 2). Dit ondanks het feit dat men wel degelijk wist dat bloed circuleert. De ontdekking in 1628 door Harvey van de bloedsomloop en de pomp functie van het hart, was algemeen bekend. Correcte Vlaams Diergeneeskundig Tijdschrift, 2021, 90 Vlaams Diergeneeskundig Tijdschrift, 2021, 90 Hoe hech tingen werden gelegd in tijden dat er nog geen catgut of nylondraad bestond komen we te weten via een citaat uit Numans handboek (editie 1875). Het hech ten ‘… geschiedt op de volgende wijze: men steekt in het midden der wond door hare beide randen of lippen eene naald of stevige ijzeren speld; neemt alsdan vijf of zes lange haren uit de manen of den staart, voegt deze tezamen en windt ze van achteren om de uitste kende einden der speld, zodat de wond overkruisd wordt (Figuur 6). De beide einden dezer, te zamen ge nomene haren, worden aan elkander geknoopt, zodat zij vast blijven zitten. Na een of twee dagen neemt men de speld uit de wond.’ Het hechtmateriaal kon ook een textieldraad zijn. Er werden gewone rechte stopnaalden gebruikt. Goed geëquipeerde vaklui be schikten over speciale naaldhouders om de rechte naalden doorheen de taaie huid te drijven (Figuur 7). Figuur 3. Meest voorkomend model van vlijm met kloppers (Museumcollectie Diergeneeskundig Verleden (Faculteit Diergeneeskunde, UGent), Merelbeke). Figuur 4. Snepper in gesloten toestand uit de Museum collectie Diergeneeskundig Verleden (Faculteit Dierge neeskunde, UGent). In de eerste decennia van de jaren 1900 werd dit instrumentarium naar de rommelzolder verwezen. Men gebruikte en gebruikt enkel nog kleine trocarts om bloed af te laten. Het volstaat dan de huid een tijd lang stevig toe te klemmen om het bloeden te stoppen. Figuur 4. Snepper in gesloten toestand uit de Museum collectie Diergeneeskundig Verleden (Faculteit Dierge neeskunde, UGent). Vlaams Diergeneeskundig Tijdschrift, 2021, 90 317 Figuur 5. Open snepper. Figuur 6. Aanbrengen van afzonderlijke hechtingen. Hechting van de vlijmsnede (verticaal) met behulp van een stopnaald (horizontaal links door de huid gestoken (uit: Hutrel d’Arboval, 1877). Figuur 5. Open snepper. Figuur 5. Open snepper. Figuur 6. Aanbrengen van afzonderlijke hechtingen. Hechting van de vlijmsnede (verticaal) met behulp van een stopnaald (horizontaal links door de huid gestoken (uit: Hutrel d’Arboval, 1877). ANDERE TECHNIEKEN Nu nog zelfs is de ingreep aangewezen bij een viertal vrij zeldzame ziekten: hemochromatose, acuut long oedeem, polyglobulie en porfyrie. De huid van de huisdieren is minder geschikt voor ‘laatkoppen’, in Vlaanderen beter bekend als ‘ven tousen’, destijds populair in de humane geneeskunde. Dat waren strak op de huid geplaatste bokalen waarin onderdruk tot stand gebracht werd met pompjes of door er iets in te laten verbranden. Daarmee werd lo kale hyperemie opgewekt. Men kon dit zo laten of via een snede uit de kunstmatig opgezwollen zone slag aderbloed laten wegvloeien. In de meest verspreide negentiende-eeuwse handboeken zoals die van Nu man staat aangegeven dat dit bij dieren enkel kon toegepast worden op plaatsen waar voldoende egale spiermassa onder de huid aanwezig was. Of dit veel gebeurde mag betwijfeld worden. De ingreep was pijnlijk en het enige zekere resultaat was een afsto telijk huidletsel. Dat genas weliswaar snel, tenminste als de patiënt zowel de ziekte als de behandeling over leefde. DANKBETUIGING Met dank aan Piet Deprez en Paul Desmet. Figuur 7. Naaldhouder voor kopnaalden (uit: Hutrel d’Arboval, 1877). Brogniez (1845) gaf aan dat ook bloedzuigers (Hirudo officinalis) konden geplaatst worden op li chaamsstreken met fijne huid. Hij vermeldde bij klei nere dieren zelfs de mogelijkheid geringe hoeveelhe den bloed te laten wegvloeien via prikken (mouche tures: kunstmatige muggenbeten), kleine insneden of scarificaties. Ter verduidelijking moet hieraan toege voegd worden dat Brogniez, coryfee van de pasop gerichte ‘Ecole Vétérinaire’ van Kuregem, voorloper van de twee Belgische faculteiten diergeneeskunde, veruit de meeste aandacht besteedde aan ‘la saignée’. Hoewel uitgesproken voorstander, beschreef hij als enige in detail de complicaties en zelfs dodelijke ac cidenten die zich bij aderlating konden voordoen. Een van de vele instrumenten die hij ontwierp en hielp re aliseren, was trouwens de snepper (Figuur 2, 3 en 4). SLOT Het vroeger overmatige gebruik en de excessen zijn al lang verdwenen. Aderlaten is ook geen on derdeel van de dagelijkse praktijk meer, maar werd nooit helemaal verlaten. De ingreep werd wellicht het langst toegepast bij hoefbevangenheid bij het paard. Figuur 7. Naaldhouder voor kopnaalden (uit: Hutrel d’Arboval, 1877). LITERATUUR Numan, A. (1875). Handboek der Genees- en Verloskunde van het Vee. Zevende uitgave, door F. Hekmeijer ver meerderd, van Goor, Gouda. Brogniez, A.J. (1839-1845). Traité de Chirurgie Vétéri- naire. Derde volume en Atlas. Société encyclographique des Sciences médicales, Brussel. Percivall, W. (1855). Hippopathology, a Systematic Trea tise on the Disorders and Lameness of the Horse. Tweede uitgave, Longman, Londen. De Smet, J. (1651). Den Lusthof van het Cureren der Peer den. Eerste druk, Verhulst, Antwerpen. Thomas, K. (1971). Religion and the Decline of Magic: Studies in Popular Beliefs in Sixteenth- and Seventeenth- Century England. Weidenfeld & Nicolson, London. Ver taling (1989): De Ondergang van de Magische Wereld. Godsdienst en Magie in Engeland 1500-1700. Agon, Amsterdam. Devriese, L., De porte, H.M.F., Bols, P. (2015). Aderlaten en etterdrachten verdrijven het ‘kwaad’ uit het lichaam. Vlaams Diergeneeskundig Tijdschrift 84, 101-109. d’Houdain – Doniol – Valcrose, G. (2001). Histoire de la saignée vétérinaire. Thèse, Alfort. Wagenfeld, L. (1841). Allgemeines Vieharzneibuch. Born träger, Königsberg. Nederlandse vertaling: W.F. Steiger walt. De bekwame Veearts. Tweede uitgave, Noothoven van Goor, Leiden. Hutrel d’Arboval, L.H.J. (1838). Dictionnaire de Méde cine, de Chirurgie et d’Hygiène Vétérinaires. Eerste uit gave, Baillière, Parijs. Figuren overgenomen uit de editie 1877. Mammerickx, M. (1997). Hoefsmeden-veeartsen in de 19de eeuw in België. Vlaams Diergeneeskundig Tijdschrift 66, 262-265. © 2021 by the authors. Licensee Vlaams Dier- geneeskundig Tijdschrift, Ghent University, Belgium. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecom mons.org/licenses/by/4.0/). Vlaams Diergeneeskundig Tijdschrift, 2021, 90 318 LITERATUUR © 2021 by the authors. Licensee Vlaams Dier- geneeskundig Tijdschrift, Ghent University, Belgium. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecom mons.org/licenses/by/4.0/). Nederbragt, B. (2018). Kort zamenstel der algemeene veeartsenijkundige ziektekunde. Het collegedictaat van Alexander Numan (1780-1852). Deel 2: etiologie. Argos 58, 304-312. 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https://openalex.org/W2948089261	https://europepmc.org/articles/pmc6549148?pdf=render	English	null	TRAV1-2+ CD8+ T-cells including oligoconal expansions of MAIT cells are enriched in the airways in human tuberculosis	Communications biology	2,019	cc-by	15,621	Correspondence and requests for materials should be addressed to D.M.L. (email: lewinsod@ohsu.edu). #A full list of authors and their afﬁliations appears at the end of the paper TRAV1-2+ CD8+ T-cells including oligoconal expansions of MAIT cells are enriched in the airways in human tuberculosis https://doi.org/10.1038/s42003-019-0442-2 OPEN Emily B. Wong et al.# Mucosal-associated invariant T (MAIT) cells typically express a TRAV1-2+ semi-invariant TCRα that enables recognition of bacterial, mycobacterial, and fungal riboﬂavin metabolites presented by MR1. MAIT cells are associated with immune control of bacterial and myco- bacterial infections in murine models. Here, we report that a population of pro-inﬂammatory TRAV1-2+ CD8+ T cells are present in the airways and lungs of healthy individuals and are enriched in bronchoalveolar ﬂuid of patients with active pulmonary tuberculosis (TB). High- throughput T cell receptor analysis reveals oligoclonal expansions of canonical and donor- unique TRAV1-2+ MAIT-consistent TCRα sequences within this population. Some of these cells demonstrate MR1-restricted mycobacterial reactivity and phenotypes suggestive of MAIT cell identity. These ﬁndings demonstrate enrichment of TRAV1-2+ CD8+ T cells with MAIT or MAIT-like features in the airways during active TB and suggest a role for these cells in the human pulmonary immune response to Mycobacterium tuberculosis. Correspondence and requests for materials should be addressed to D.M.L. (email: lewinsod@ohsu.edu). #A full list of authors and their afﬁliations appears at the end of the paper. COMMUNICATIONS BIOLOGY \| (2019) 2:203 \| https://doi.org/10.1038/s42003-019-0442-2 \| www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 M mucosal sites, associated lymphoid tissues and unmatched per- ipheral blood samples (n = 6) (Fig. 1d). In contrast, signiﬁcantly higher frequencies of TRAV1-2+ cells from the lung produced TNF in response to M. smegmatis-infected cells compared with TRAV1-2+ cells from lymphoid tissues, small intestine, or per- ipheral blood (P = 0.035, 0.0025, 0.0023 and 0.0005 (Mann–Whitney U test), Fig. 1e). Cell yields from these tissues were insufﬁcient to establish functional dependence on MR1 as has been shown previously with this assay4. Nonetheless, these data demonstrate that mycobacterial stimulation results in TNF production by donor-unrestricted, lung resident TRAV1-2+ CD8+ T cells. M ucosal-associated invariant T (MAIT) cells are uncon- ventional lymphocytes that use semi-invariant T cell receptor-alpha (TCRα) chains to recognize non-peptide small molecule ligands presented by the HLA-Ib molecule MR11–6. In mice, MAIT cells have been shown to play a protective role in models of respiratory infection7–10. In humans, MAIT cells are abundant in the peripheral blood of healthy individuals, where they produce cytolytic enzymes and pro-inﬂammatory cytokines and typically express a TRAV1-2+ TCRα chain and the CD8 coreceptor1–4,11–13. MAIT cells are depleted in the blood of humans with TB4,5,14. TRAV1-2+ CD8+ T-cells including oligoconal expansions of MAIT cells are enriched in the airways in human tuberculosis https://doi.org/10.1038/s42003-019-0442-2 OPEN However, little is known about the function and phenotype of MAIT cells in the human lung, especially in the setting of pulmonary tuberculosis (TB). We postulated that MAIT cells are recruited to and/or expand at sites where Myco- bacterium tuberculosis (Mtb) antigens are present, potentially acting as sentinels of infection in the respiratory mucosa. TRAV1-2+ CDR3α usage in Mtb-infected lung tissue. On the basis of these results, we hypothesized that pulmonary infection with Mtb leads to the migration to and/or expansion of TRAV1-2+ CD8+ cells in the lung, potentially driven by Mtb-derived MR1 ligands. A hallmark of the human immune response to Mtb is the formation of lung granulomas. We therefore sought to determine the relevance of TRAV1-2+ T cell receptor (TCR) usage in lung granulomas from patients with TB. Single cell suspensions were prepared from diseased lung parenchyma from individuals (n = 5) undergoing clinically indicated surgical resection for complica- tions of TB18. The most highly diseased lung granuloma (LG) tissues were designated “A” and the least diseased tissues desig- nated “C.” CD4- T cells from these samples were sorted by ﬂow cytometry and subjected to high-throughput repertoire analysis using the bias-controlled immunoSEQ TCR sequencing plat- form19. In the 12 samples that yielded the minimal necessary sequencing data for analysis (>104 productive reads, yielding a median of 3,919 unique productive TCRα reads (range 397–28,792) and a median of 167 TRAV1-2-utilizing unique productive TCRα reads (range 19–1081), the overall frequency of TRAV1-2+ TCR sequences in granulomas ranged from 3.1 to 5.9% across all donors and tissue samples (Fig. 2a and Supple- mentary Table 1). These frequencies are similar to those observed in peripheral blood and lymph nodes. We then developed an algorithm based on published MAIT CDR3α amino acid (aa) sequences16,20 to determine which of these TRAV1-2+ CDR3α sequences represented MAIT cell-consistent TCRαs. A CDR3α sequence similarity analysis was performed using “MAIT Match” (http://www.cbs.dtu.dk/services/MAIT_Match), a tool based on the method described by Shen et al.21, where a score of 1 reﬂects a perfect match and a score of 0 a perfect mismatch with published MAIT cell CDR3α sequences. To determine the validity of this tool, we compared the proportion of TRAV1-2+ sequences with the proportion of TRAV12-2+ sequences (an unrelated control) for TCRs with scores ranging from 0.85 to 1. TRAV1-2+ CD8+ T-cells including oligoconal expansions of MAIT cells are enriched in the airways in human tuberculosis https://doi.org/10.1038/s42003-019-0442-2 OPEN MAIT Match scores of 0.95 to 1 were signiﬁcantly increased among the in TRAV1-2+ but not TRAV12-2+ TCR sequences (P = 0.0035, P = 0.00046, t test; Fig. 2b). We therefore chose a MAIT Match score of 0.95 as a conservative threshold to deﬁne MAIT cell-consistent TCRs (Fig. 2b). In one individual with paired samples from the lung and mediastinal lymph node (LN), TRAV1-2 usage was comparable at both sites, but similarity analysis revealed MAIT cell-consistent TCR enrichment in the lung (P < 0.0001; 2-way ANOVA; Fig. 2c). To address the possibility that Mtb drives the recruitment and/ i f TRAV1 2+ T ll ith MAIT i t t Here we report that a population of pro-inﬂammatory TRAV1- 2+ CD8+ T cells are present in the airways and lungs of healthy individuals and are enriched in bronchoalveolar ﬂuid of patients with active pulmonary TB. Some of these cells demonstrate MR1- restricted mycobacterial reactivity, phenotypic features and/or TCRα chain usage suggestive of MAIT cell identity. We conclude that TRAV1-2+ CD8+ T cells with MAIT or MAIT-like features are oligoclonally expanded in the airways during active TB, suggesting that they play a role in the human pulmonary immune response to Mycobacterium tuberculosis. Results T TRAV1-2+ CD8+ T-cells in human lung and intestine tissues. To explore the role of MAIT cells in healthy mucosal tissues, we ﬁrst determined the frequency of TRAV1-2+ CD8+ cells in the respiratory tract of an individual organ donor (Fig. 1a). Dramatic enrichment was observed in the trachea, where nearly half of all CD8+ T cells expressed TRAV1-2 (Fig. 1a). TRAV1-2+ cells were also enriched in the proximal and distal bronchi (35 and 22% of CD8+ T cells, respectively) and in the lung parenchyma (17% of CD8+ T cells), relative to the draining mediastinal lymph node where the frequency (6% of CD8+ T cells) approximated levels typically found in peripheral blood11,15. To determine the ana- tomical localization of TRAV1-2+ CD8+ cells in the airway, we used immunohistochemistry to quantify CD8+ and TRAV1-2+ cells in 1st and 2nd order bronchial sections from three additional organ donors (Fig. 1b, left). Although the number of CD8+ cells was similar in tissue sections from the proximal and distal air- ways, TRAV1-2+ cells were more frequent in the proximal compared to distal airway (Fig. 1b, right). As expression of TRAV1-2+ TCRs is insufﬁcient to deﬁne MAIT cells, we also performed ex vivo functional assays in which cytokine- production by TRAV1-2+ CD8+ cells upon exposure to HLA mismatched M. smegmatis-infected antigen-presenting cells is used to deﬁne mycobacterial-reactive MAIT cells4,12,16,17. In a single donor for whom paired tissues were available, we evaluated lymphocytes from lung parenchyma, the small intestinal lamina propria (LP), and the small intestinal intraepithelial lymphocytes (IEL) for M. smegmatis-dependent release of the pro- inﬂammatory cytokine TNF. Interestingly, TNF-producing TRAV1-2+ cells were found only in the lung (Fig. 1c, left). It is also notable that CD161, a C-type lectin highly expressed on peripheral MAIT cells11,13,17, was not detected on TRAV1-2+ CD8+ T cells from the lung but was found in abundance on small intestinal TRAV1-2+ CD8+ T cells (Fig. 1c, right). We next compared the frequencies of TRAV1-2+ and TNF-producing cells in the lung (n = 9) and intestinal mucosa (n = 8, unmatched samples) where MAIT cells were initially found to be enriched3. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 Having found th TRAV1-2+ CD8+ cells are enriched in healthy airways a CD8 - APC-Cy7 TRAV1-2 - APC b c d e 8 6 4 2 0 × 104 cells/mm3 CD161 - PerCPCy5.5 TNF - PE TRAV1-2+ CD8+ T cells Lung LP IEL 1st order bronchus 2nd order bronchus 5.68 21.3 43.6 29.5 6.56 15.2 50.8 27.5 4.54 5.5 70.1 19.9 0.87 1.32 1.3 8.92 46 43.8 TRAV1-2 CD8 Epithelium Basement membrane Lamina propria 1st 2nd 1st 2nd CD8+ TRAV1-2+ 100 80 60 40 20 0 100 80 60 40 20 0 10 1 10 2 10 3 10 4 –10 3 10 3 10 4 10 5 0 20 15 10 5 0 Lung Med LN IEL LP Mes LN PBMC 100 80 60 40 20 0 Lung Med LN IEL LP Mes LN PBMC % TRAV1-2+ of CD8+ % TNF+ of TRAV1-2+ CD8+ * *** DAPI 77 20.8 UNICATIONS BIOLOGY \| (2019)2:203 \| https://doi org/10 1038/s42003-019-0442-2 \| www nature com/commsbio b c 8 6 4 2 0 × 104 cells/mm3 1st order bronchus 2nd order bronchus TRAV1-2 CD8 Epithelium Basement membrane Lamina propria 1st 2nd 1st 2nd CD8+ TRAV1 2+ DAPI b DA c T c CD161 - PerCPCy5.5 TNF - PE TRAV1-2+ CD8+ T cells Lung LP IEL 100 80 60 40 20 0 100 80 60 40 20 0 10 1 10 2 10 3 10 4 –10 3 10 3 10 4 10 5 0 uences were detected among the MAIT cell-consistent CDR3α some tissue s d e CD161 - PerCPCy5.5 TNF - PE 20 15 10 5 0 Lung Med LN IEL LP Mes LN PBMC 100 80 60 40 20 0 Lung Med LN IEL LP Mes LN PBMC % TRAV1-2+ of CD8+ % TNF+ of TRAV1-2+ CD8+ * *** d 20 15 10 5 0 Lung Med LN IEL LP Mes LN PBMC % TRAV1-2+ of CD8+ e 10 5 0 Lung Med LN IEL LP Mes LN PBMC 100 80 60 40 20 0 Lung Med LN IEL LP Mes LN PBMC % TRAV1-2 % TNF+ of TRAV1-2+ CD8+ * *** e 100 80 60 40 20 0 Lung Med LN IEL LP Mes LN PBMC % TNF+ of TRAV1-2+ CD8+ * * e some tissue samples occurred as the dominant MAIT cell- consistent TCR. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 CD8 - APC-Cy7 TRAV1-2 - APC Trachea 1st order bronchus Lung parenchyma Mediastinal lymph node 2nd order bronchus a 5.68 21.3 43.6 29.5 6.56 15.2 50.8 27.5 4.54 5.5 70.1 19.9 0.87 1.32 1.3 8.92 46 43.8 77 20.8 a nces were detected among the MAIT cell-consistent CDR3α nces present in granulomatous lung tissue isolated from ts with TB. Notably, public MAIT cell-consistent CDR3α frequently encoded by multiple synonymous nucleotide nces within individuals suggesting the expansion of multiple with the same CDR3α amino acid sequences (Fig. 2e, In contrast, private MAIT cell-consistent CDR3α nces were encoded by individual nucleotide sequences sting that these were the result of expansions of a single cell clone in each donor (Fig. 2e, left). Private CDR3α nces were not restricted to infrequent clonotypes and in some tissue samples occurred as the dominant MAIT ce consistent TCR. Bronchoalveolar TRAV1-2+ CD8+ T cells in active pulmona TB. Diminished frequencies of circulating MAIT cells ha consistently been observed in people with TB4,5. This appare peripheral depletion may occur as a consequence of select MAIT cell migration to the lung or may reﬂect increas host vulnerability to infection with Mtb. Results T The frequencies of TRAV1-2+ CD8+ T cells were similar across To address the possibility that Mtb drives the recruitment and/ or expansion of TRAV1-2+ T cells with MAIT-consistent CDR3α‘s in granulomatous tissue, we analyzed the MAIT cell- consistent CDR3α sequences (MAIT Match score 0.95-1) found in diseased lung parenchyma (n = 5 individuals, 11 samples). It is established that certain MAIT cell TCRα chains can be shared among individuals (public sequences)22, while donor-unique (private) CDR3α sequences can be selected in response to distinct microbes16. As shown in Fig. 2d, both private and public CDR3α MUNICATIONS BIOLOGY \| (2019) 2:203 \| https://doi.org/10.1038/s42003-019-0442-2 \| www.nature.com/commsbio 2 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 MAIT cells can be deﬁned in peripheral blood by TRAV1- 2 usage in conjunction with high-level expression of the c-type lectin CD161, and the di-peptidase CD2613,26. In BAL ﬂuid obtained from patients with TB, TRAV1-2+ CD8+ T cells expressed low levels of CD161 compared with peripheral blood TRAV1-2+ CD8+ T cells (Fig. 3d), which is consistent with the data from healthy lung tissue (Fig. 1c) and the prior demon- stration that CD161 can be down-regulated as a result of MAIT cell activation17,28,29. In contrast, TRAV1-2+ CD8+ T cells in the BAL ﬂuid more consistently expressed CD26, which is abun- dantly present on all functional MR1-restricted MAIT cells in Although TRAV1-2 usage is a deﬁning feature of MAIT cells, the same gene segment can be expressed by T cells recognizing mycobacterial ligands presented in the context of HLA-Ia molecules and CD1b31. On the basis that TRAV1-2+ CD8+ T cells display a surface phenotype suggestive of tissue-resident MAIT cells in BAL ﬂuid isolated from patients with active TB, we postulated that the corresponding CDR3α sequences would provide a molecular signature reﬂecting MAIT cell enrichment relative to TRAV1-2+ CD8+ T cells in matched peripheral blood samples. To test this hypothesis, we performed high-throughput TCR repertoire analysis of TRAV1-2+ CD4−T cells sorted by ﬂow cytometry from cryopreserved BAL ﬂuid and matched peripheral blood specimens obtained from three donors with active TB (Supplementary Table 3). MAIT cell-consistent CDR3α sequences comprised a higher percentage of the TRAV1-2+ repertoire in BAL ﬂuid compared with peripheral blood, irrespective of the parameter used to deﬁne MAIT cell- consistent CDR3α sequences, including assessment of similarity to published MAIT cell CDR3α sequences (MAIT Match score = 0.95 or 1) or according to usage of TRAJ12, TRAJ20 or TRAJ33 (Fig. 3e; P = 0.0036; 2-way ANOVA). Among the patients with TB, CDR3α sequences with the highest MAIT Match scores (≥0.95) were enriched in BAL ﬂuid, while those with the lowest MAIT Match scores (<0.85), were more frequent in peripheral blood (Fig. 3f ). To determine the extent to which individual MAIT cell- consistent CDR3α sequences (MAIT Match Score ≥0.95) were shared between these two anatomical compartments, we created a TCR Enrichment Analysis (TEA) webtool (https://github.com/ eisascience/Wong-Gold-Lewinsohn) to enable visualization and weighted frequency analysis of the most common MAIT cell- consistent CDR3α sequences in matched samples (Fig. 3g and Supplementary Table 4). COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 1c) and the prior demon- stration that CD161 can be down-regulated as a result of MAIT cell activation17,28,29. In contrast, TRAV1-2+ CD8+ T cells in the BAL ﬂuid more consistently expressed CD26, which is abun- dantly present on all functional MR1-restricted MAIT cells in respond to mycobacteria (Fig. 1e)4, we hypothesized that pul- monary infection with Mtb drives the accumulation and expan- sion of TRAV1-2+ CD8+ cells in the lung in response to Mtb- derived MR1 ligands. To address this possibility, we measured the frequency of TRAV1-2+ CD8+ T cells in bronchoalveolar (BAL) ﬂuid samples obtained from individuals with untreated, active pulmonary TB and controls with no evidence of infectious or inﬂammatory pulmonary disease (Supplementary Table 2). In BAL ﬂuid, TRAV1-2+ CD8+ T cells were signiﬁcantly enriched in patients with TB at frequencies approximately 3-fold higher than controls (P = 0.0022, Mann–Whitney U test, Fig. 3a). Conversely, in matched peripheral blood samples, TRAV1-2+ CD8+ T cells were signiﬁcantly diminished in patients with TB at frequencies approximately 2-fold lower compared to healthy controls (P = 0.0028, Mann–Whitney U test, Fig. 3a). To assess the functional capacity of TRAV1-2+ CD8+ T cells in the BAL ﬂuid and matched peripheral blood samples, we utilized α-CD2/ CD3/CD28 beads as a stimulant to trigger responses via the TCR. Cell yields were insufﬁcient to explore ligand-speciﬁc activation, which may also be subject to bias arising from compartment- speciﬁc differences in MR1-expression by antigen-presenting cells23. MAIT cells have been reported to produce IFN-γ, TNF, granzymes, granulysin, IL-17 and IL-2224–26. Among these, we chose to measure TNF, a representative Th1 effector cytokine essential for immune control of Mtb27 and IL-17, an immuno- modulatory cytokine reportedly produced in a TCR-independent manner by MAIT cells28. A signiﬁcantly greater proportion of TRAV1-2+ CD8+ T cells in BAL ﬂuid produced TNF (median 40%, range 36–91%) compared with TRAV1-2+ CD8+ T cells in matched peripheral blood samples (median 15%, range 4.7–27%) (P = 0.004, Mann–Whitney U test, Fig. 3b, c and Supplementary Fig. 1). In contrast fewer than 1% of TRAV1-2+ CD8+ T cells in the BAL ﬂuid and only 2% in matched peripheral blood samples produced IL-17 (Supplementary Fig. 2). We therefore concluded that TCR triggering of these BAL-resident TRAV1-2+ CD8+ T cells does not evoke IL-17 production, though other mitogenic or cytokine-associated stimulations may do so. Next, we char- acterized the phenotype of BAL-resident TRAV1-2+ CD8+ T cells. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 Fig. 1 TRAV1-2+ CD8+ T cells from the lung but not the intestine of healthy organ donors respond to mycobacterial infection by producing TNF. a Dot plots showing the frequency of TRAV1-2+ CD8+ T cells among live CD3+ cells in the indicated tissue samples from one donor. b Tissue sections from the 1st and 2nd order bronchi were obtained from healthy individuals (n = 3 biologically independent samples). Immunohistochemistry was performed to quantify CD8+ (median 1.6 × 104 vs. 2 × 104 cells /mm3) and TRAV1-2+ cells (7,000 vs. 4,000 cells/mm3, Supplementary Data). Representative sections from 1st and 2nd order bronchi are depicted (left), showing CD8+ cells (red), TRAV1-2+ cells (green) and cell nuclei (DAPI; blue). c Histograms depicting TNF production (left) and CD161 expression (right) by TRAV1-2+ CD8+ T cells from matched lung parenchyma (green), small intestine lamina propria (LP; blue) and the small intestinal intraepithelial layer (IEL; violet) after overnight stimulation with M. smegmatis-infected antigen-presenting cells (dotted black line indicates the unstimulated control). d Frequency of TRAV1-2+ cells among CD8+ T cells from lung (n = 9 biologically independent samples), mediastinal lymph node (Med LN; n = 11 biologically independent samples), IEL (n = 7 biologically independent samples), LP (n = 8 biologically independent samples), mesenteric lymph node (Mes LN, n = 5 biologically independent samples), and peripheral blood (PBMC; n = 6 biologically independent samples, Supplementary Data). Medians and interquartile ranges are displayed. e Frequency of TNF-producing TRAV1-2+ CD8+ T cells after exposure to M. smegmatis-infected antigen-presenting cells: lung (n = 7 biologically independent samples), Med LN (n = 6 biologically independent samples), IEL (n = 5 biologically independent samples), LP (n = 6 biologically independent samples), Mes LN (n = 2 biologically independent samples), PBMC (n = 12 biologically independent samples, Supplementary Data). From top to bottom, P = 0.035, 0.0025, 0.0023 and 0.0005 (Mann–Whitney U test). Medians and interquartile ranges are displayed peripheral blood17,24. CD103, the αE integrin associated with tissue-resident memory T cells30 was expressed variably but exclusively on BAL TRAV1-2+ CD8+ T cells. respond to mycobacteria (Fig. 1e)4, we hypothesized that pul- monary infection with Mtb drives the accumulation and expan- sion of TRAV1-2+ CD8+ cells in the lung in response to Mtb- derived MR1 ligands. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 To address this possibility, we measured the frequency of TRAV1-2+ CD8+ T cells in bronchoalveolar (BAL) ﬂuid samples obtained from individuals with untreated, active pulmonary TB and controls with no evidence of infectious or inﬂammatory pulmonary disease (Supplementary Table 2). In BAL ﬂuid, TRAV1-2+ CD8+ T cells were signiﬁcantly enriched in patients with TB at frequencies approximately 3-fold higher than controls (P = 0.0022, Mann–Whitney U test, Fig. 3a). Conversely, in matched peripheral blood samples, TRAV1-2+ CD8+ T cells were signiﬁcantly diminished in patients with TB at frequencies approximately 2-fold lower compared to healthy controls (P = 0.0028, Mann–Whitney U test, Fig. 3a). To assess the functional capacity of TRAV1-2+ CD8+ T cells in the BAL ﬂuid and matched peripheral blood samples, we utilized α-CD2/ CD3/CD28 beads as a stimulant to trigger responses via the TCR. Cell yields were insufﬁcient to explore ligand-speciﬁc activation, which may also be subject to bias arising from compartment- speciﬁc differences in MR1-expression by antigen-presenting cells23. MAIT cells have been reported to produce IFN-γ, TNF, granzymes, granulysin, IL-17 and IL-2224–26. Among these, we chose to measure TNF, a representative Th1 effector cytokine essential for immune control of Mtb27 and IL-17, an immuno- modulatory cytokine reportedly produced in a TCR-independent manner by MAIT cells28. A signiﬁcantly greater proportion of TRAV1-2+ CD8+ T cells in BAL ﬂuid produced TNF (median 40%, range 36–91%) compared with TRAV1-2+ CD8+ T cells in matched peripheral blood samples (median 15%, range 4.7–27%) (P = 0.004, Mann–Whitney U test, Fig. 3b, c and Supplementary Fig. 1). In contrast fewer than 1% of TRAV1-2+ CD8+ T cells in the BAL ﬂuid and only 2% in matched peripheral blood samples produced IL-17 (Supplementary Fig. 2). We therefore concluded that TCR triggering of these BAL-resident TRAV1-2+ CD8+ T cells does not evoke IL-17 production, though other mitogenic or cytokine-associated stimulations may do so. Next, we char- acterized the phenotype of BAL-resident TRAV1-2+ CD8+ T cells. MAIT cells can be deﬁned in peripheral blood by TRAV1- 2 usage in conjunction with high-level expression of the c-type lectin CD161, and the di-peptidase CD2613,26. In BAL ﬂuid obtained from patients with TB, TRAV1-2+ CD8+ T cells expressed low levels of CD161 compared with peripheral blood TRAV1-2+ CD8+ T cells (Fig. 3d), which is consistent with the data from healthy lung tissue (Fig. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 sequences were detected among the MAIT cell-consistent CDR3α sequences present in granulomatous lung tissue isolated from patients with TB. Notably, public MAIT cell-consistent CDR3α were frequently encoded by multiple synonymous nucleotide sequences within individuals suggesting the expansion of multiple clones with the same CDR3α amino acid sequences (Fig. 2e, right). In contrast, private MAIT cell-consistent CDR3α sequences were encoded by individual nucleotide sequences suggesting that these were the result of expansions of a single MAIT cell clone in each donor (Fig. 2e, left). Private CDR3α sequences were not restricted to infrequent clonotypes and in some tissue samples occurred as the dominant MAIT cell- consistent TCR. Bronchoalveolar TRAV1-2+ CD8+ T cells in active pulmonary TB. Diminished frequencies of circulating MAIT cells have consistently been observed in people with TB4,5. This apparent peripheral depletion may occur as a consequence of selective MAIT cell migration to the lung or may reﬂect increased host vulnerability to infection with Mtb. Having found that TRAV1-2+ CD8+ cells are enriched in healthy airways and MMUNICATIONS BIOLOGY \| (2019) 2:203 \| https://doi.org/10.1038/s42003-019-0442-2 \| www.nature.com/commsbio 3 ARTICLE ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 To evaluate whether TRAV1-2+ CD8+ Donor and site e Nucleotide sequences/CDR3α/Donor Donor Unique Shared Synonymous nucleotide sequences used to generate the five most frequenct unique or shared MAIT CDR3α amino acid sequences CAVLDSNYQLIW (13) CAVTDSNYQLIW (7) CAVMDSNYQLIW (5) CAVRDSNYQLIW (4) CAVIDSNYQLIW (3) CAVRDGDYQLIW (1) CAILDSNYQLIW (1) CASLDSNYQLIW (1) CAVREDNYGQNFVF (1) CAVRESNYQLIW (1) C A V L D S N Y Q L I W CTC TGT GCT GTC TTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTC CTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTG CTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTG TTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTT CTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTT CTC GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTT TTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTA TTA GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTA TTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTC CTA GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTC CTC GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTT CTT GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTC CTA GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT TRAV1-2 TGT GCT GTG AGA GAT GAT AGC AAC TRAJ33 Donor 27 26 24 23 21 27 26 24 23 21 COMMUNICATIONS BIOLOGY \| (2019)2 203 \| htt //d i /10 1038/ 42003 019 0442 2 \| t / bi e Nucleotide sequences/CDR3α/Donor Donor Unique Shared Synonymous nucleotide sequences used to generate the five most frequenct unique or shared MAIT CDR3α amino acid sequences CAVLDSNYQLIW (13) CAVTDSNYQLIW (7) CAVMDSNYQLIW (5) CAVRDSNYQLIW (4) CAVIDSNYQLIW (3) CAVRDGDYQLIW (1) CAILDSNYQLIW (1) CASLDSNYQLIW (1) CAVREDNYGQNFVF (1) CAVRESNYQLIW (1) C A V L D S N Y Q L I W CTC TGT GCT GTC TTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTC CTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTG CTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTG TTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTT CTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTT CTC GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTT TTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTA TTA GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTA TTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTC CTA GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTC CTC GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTT CTT GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTC CTA GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT TRAV1-2 TGT GCT GTG AGA GAT GAT AGC AAC TRAJ33 Donor 27 26 24 23 21 27 26 24 23 21 A3 A6 A8 and A10) expressed MAIT cell consistent CDR3α mismatched M smegmatis infected or Mtb infected e Nucleotide sequences/CDR3α/Donor Donor Unique Shared Synonymous nucleotide sequences used to generate the five most frequenct unique or shared MAIT CDR3α amino acid sequences CAVLDSNYQLIW (13) CAVTDSNYQLIW (7) CAVMDSNYQLIW (5) CAVRDSNYQLIW (4) CAVIDSNYQLIW (3) CAVRDGDYQLIW (1) CAILDSNYQLIW (1) CASLDSNYQLIW (1) CAVREDNYGQNFVF (1) CAVRESNYQLIW (1) C A V L D S N Y Q L I W CTC TGT GCT GTC TTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTC CTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTG CTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTG TTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTT CTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTT CTC GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTT TTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTA TTA GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTA TTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTC CTA GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTC CTC GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTT CTT GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTC CTA GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT TRAV1-2 TGT GCT GTG AGA GAT GAT AGC AAC TRAJ33 Donor 27 26 24 23 21 27 26 24 23 21 e Nucleotide sequences/CDR3α/Donor Unique Synonymous nucleotide sequenc CAVRDGDYQLIW (1) CAILDSNYQLIW (1) CASLDSNYQLIW (1) CAVREDNYGQNFVF (1) CAVRESNYQLIW (1) Donor 27 26 24 23 21 e Synonymous nucleotide sequences used to generate the five most frequenct unique or shared MAIT CDR3α amino acid sequences Synonymous nucleotide sequences used to generate the five most frequenct unique or shared MAIT CDR3α amino acid sequences Donor Shared sequences used to generate the five most frequenct unique or shared MAIT CDR3α amino acid sequences CAVLDSNYQLIW (13) CAVTDSNYQLIW (7) CAVMDSNYQLIW (5) CAVRDSNYQLIW (4) CAVIDSNYQLIW (3) IW (1) W (1) W (1) QNFVF (1) W (1) C A V L D S N Y Q L I W CTC TGT GCT GTC TTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTC CTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTG CTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTG TTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTT CTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTT CTC GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTT TTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTA TTA GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTA TTG GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTC CTA GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTC CTC GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTT CTT GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT CTC TGT GCT GTC CTA GAT AGC AAC TAT CAG TTA ATC TGG GGC GCT TRAV1-2 TGT GCT GTG AGA GAT GAT AGC AAC TRAJ33 27 26 24 23 21 A3, A6, A8 and A10) expressed MAIT cell-consistent CDR3α sequences (MAIT Match score = 0.98-1; Table 1). COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 In all three patients, the most frequent MAIT cell-consistent CDR3α sequences were present in both compartments, with disproportionate expansion in the BAL ﬂuid compared with the peripheral blood. In contrast, CDR3α sequences with low MAIT Match scores (<0.85) were generally expanded only in one anatomical compartment (Supplementary Fig. 3). The selective expansion of MAIT cell-consistent CDR3α sequences in the lung compartment relative to peripheral blood suggests antigen-driven clonal expansion in response to pulmon- ary infection with Mtb. y To determine if TRAV1-2+ CD8+ T cells present in BAL ﬂuid contained MAIT cells, we examined the MR1-restricted function of T cell clones generated from a BAL ﬂuid sample obtained from a patient with TB. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 Six of these TRAV1-2+ clones (D0033-A1, A2, COMMUNICATIONS BIOLOGY \| (2019) 2:203 \| https://doi.org/10.1038/s42003-019-0442-2 \| www.nature.com/commsbio 4 4 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 Frequency of TRAV1-2+ sequences (%) Donor and site CDR3α sequences Denotes a sequence unique to 1 donor 0 2 4 6 8 21LG 23LG-A 23LG-B 23LG-C 23LN 24LG-A 24LG-B 24LG-C 26LG-A 26LG-B 27LG-A 27LG-B % Sequences with TRAV1-2 1 1 0.95 0.95 0.9 0.9 0.85 0.85 0.01 0.1 1 10 Similarity score Frequency of TRAV chain (%) TRAV12-2 TRAV1-2 a b c d e Synonymous nucleotide sequences used to generate the five most frequenct unique or shared MAIT CDR3α amino acid sequences Donor 23 Total 1 0.95 0.01 0.1 1 10 Frequency of TRAV1-2 sequences (%) Lung LN Similarity score 5 10 15 20 8.8 8.1 5.8 5.4 2.7 1.6 1.3 0.9 0.9 0.4 0.9 10.2 2.8 2.3 0.9 13 7 7 7 2.8 2.3 1.9 1.9 1.4 0.9 0.9 0.9 0.8 9.2 8.8 3 0.6 5.3 1.9 1.6 7 0.3 1.3 0.4 2.7 1.9 1.8 0.7 0.6 0.4 0.1 1.8 0.4 0.3 4.8 6.6 1.6 0.7 1.7 0.8 0.8 0.8 1.3 2.7 0.5 0.3 0.3 0.3 0.2 0.2 0.2 0.2 0.2 0.1 0.5 0.3 0.3 0.3 0.3 0.2 0.2 0.1 0.1 0.2 0.2 0.6 1.2 1.7 1.4 0.8 0.7 0.9 1.1 1.2 1.7 2 5 15.6 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.2 0.2 0.2 0.3 0.4 0.5 0.1 0.6 0.7 0.7 0.9 1 1.1 1.1 1.3 1.7 1.9 2 0.2 1.2 2 2 1 1.3 1.9 1.1 1 1.6 24.6 1 0.7 0.7 2 1.3 1.6 5.2 2.7 3.1 1.4 1.4 2.4 0.1 1.5 1.1 12.1 0.7 1.1 4.4 6.3 6.6 8.1 0.3 2.5 5.3 6.9 0.1 0.3 1.8 2.2 0.1 1.9 3.9 3.3 1.4 CAVIDSNYQLIW CAVMDSNYQLIW CAVRDSNYQLIW CAVRESNYQLIW CAVKDSNYQLIW CAVRDRDYKLSF CAVRDSDYKLSF CAALDSNYQLIW CAVTDSNYQLIW CAVQDSNYQLIW CAVREDNYGQNFVF CAVKNSNYQLIW CAVLDSNYQLIW CAVRDGDYKLSF CAVRNTGGFKTIF CAPMDSNYQLIW CADMDSNYQLIW CASMDSNYQLIW CAVRSDRGSTLGRLYF CAAKAAGNKLTF CAALDSDYQLIW CAVDSNYQLIW CAAMDSNYQLIW CAVSDSNYQLIW CAVLDSSYKLIF CAVGDSSYKLIF CACMDSNYQLIW CAGIDSNYQLIW CAVIDGDYKLSF CAVVDSNYQLIW CAPLDSSYKLIF CALLDSNYQLIW CAVMDSSYKLIF CAVRDRNYQLIW CAVNRDDKIIF CAVHDSNYQLIW CVVMDSSYKLIF CATRDSNYQLIW CAVGNNNDMRF CAVRDQDYQLIW CVQMDSNYQLIW CAVTNSNYQLIW CAFMDSNYQLIW CALGDSNYQLIW CAVSSNDYKLSF CAVRDEDYKLSF CAVVTNDYKLSF CAASDSNYQLIW CAVRGATDSWGKLQF CAAIDSNYQLIW CAVRDRDYQLIW CASADSNYQLIW CASLDSNYQLIW CAVKDSSYKLIF CAVADSNYQLIW CAAMDSDYQLIW CAVNRDDKII CVLMDSNYQLIW CAGMDSNYQLIW CAYMDSNYQLIW CAVSGSARQLTF CAVRDNDYKLSF CAVARDDKIIF CAVVDSSYKLIF CAALDSSYKLIF CAVKGSDGQKLLF CAAMDSSYKLIF CAILDSNYQLIW CGFMDSNYQLIW CAEQAGTALIF CAVGSSGTYKYIF CAVTDGDYKLSF CACLDSNYQLIW CANMDSNYQLIW CAGFDSNYQLIW CASKAAGNKLTF CAVSDTGFQKLVF CAVNKAAGNKLTF CAAADSNYQLIW CAVKGDYKLSF CAVRDGNYQLIW CAVRDGDYQLIW CAGLDSNYQLIW CAAGDSNYQLIW CAGVDSNYQLIW CAVGDSNYQLIW CVFMDSNYQLIW 21LG 23LGA 23LGB 23LGC 24LGA 24LGB 24LGC 26LGA 26LGC 27LGA 27LGB 1.8 0.6 1.5 6.1 0 2 4 6 8 21LG 23LG-A 23LG-B 23LG-C 23LN 24LG-A 24LG-B 24LG-C 26LG-A 26LG-B 27LG-A 27LG-B % Sequences with TRAV1-2 a d a a % Sequences with TRAV1-2 1 1 0.95 0.95 0.9 0.9 0.85 0.85 0.01 0.1 1 10 Similarity score Frequency of TRAV chain (%) TRAV12-2 TRAV1-2 b Similarity score c Donor 23 Total 1 0.95 0.01 0.1 1 10 Frequency of TRAV1-2 sequences (%) Lung LN Similarity score c A3, A6, A8 and A10) expressed MAIT cell-consistent CDR3α sequences (MAIT Match score = 0.98-1; Table 1). COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 Stimulation of these clones with HLA-mismatched M. smegmatis-infected or Mtb-infected antigen-presenting cells induced robust IFN-γ, while two control clones from the same patient (D0033-D7 and E7) failed to produce IFNγ under identical conditions. In contrast, stimulation of the TRAV1-2+ clones with HLA- mismatched M. smegmatis-infected or Mtb-infected antigen-presenting cells32 resulted in negligible IFN tion, thereby demonstrating MR1-dependent cytokine consistent with MAIT cell function (Fig. 3h). MR1 tetramer loaded with 5-OP-RU ligand has been identify functional MAIT cells in the human circulation13. Discussion Collectively, our data indicate that donor-unrestricted myco- bacterial-reactive TRAV1-2+ CD8+ T cells are present in the human respiratory mucosa and that pulmonary infection with Mtb leads to an enrichment of airway resident, pro-inﬂammatory TRAV1-2+ CD8+ cells including oligoclonal expansions of MAIT cells. In contrast to MR1/5-OP-RU tetramer staining in the peripheral blood where positive and negative populations were clearly discernable, the MR1/5-OP-RU tetramer staining of BAL cells was of heterogeneous intensity and did not allow unambiguous delineation of MR1/5-OP-RU tetramer negative and positive populations. As a result, we sorted TRAV1-2+ cells based on MR1/5-OP-RU tetramer staining, subjected both positive and negative subsets to TCR sequencing, and analyzed MAIT cell-consistent CDR3α usage in each population (Table 2). CDR3α chain sequencing of MR1/5-OP-RU tetramer positive cells from BAL and peripheral blood revealed that 93.9% and 89.2% of these respectively utilized MAIT cell-consistent TCRs. CDR3α chain sequencing of the MR1/5-OP-RU tetramer negative TRAV1-2+ populations demonstrated that a substantial propor- tion (13.7%) of the MR1/5-OP-RU tetramer negative cells in the BAL utilized MAIT cell-consistent CDR3α chains. In contrast, only 2.7% of MR1/5-OP-RU tetramer negative cells from the peripheral blood utilized MAIT cell-consistent CDR3α chains. These data suggest that MR1/5-OP-RU tetramer may perform less efﬁciently in BAL ﬂuid than in peripheral blood. Notably, in the other donor (91), in whom 28.5% of the TRAV1-2+ cells had a MAIT cell-consistent CDR3α, only 5.09% of the TRAV1-2+ cells from the BAL stained MR1/5-OP-RU positive. In this donor, 24.7% of the MR1/5-OP-RU tetramer negative cells had MAIT cell-consistent CDR3α chains, suggesting that MR1/5-OP-RU tetramer staining of BAL cells may underestimate the presence of MAIT cells as determined by CDR3α usage. In lung tissue explanted from healthy organ donors, we ﬁnd that TRAV1-2+ CD8+ T cells localize to the respiratory tract mucosal surface. In contrast to their counterparts in the gut mucosa, TRAV1-2+ CD8+ T cells from the respiratory mucosa produce TNF in response to mycobacterial stimulation by donor-unrestricted antigen-presenting cells. This suggests that TRAV1-2+ CD8+ T cells in the airway mucosa may play a role in anti-Mtb immunity by initiating a local pro-inﬂammatory response upon exposure to aerosolized Mtb. p p p In the setting of active pulmonary tuberculosis, we observed striking expansions of TRAV1-2+ CD8+ T cells in the bronch- oalveolar compartment. Compared to paired peripheral blood TRAV1-2+ CD8+ T cells, the bronchoalveolar TRAV1-2+ CD8+ T cells produced signiﬁcantly more TNF. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 Fig. 2 Expansions of MAIT cell-consistent CDR3α‘s are present in tuberculous lung granulomas. a Frequency of TRAV1-2+ sequences as a percentage of all productive TCRα sequences. In some cases, multiple areas of tissue were sampled, ranging from closest (A) to furthest (C) from the site of disease. b MAIT cell TCRα sequences are consistent with similarity scores of 0.95 and 1. Each symbol represents the frequency of TRAV1-2+ or TRAV12-2+ sequences within each similarity score for each donor sample (n = 12 biologically independent samples). c Frequency of total TRAV1-2+ sequences or those with similarity scores of 0.95 and 1 in the lung (n = 3 biologically independent samples) and mediastinal lymph node (LN, 1 sample) from donor 23. Height represents mean, error bars represent standard error. d Frequencies among TRAV1-2+ sequences of the top 10 public and private MAIT cell CDR3α sequences (MAIT Match score ≥0.95) across individual donors and lung samples. e Variation in the number of synonymous nucleotide sequences encoding the ﬁve most frequent private (left) and public (right) MAIT cell CDR3α amino acid (aa) sequences from all samples displayed in Fig. 3d. For each aa sequence, each colored bar represents a different nucleotide sequence. The 13 different nucleotide sequences used to generate the shared MAIT cell CDR3α aa sequence CAVLDSNYQLIW are displayed. Text color represents nucleotide origin: purple (TRAV), black (TRAD or n insertion), red (TRAJ). LG lung granuloma, LN lymph node. Source data are provided in Supplementary Data the BAL of humans with TB could be stained by MR1/5-OP-RU tetramer, as well as the relationship between MAIT cell-consistent CDR3α usage and MR1/5-OP-RU tetramer staining, we took advantage of two donors with TB with available cryopreserved specimens. We stained cells from paired BAL and peripheral blood samples with TRAV1-2 antibody, MR1/5-OP-RU tetramer and MR1/6-FP tetramer (negative control). As shown in Fig. 4a, BAL cells from donor 1020 demonstrated MR1/5-OP-RU tetramer staining of 33.7% of the TRAV1-2+ cells, supporting the TCRα sequencing analysis that found that 40.7% of BAL TRAV1-2 CDR3α sequences were MAIT cell-consistent (MAIT Match score >.95). In the peripheral blood of this participant, only 3.06% of the TRAV1-2+ peripheral cells demonstrated MR1/ 5-OP-RU tetramer staining, in line with the TCRα sequencing analysis that had found that 5.09% of peripheral TRAV1-2 CDR3α‘s were MAIT cell-consistent. antigen-presenting cells (A549) and abrogated IFN-γ production when stimulated with identically infected MR1-KO antigen- presenting cells32. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 TCR sequencing demonstrated that each of these clones utilized a MAIT cell-consistent CDR3α (Fig. 4b). Surprisingly, despite clear evidence of MR-1 restricted function, usage of MAIT cell-consistent CDR3αʼs (Table 3) and TRAV1-2 staining of similar intensity (Fig. 4c), these clones demonstrated considerable heterogeneity in MR1/5-OP-RU tetramer staining, with two of the four clones staining weakly (Fig. 4d). COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 Stimulation of these clones with HLA-mismatched M. smegmatis-infected or Mtb-infected antigen-presenting cells induced robust IFN-γ, while two control clones from the same patient (D0033-D7 and E7) failed to produce IFNγ under identical conditions. In contrast, stimulation of the TRAV1-2+ clones with HLA- mismatched M. smegmatis-infected or Mtb-infected MR1-KO antigen-presenting cells32 resulted in negligible IFN-γ-produc- tion, thereby demonstrating MR1-dependent cytokine production consistent with MAIT cell function (Fig. 3h). MR1 tetramer loaded with 5-OP-RU ligand has been shown to identify functional MAIT cells in the human peripheral circulation13. To evaluate whether TRAV1-2+ CD8+ T cells in 5 MMUNICATIONS BIOLOGY \| (2019) 2:203 \| https://doi.org/10.1038/s42003-019-0442-2 \| www.nature.com/commsbio COMMUNICATIONS BIOLOGY \| (2019) 2:203 \| https://doi.org/10.1038/s42003-019-0442-2 \| www.nature.com/commsbio Discussion smegmatis-infected We postulate that variable MR1-tetramer staining observed on bronchoalveolar TRAV1-2+ CD8+ cells could reﬂect a state of COMMUNICATIONS BIOLOGY \| (2019) 2:203 \| https://doi.org/10.1038/s42003-019-0442-2 \| www.nature.com/commsbio 6 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 ARTICLE BAL PBMC BAL PBMC 0 20 40 60 80 100 % cytokine producing of TRAV1-2 +CD8+ TNF IL-17 0.1 1 10 < 0.85 0.85–0.89 0.90–0.94 0.95–0.99 1.00 Enriche din BAL Enriched in PBMC MAIT match score Relative frequencyof TRAV1-2+ (FC) BAL PBMC CD161-PE-Cy7 CD26-FITC CD103-PE-Cy7 TRAV1-2+ CD8+ a b c d e f g h 1016 0091 1020 Frequency in PBMC (log2) Frequency in BAL (log2) TRAV1-2+ CDR3α 2.0% 4.0% 6.0% 8.0% BAL + PBMC frequency TRAJ 12 / 20 / 33 TRAV 1-2 - APC TNF - PE TB BAL TB PBMC Unstimulated CD8+ T cells Stimulated TB Control TB Healthy 0 10 20 30 40 50 % TRAV1-2+ of CD8+ BAL PBMC 0 –103 103 104 105 0 20 40 60 80 100 8.47 0.078 0.47 91.0 0 0 –10 3 10 5 4.75 1.67 9.01 84.6 1.97 35.4 21.1 41.6 49.8 0.86 0.99 48.4 0 20 40 60 80 % of TRAV1-2+ population BAL PBMC A1 A2 A3 A6 A8 A10 D7 E7 0 10 200 400 600 800 1000 SFU-IFNy Unstimulated M. smeg infection of MR1-KO A549 M. smeg infection of WT A549 M. tb infection of MR1-KO A549 M. Discussion Some, but not all, of these expanded bronchoalveolar TRAV1-2+ CD8+ T cells could be identiﬁed as MAIT cells based on their utilization of MAIT cell-consistent CDR3α chains, demonstration of MR1-restricted function or selective binding of the MR1/5-OP-RU tetramer. It should be noted that among the TRAV1-2+ CD8+ T cells that could not be unequivocally conﬁrmed as MAIT cells we identiﬁed subpopulations that displayed certain “MAIT-like” features, such as high-level expression of CD26 or oligoclonal expansions of TRAV1-2+ TCRα chains with features similar to MAIT cell CDR3α sequences (incorporation of the TRAJ12, TRAJ20 or TRAJ33 segments, or the presence of the Tyr95 which is known to be critical for MAIT cell TCR binding to MR1-restricted ligands34). Our attempts to clone these populations have been unsuccessful to date, such that further work will be required to determine if these TRAV1-2+ CD8+ T cells with “MAIT-like” features are restricted by MR1. It is also notable that MR1/5-OP- RU tetramers identiﬁed only a subset of TRAV1-2+ CD8+ T cells with MAIT cell-consistent CDR3a’s. To better understand the relationship between MR1/5-OP-RU tetramer staining, CDR3α usage and MR1-dependent T cell activity, we sorted MR1/5-OP-RU positive cells from the BAL of an available individual with non-TB pneumonia and performed limiting dilution cloning using anti-CD3 and IL-2 stimulation. Following rapid expansion33, each clone was characterized functionally for MR1-restriction and antigenic speciﬁcity. As shown in Fig. 4c, four clones (D1004-B3, E1, E5, and H3) produced IFN-γ when stimulated with M. Discussion Further work will be required to better understan the relationship between TCR-dependent MR1-dependen activation, MR1/5-OP-RU tetramer staining, and ligan BAL PBMC BAL PBMC 0 % TNF IL-17 0.1 1 10 < 0.85 0.85–0.89 0.90–0.94 0.95–0.99 1.00 Enriche din BAL Enriched in PBMC MAIT match score Relative frequencyof TRAV1-2+ (FC) BAL PBMC CD161-PE-Cy7 CD26-FITC CD103-PE-Cy7 TRAV1-2+ CD8+ d e f g h 1016 0091 1020 Frequency in PBMC (log2) Frequency in BAL (log2) TRAV1-2+ CDR3α 2.0% 4.0% 6.0% 8.0% BAL + PBMC frequency TRAJ 12 / 20 / 33 TRAV 1-2 - AP TNF - PE TB Control TB Healthy 0 10 % BAL PBMC 0 –103 103 104 105 0 20 40 60 80 100 0.47 91.0 0 0 –10 3 9.01 84.6 0 20 40 60 80 % of TRAV1-2+ population BAL PBMC A1 A2 A3 A6 A8 A10 D7 E7 0 10 200 400 600 800 1000 SFU-IFNy Unstimulated M. smeg infection of MR1-KO A549 M. smeg infection of WT A549 M. tb infection of MR1-KO A549 M. Discussion tb infection of WT A549 BAL-derived T cell clones 10 3 –10 3 10 3 10 4 10 5 –4 –6 –8 10 –10 –8 –6 –4 –10 –8 –6 –4 –10 –8 –6 –4 –4 –6 –8 10 –4 –6 –8 10 CAAMDSNYQLIW: (5,2) CAVKDSNYQLIW: (3,2) CAVSDSNYQLIW: (4,1) CAASDSNYQLIW: (1,2) CAVRDGDYKLSF: (2,2) CAVLDSNYQLIW: (11,7) CAVRDGDYQLIW: (1,2) CAVTDSNYQLIW: (3,2) CAFTDSNYQLIW: (2,1) CAVMDSNYQLIW: (3,3) CAVRDSNYQLIW: (3,1) CAVVDSNYQLIW: (3,1) CAALDSNYQLIW: (1,1) CAHMDSNYQLIW: (1,0) CASVDSNYQLIW: (0,1) CAVKDSNYQLIW: (2,1) CAVMDSSYKLIF: (1,2) CAVTDSNYQLIW: (4,4) CAAMDSNYQLIW: (3,0) CAIMDSNYQLIW: (0,1) CAVHDSNYQLIW: (0,1) CAVLDSNYQLIW: (5,2) CAVRDGDYKLSF: (1,1) CAVVDSNYQLIW: (5,1) CAGMDSNYQLIW: (0,1) CAPRDSNYQLIW: (0,1) CAVIDSNYQLIW: (3,0) CAVMDSNYQLIW: (5,4) CAVRDSNYQLIW: (2,4) CAAKDSNYQLIW: (2,1) CAAMDSNYQLIW: (2,3) CAVKDSNYQLIW: (6,5) CAVRDSNYQLIW: (4,7) CAVTDSNYQLIW: (5,10) CAALDSNYQLIW: (4,6) CAGMDSNYQLIW: (4,6) CAVLDSNYQLIW: (10,14) CAVSDSNYQLIW: (5,15) CAVVDSNYQLIW: (3,9) CAALNDYKLSF: (1,0) CAVISGFGNVLHC: (1,0) CAVMDSNYQLIW: (5,6) CAVSGGFGNVLHC: (2,1) 1.00 ≥ 0.95 MAIT match score COMMUNICATIONS BIOLOGY \| (2019)2:203 \| https://doi.org/10.1038/s42003-019-0442-2 \| www.nature.com/commsbio 0.1 1 10 < 0.85 0.85–0.89 0.90–0.94 0.95–0.99 1.00 Enriche din BAL Enriched in PBMC MAIT match score Relative frequencyof TRAV1-2+ (FC) f e TRAJ 12 / 20 / 33 0 20 40 60 80 % of TRAV1-2+ population BAL PBMC 1.00 ≥ 0.95 MAIT match score BAL PBMC CD161-PE-Cy7 CD26-FITC CD103-PE-Cy7 TRAV1-2+ CD8+ d 0 –103 103 104 105 0 20 40 60 80 100 f d e e g g 1016 0091 1020 Frequency in PBMC (log2) Frequency in BAL (log2) TRAV1-2+ CDR3α 2.0% 4.0% 6.0% 8.0% BAL + PBMC frequency –4 –6 –8 10 –10 –8 –6 –4 –10 –8 –6 –4 –10 –8 –6 –4 –4 –6 –8 10 –4 –6 –8 10 CAAMDSNYQLIW: (5,2) CAVKDSNYQLIW: (3,2) CAVSDSNYQLIW: (4,1) CAASDSNYQLIW: (1,2) CAVRDGDYKLSF: (2,2) CAVLDSNYQLIW: (11,7) CAVRDGDYQLIW: (1,2) CAVTDSNYQLIW: (3,2) CAFTDSNYQLIW: (2,1) CAVMDSNYQLIW: (3,3) CAVRDSNYQLIW: (3,1) CAVVDSNYQLIW: (3,1) CAALDSNYQLIW: (1,1) CAHMDSNYQLIW: (1,0) CASVDSNYQLIW: (0,1) CAVKDSNYQLIW: (2,1) CAVMDSSYKLIF: (1,2) CAVTDSNYQLIW: (4,4) CAAMDSNYQLIW: (3,0) CAIMDSNYQLIW: (0,1) CAVHDSNYQLIW: (0,1) CAVLDSNYQLIW: (5,2) CAVRDGDYKLSF: (1,1) CAVVDSNYQLIW: (5,1) CAGMDSNYQLIW: (0,1) CAPRDSNYQLIW: (0,1) CAVIDSNYQLIW: (3,0) CAVMDSNYQLIW: (5,4) CAVRDSNYQLIW: (2,4) CAAKDSNYQLIW: (2,1) CAAMDSNYQLIW: (2,3) CAVKDSNYQLIW: (6,5) CAVRDSNYQLIW: (4,7) CAVTDSNYQLIW: (5,10) CAALDSNYQLIW: (4,6) CAGMDSNYQLIW: (4,6) CAVLDSNYQLIW: (10,14) CAVSDSNYQLIW: (5,15) CAVVDSNYQLIW: (3,9) CAALNDYKLSF: (1,0) CAVISGFGNVLHC: (1,0) CAVMDSNYQLIW: (5,6) CAVSGGFGNVLHC: (2,1) 0091 –10 –8 –6 –4 –4 –6 –8 10 h A1 A2 A3 A6 A8 A10 D7 E7 0 10 200 400 600 800 1000 SFU-IFNy Unstimulated M. smeg infection of MR1-KO A549 M. smeg infection of WT A549 M. tb infection of MR1-KO A549 M. Discussion tb infection of WT A549 10 4 10 3 –10 3 10 3 10 4 10 5 –4 –6 –8 10 –10 –8 –6 –4 –10 –8 –6 –4 –10 –8 –6 –4 –4 –6 –8 10 –4 –6 –8 10 CAAMDSNYQLIW: (5,2) CAVKDSNYQLIW: (3,2) CAVSDSNYQLIW: (4,1) CAASDSNYQLIW: (1,2) CAVRDGDYKLSF: (2,2) CAVLDSNYQLIW: (11,7) CAVRDGDYQLIW: (1,2) CAVTDSNYQLIW: (3,2) CAFTDSNYQLIW: (2,1) CAVMDSNYQLIW: (3,3) CAVRDSNYQLIW: (3,1) CAVVDSNYQLIW: (3,1) CAALDSNYQLIW: (1,1) CAHMDSNYQLIW: (1,0) CASVDSNYQLIW: (0,1) CAVKDSNYQLIW: (2,1) CAVMDSSYKLIF: (1,2) CAVTDSNYQLIW: (4,4) CAAMDSNYQLIW: (3,0) CAIMDSNYQLIW: (0,1) CAVHDSNYQLIW: (0,1) CAVLDSNYQLIW: (5,2) CAVRDGDYKLSF: (1,1) CAVVDSNYQLIW: (5,1) CAGMDSNYQLIW: (0,1) CAPRDSNYQLIW: (0,1) CAVIDSNYQLIW: (3,0) CAVMDSNYQLIW: (5,4) CAVRDSNYQLIW: (2,4) CAAKDSNYQLIW: (2,1) CAAMDSNYQLIW: (2,3) CAVKDSNYQLIW: (6,5) CAVRDSNYQLIW: (4,7) CAVTDSNYQLIW: (5,10) CAALDSNYQLIW: (4,6) CAGMDSNYQLIW: (4,6) CAVLDSNYQLIW: (10,14) CAVSDSNYQLIW: (5,15) CAVVDSNYQLIW: (3,9) CAALNDYKLSF: (1,0) CAVISGFGNVLHC: (1,0) CAVMDSNYQLIW: (5,6) CAVSGGFGNVLHC: (2,1) 1.00 ≥ 0.95 MAIT match score COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 ARTICLE a TB Control TB Healthy 0 10 20 30 40 50 % TRAV1-2+ of CD8+ BAL PBMC b TRAV 1-2 - APC TNF - PE TB BAL TB PBMC Unstimulated CD8+ T cells Stimulated 8.47 0.078 0.47 91.0 0 0 –10 3 10 5 4.75 1.67 9.01 84.6 1.97 35.4 21.1 41.6 49.8 0.86 0.99 48.4 10 4 10 3 –10 3 10 3 10 4 10 5 BAL PBMC BAL PBMC 0 20 40 60 80 100 % cytokine producing of TRAV1-2 +CD8+ TNF IL-17 c c a activation among tissue-resident cells. Supporting this, we note that differential tetramer staining can be observed following expansion of MAIT cell clones with activating cytokines (Sup- plementary Figure 4). Alternatively, we postulate that TRAV1-2+ CD8+ T cells with MAIT cell-consistent CD3α‘s may have altered tetramer-binding avidity as a result of differential afﬁnity of their TCRs for MR1-ligands. This possibility is suggested by the vari able magnitude of response to M. smegmatis in the functiona assay, and has recently been demonstrated for the photolumazin I ligand35. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 Fig. 3 TNF-producing TRAV1-2+ CD8+ cells including oligoclonally expanded MAIT cells are enriched in bronchoalveolar lavage ﬂuid from patients with TB. a Frequency of TRAV1-2+ cells among CD8+ T cells from the bronchoalveolar lavage (BAL) ﬂuid from patients with TB (n = 6 biologically independent samples) and cancer controls (n = 6 biologically independent samples), and among CD8+ T cells in matched peripheral blood samples (PBMC) from patients with TB (n = 5 biologically independent samples) and unmatched peripheral blood samples from healthy controls (n = 13 biologically independent samples). Medians and interquartile ranges are displayed. P < 0.01; Mann–Whitney U test. b Dot plots showing TNF production by TRAV1-2+ CD8+ T cells in matched BAL and peripheral blood samples (PBMC) from a patient with TB. Cells were stimulated with α−CD2/CD3/CD28 beads. c Frequency of TNF or IL-17 production by TRAV1-2+ CD8+ T cells in matched BAL and peripheral blood samples (PBMC; n = 5 biologically independent samples). Medians and interquartile ranges are displayed. **P < 0.01; Mann–Whitney U test. d Expression of CD161, CD26 and CD103 on TRAV1-2+ CD8+ T cells in matched BAL and peripheral blood samples (PBMC) from patients with TB (n = 4 biologically independent samples). Histograms are mode-normalized. e Frequency of MAIT cell-consistent CDR3α sequences within TRAV1-2+ CD4- T cells in BAL ﬂuid and peripheral blood samples (PBMC) from patients with TB (n = 3 biologically independent samples). Height represents the mean, error bars represent the range. f Relative frequency of CDR3α sequences by MAIT Match Score category in BAL ﬂuid vs. matched peripheral blood (PBMC; n = 3 biologically independent samples). g Depiction of the top 10 most frequent MAIT cell-consistent CDR3α sequences (MAIT Match score ≥0.95) among TRAV1-2+ sequences in each compartment. Legend format: CDR3α aa (# of synonymous nucleotide sequences in peripheral blood, # of synonymous nucleotide sequences in BAL ﬂuid). h IFNγ spot-forming units (SFU) produced by BAL T cell clones stimulated with M. smegmatis-infected or Mtb-infected wildtype (WT) or MR1-KO A549 cells (n=8 biologically independent clones). Height represents the mean of two independent replicates per stimulation, error bars represent the standard deviation. Source data are available in Supplementary Data. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 Table 1 TCRα/β sequences and MAIT Match scores for six MAIT cell clones and two control clones derived from bronchoalveolar ﬂuid from a patient with tuberculosis Clone TCRα sequencing TCRβ sequencing TRAV CDR3α TRAJ MAIT Match score TRBV CDR3β TRBJ MAIT cell clones D0033-A1 1-2 CAALDSNYQLIW 33 1.00 4-3 CASSQDMVSITDTQY 2-3 D0033-A2 1-2 CAVTDSNYQLIW 33 1.00 3-1 CASSQAETELNTGELF 2-2 D0033-A3 1-2 CVTMDSNYQLIW 33 0.98 6-1 CASSEAGGGYNEQF 2-1 D0033-A6 1-2 CAVVDSNYQLIW 33 1.00 4-2 CASSHSSGTGGNEQF 2-1 D0033-A8 1-2 CVTMDSNYQLIW 33 0.98 6-1 CASSEAGGGYNEQF 2-1 D0033-A10 1-2 CAVTDSNYQLIW 33 1.00 3-1 CASSSGLEVTGELF 2-2 Control T cell clones D0033-D7 20 CAARFSDGQKLL 16 0.92 7-9 CASSEGTGVEWDGYT 1-2 D0033-E7 39 CAVPGGGADGLT 45 0.85 2 CASVASGVRDTQY 2-3 Table 1 TCRα/β sequences and MAIT Match scores for six MAIT cell clones and two control clones derived from bronchoalveolar ﬂuid from a patient with tuberculosis of clones in the lung compartment. Further understanding of this will require additional organ-speciﬁc datasets to allow compar- ison of diseased and reference TCR repertoires. We found both public and private MAT cell-consistent CDR3α chains in the TB- infected human lung tissues we analyzed. Interestingly, public MAIT cell-consistent CDR3α chains were frequently encoded by multiple synonymous nucleotide sequences within an individual sample. This ﬁnding is consistent with a previous report impli- cating convergent recombination as a determinative process in the generation of public MAIT cell CDR3α sequences22, and suggests that tissue-resident public MAIT cell-consistent CDR3α expansions are the result of multiple individual MAIT cells clonally expanding in infected tissues. The signiﬁcance of cells with private MAIT cell-consistent CDR3α chains in the context of Mtb-infected tissue remains uncertain. One possibility is that public and private MAIT cell-consistent CDR3α chains have similar ligand-binding properties, such that utilization and expansion of speciﬁc clonotypes in individual hosts is the result of differences in the naive TCR repertoire and is not driven by speciﬁc microbial exposures. Alternatively, the observed clonal expansion of private MAIT cell-consistent clonotypes within Mtb-infected tissue may reﬂect selective expansions in response to the local presence of microbe-derived ligands presented by MR135. A third possibility is that because our sample set is small, sequences that appear to be private in this analysis could in fact turn out to be public when larger numbers of individual donors are sampled. In order to determine the signiﬁcance of private and selectivity among bronchoalveolar TRAV1-2+ CD8+ T cells. COMMUNICATIONS BIOLOGY \| (2019) 2:203 \| https://doi.org/10.1038/s42003-019-0442-2 \| www.nature.com/commsbio Discussion tb infection of WT A549 BAL-derived T cell clones h activation among tissue-resident cells. Supporting this, we note that differential tetramer staining can be observed following expansion of MAIT cell clones with activating cytokines (Sup- plementary Figure 4). Alternatively, we postulate that TRAV1-2+ CD8+ T cells with MAIT cell-consistent CD3α‘s may have altered tetramer-binding avidity as a result of differential afﬁnity of their TCRs for MR1-ligands. This possibility is suggested by the vari- able magnitude of response to M. smegmatis in the functional assay, and has recently been demonstrated for the photolumazine I ligand35. Further work will be required to better understand the relationship between TCR-dependent MR1-dependent activation, MR1/5-OP-RU tetramer staining, and ligand 7 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 Tetramer + 0.71 Tetramer - 96.4 Tetramer + 3.06 Tetramer - 96.2 Tetramer + 0.46 Tetramer - 98.2 MR1 - 6FP tetramer - PE MR1 - 5OPRU tetramer - PE Bronchoalveolar cells Peripheral blood cells Tetramer + 33.7 Tetramer - 62.5 10 0 10 2 10 4 10 6 0 –103 10 3 10 4 10 5 93.9% MAIT cell- consistent CDR3a 13.7% MAIT cell- consistent CDR3a 89.2% MAIT consistent CDR3a 2.7% MAIT consistent CDR3a CD3 - PE-CF594 a b c d MR1/5-OPRU tetramer - PE D1004-B3 D1004-E1 D1004-E5 D1004-H3 D1004-B3, MR1/5-OPRU tetramer D1004-B3, MR1/6-FP tetramer D1004-E5, MR1/5-OPRU tetramer D1004-E5, MR1/6-FP tetramer MR1 tetramer - PE D1004-B3 D1004-E1 D1004-E5 D1004-H3 TRAV1-2 - APC 0 10 4 105 0 20 40 60 80 100 Normalized to mode 0 10 4 10 5 0 20 40 60 80 100 Normalized to mode 0 20 40 60 80 100 0 –10 3 10 3 10 4 10 5 0 –103 10 3 10 4 105 0 –10 3 103 10 4 10 5 10 0 10 2 10 4 10 6 100 102 104 106 100 102 104 106 0 10 4 10 5 800 600 400 200 0 WT MR1-KO WT MR1-KO WT MR1-KO WT MR1-KO D1004-B3 D1004-E1 D1004-E5 D1004-H3 SFU-IFNy erogeneous MR1/5-OP-RU staining of bronchoalveolar TRAV1-2+ CD8+ T cells with MAIT cell-consistent CDR3α‘s and MR1-restri cy of MR1-tetramer+ cells (loaded with active (5-OP-RU) and control (6FP) ligand) in TRAV1-2+ T cells (gated on live, CD3+, C tes) from the BAL ﬂuid and peripheral blood of a patient with TB. The proportion of cells utilizing MAIT cell-consistent CDR3α‘s ( 5) in MR1/5-OP-RU tetramer positive and negative populations are shown. b IFNγ spot-forming units (SFU) produced by four T from BAL ﬂuid and stimulated with M. smegmatis-infected wildtype (WT) or MR1-KO A549 cells, Supplementary Data. c α-TRAV1 l clones generated from BAL ﬂuid demonstrates consistent staining. Histograms are mode-normalized. d Binding of MR1/5-OP-RU four T cell clones generated from BAL ﬂuid demonstrates heterogenous MR1/5-OPRU tetramer staining (left). Binding of MR1/6-FP PRU tetramer is shown for two clones (right). COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 Histograms are mode-normalized NICATIONS BIOLOGY \| https://doi.org/10.1038/s42003 019 0442 2 A Tetramer + 0.71 Tetramer - 96.4 Tetramer + 3.06 Tetramer - 96.2 Tetramer + 0.46 Tetramer - 98.2 MR1 - 6FP tetramer - PE MR1 - 5OPRU tetramer - PE Bronchoalveolar cells Peripheral blood cells Tetramer + 33.7 Tetramer - 62.5 10 0 10 2 10 4 10 6 0 –103 10 3 10 4 10 5 93.9% MAIT cell- consistent CDR3a 13.7% MAIT cell- consistent CDR3a 89.2% MAIT consistent CDR3a 2.7% MAIT consistent CDR3a CD3 - PE-CF594 a 0 –10 3 10 3 10 4 10 5 0 –103 10 3 10 4 105 0 –10 3 103 10 4 10 5 10 0 10 2 10 4 10 6 100 102 104 106 100 102 104 106 MR1 - 5OPRU tetramer - PE Bronchoalveolar cells Tetramer + 33.7 Tetramer - 62.5 10 0 10 2 10 4 10 6 0 –103 10 3 10 4 10 5 93.9% MAIT cell- consistent CDR3a 13.7% MAIT cell- consistent CDR3a a a Tetramer + 3.06 Tetramer - 96.2 Peripheral blood cells 89.2% MAIT consistent CDR3a 2.7% MAIT consistent CDR3a 0 –10 3 10 3 10 4 10 5 10 0 10 2 10 4 10 6 Tetramer + 0.71 Tetramer - 96.4 Tetramer + 0.46 Tetramer - 98.2 MR1 - 6FP tetramer - PE CD3 - PE-CF594 0 –103 10 3 10 4 105 0 –10 3 103 10 4 10 5 100 102 104 106 100 102 104 106 b 800 600 400 200 0 WT MR1-KO WT MR1-KO WT MR1-KO WT MR1-KO D1004-B3 D1004-E1 D1004-E5 D1004-H3 SFU-IFNy b c D1004-B3 D1004-E1 D1004-E5 D1004-H3 TRAV1-2 - APC 0 10 4 105 0 20 40 60 80 100 Normalized to mode c d MR1/5-OPRU tetramer - PE D1004-B3 D1004-E1 D1004-E5 D1004-H3 0 10 4 10 5 0 20 40 60 80 100 Normalized to mode d D1004-B3, MR1/5-OPRU tetramer D1004-B3, MR1/6-FP tetramer D1004-E5, MR1/5-OPRU tetramer D1004-E5, MR1/6-FP tetramer MR1 tetramer - PE 0 20 40 60 80 100 0 10 4 10 5 Fig. 4 Heterogeneous MR1/5-OP-RU staining of bronchoalveolar TRAV1-2+ CD8+ T cells with MAIT cell-consistent CDR3α‘s and MR1-restricted function. a Frequency of MR1-tetramer+ cells (loaded with active (5-OP-RU) and control (6FP) ligand) in TRAV1-2+ T cells (gated on live, CD3+, CD8+ lymphocytes) from the BAL ﬂuid and peripheral blood of a patient with TB. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 At this point we conclude that MR1/5-OP-RU tetramer staining of bronchoalveolar MAIT cells is weaker and more variable than MR1/5-OP-RU tetramer staining of peripheral blood MAIT cells and hence may underestimate MAIT cell prevalence in the BAL. In contrast to the bronchoalveolar ﬂuid of active TB patients, analysis of TCRα chain usage in granulomas of patients under- going lung-resection for clinically complicated tuberculosis did not demonstrate dramatic expansions of TRAV1-2+TCRαʼs. The contrast between the enrichment of TRAV1-2+ CD8+ T cells observed in bronchoalveolar lavage ﬂuid and the relatively low frequencies of TRAV1-2+TCRα's found in the lung granuloma tissue may be due to differences between cells present in the airway mucosal environment and in lung parenchymal tissue. It is also possible that the kinetics of expansion of TRAV1-2+ CD8+ cells with MAIT cell-consistent CDR3α‘s varied during the long course of TB disease and anti-tuberculosis therapy that preceded surgical treatment in these medically-complex lung-resection patients. It is therefore notable that even in the resected granuloma tissue, the subset of TCRα‘s with MAIT cell-consistent sequences was enriched among the TRAV1-2+ CDR3α‘s in lung granuloma tissue compared to paired mediastinal lymph node tissue. We postulate that this relative enrichment of MAIT cell-consistent TCRs among TRAV1-2+ sequences from the lung was driven by local antigen exposure, while acknowledging that tissue-speciﬁc non-antigen stimuli could also lead to the independent expansion 8 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 The proportion of cells utilizing MAIT cell-consistent CDR3α‘s (MAIT Match Score ≥.95) in MR1/5-OP-RU tetramer positive and negative populations are shown. b IFNγ spot-forming units (SFU) produced by four T cell clones generated from BAL ﬂuid and stimulated with M. smegmatis-infected wildtype (WT) or MR1-KO A549 cells, Supplementary Data. c α-TRAV1-2 staining of four T cell clones generated from BAL ﬂuid demonstrates consistent staining. Histograms are mode-normalized. d Binding of MR1/5-OP-RU tetramer on the same four T cell clones generated from BAL ﬂuid demonstrates heterogenous MR1/5-OPRU tetramer staining (left). Binding of MR1/6-FP (control) and MR1/5-OPRU tetramer is shown for two clones (right). Histograms are mode-normalized COMMUNICATIONS BIOLOGY \| (2019) 2:203 \| https://doi.org/10.1038/s42003-019-0442-2 \| www.nature.com/commsbio 9 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 Table 2 Comparison of MR1/5-OPRU tetramer staining and usage of MAIT cell-consistent CDR3α sequences within TRAV1-2+ CD8+ T cells in the bronchoalveolar (BAL) and peripheral blood (PBMC) compartments from two patients with active TB Participant ID Compartment % MAIT cell- consistent CDR3α % MR1/5-OPRU tetramer-positive % MAIT cell-consistent CDR3α of MR1/5-OPRU tetramer- positive % MAIT cell-consistent CDR3α of MR1/5-OPRU tetramer- negative 1020 BAL 40.7 33.7 93.9 13.7 1020 PBMC 5.4 3.1 89.2 2.7 0091 BAL 28.5 5.1 100.0 24.7 0091 PBMC 18.6 6.3 71.4 15.1 Table 2 Comparison of MR1/5-OPRU tetramer staining and usage of MAIT cell-consistent CDR3α s CD8+ T cells in the bronchoalveolar (BAL) and peripheral blood (PBMC) compartments from two p Table 3 TCRα/β sequences and MAIT Match scores for four MAIT cell clones derived from bronchoalveolar cells Clone TCRα sequencing TCRβ sequencing TRAV CDR3α TRAJ MAIT Match score TRBV CDR3β TRBJ D1004-B3 1-2 CAVTDSNYQLIW 33 1.00 6-5 CASSYEGGGQPQHF 1-5 D1004-E1 1-2 CAALDSNYQLIW 33 1.00 6-4 CASSDGEGQPQHF 1-5 D1004-E5 1-2 CAAMDSNYQLIW 33 1.00 30-1 CAWSHSDRDLNEQYF 2-7 D1004-H3 1-2 CAAMDSNYQLIW 33 1.00 3 CASSQASGGEETQYF 2-5 Table 3 TCRα/β sequences and MAIT Match scores for four MAIT cell clones derived from bronch β sequences and MAIT Match scores for four MAIT cell clones derived from bronchoalveolar cells public MAIT cell-consistent TCRs in the context of mycobacterial infection, further study of selective ligand speciﬁcity in larger numbers of donors is needed. Nonetheless, the convergence of multiple nucleotide rearrangements on expanded public MAIT cell-consistent TCRα chains suggests that in some instances, multiple MAIT cells with genetically unique but functionally similar TCRα chains clonally expand in the TB-infected lung, potentially in response to microbe-derived antigenic-stimulation. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 allowing adults undergoing clinically indicated lung resection for complicated tuberculosis at Inkosi Albert Luthuli Central Hospital (IALCH) to donate excess tissue for scientiﬁc research18. Tissue was isolated from different areas of resected lungs based on the experience of the operating surgeon and the preoperative radiological data. Clinical characteristics of the individuals and samples have been described18. All donors provided written informed consent prior to surgery. BAL ﬂuid and paired peripheral blood samples were obtained under a protocol approved by the UKZN BREC and Partners Institutional Review Board allowing collection of excess ﬂuid from adult patients undergoing clinically indicated diagnostic bronchoscopies at IALCH. Active tuberculosis was deﬁned microbiologically (positive BAL Mtb culture or BAL Mtb PCR by GeneXpert) and/ or histologically (Ziehl-Neelsen positive necrotizing granulomas on transbronchial biopsy obtained at the time of BAL). Uninfected controls were deﬁned as individuals with no evidence of either infectious or inﬂammatory lung disease, as determined by a committee of study physicians on the basis of clinical history, chest x-rays, computerized tomography scans, and negative BAL microbiology (mycobacterial, bacterial and fungal cultures, and Mtb PCR). Most controls underwent bronchoscopy for suspected lung cancer, and a non-cancerous segment was lavaged in these cases. All donors provided written informed consent prior to bronchoscopy. Cryopreserved peripheral blood mononuclear cells (PBMCs) from healthy donors (deﬁned as asymptomatic and HIV-negative with no evidence of Mtb by ELISPOT) were available from the iThimba Cohort which was approved by the UKZN BREC and Partners Institutional Review Board39. All participants provided written informed consent. y g In line with recent studies that have found MAIT cell expan- sions in the lungs of mice experimentally infected with Mtb36 and S. enterica serovar Typhimurium (S. Typhimurium)37, we found expansions of TRAV1-2+ CD8+ T cells with MAIT or MAIT-like features in the BAL and lung parenchyma in patients with TB. Supported by the ﬁndings Chen et al. who found that accumu- lation of MAIT cells in the lungs of mice following challenge with S. Typhimurium is dependent on antigen derived from the microbial riboﬂavin synthesis pathway37, we postulate that these TRAV1-2+ CD8+ cell enrichments contain MAIT cells and are driven by Mtb-derived small molecular ligands. Howson et al. recently reported that MAIT cell clones with more avid ligand- binding expand during S. enterica serovar Paratyphi A infection and that these clones remain expanded after treatment of the infection38. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 This ﬁnding supports the idea that exposure to microbe-derived MR1 ligands alters the human MAIT cell TCR repertoire and suggests a role for MR1-ligand vaccine strategies. Overall our ﬁndings suggest a previously unrecognized and potentially important role for TRAV1-2+ CD8+ T cells with MAIT or MAIT-like features in the immune response to aero- solized Mtb infection, and would support exploration of these cells as targets of either vaccination or immunotherapeutic strategy. Isolation and stimulation of lung and gut T cells. Lymphocytes were isolated from fresh lung tissue as described previously4. A two-step process was used to extract cells from the small intestine. For collection of lymphocytes from the intraepithelial (IEL) layer, the tissue was washed in HBSS, stripped of muscle, and incubated with agitation for 30 min in 0.15% dithiothreitol (Sigma-Aldrich). IEL lymphocytes were then harvested, and the remaining tissue was incubated for 30 min in PBS. Lamina propria (LP) lymphocytes were released by digestion with 0.1% collagenase (CLS-3, Worthington) and 0.3% DNAse (Roche) for 30 min at 37 °C. IEL and LP preparations were further enriched over a discontinuous Percoll gradient. Lymphocyte stimulations were performed as described previously4,12,16. Brieﬂy, lymphocytes were incubated for 16 h with uninfected (control) or M. smegmatis strain mc2122-infected (multiplicity of infection = 3) A549 cells (ATCC CCL-185) at a ratio of 3:1 in the presence of α-CD28 and α-CD49d (Biolegend), together with an α-TNF mAb (Beckman Coulter) and the TNF-Processing Inhi- bitor 0 (TAPI-0, 10 μM) (Calbiochem). Cells were then stained as described above for surface expression of CD45, CD3, CD8, TRAV1-2, and CD1614. Dead cells were excluded using Aqua LIVE⁄DEAD (Invitrogen). Stained samples were acquired on a Fortessa ﬂow cytometer (BD Biosciences) and data were analyzed with FlowJo software version 10.6 (Tree Star). COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 Images were acquired using an Olympus FluoView FV1000 laser scanning confocal microscope system with a 40 × 1.3 Oil Plan Fluorite objective. Confocal images were analyzed using Imaris Analysis Software. Visualization of MAIT cell CDR3α sequences. Data were coded in R using the packages RColorBrewer, Shiny, data.table, ggplot2, and dplyr. Synonymous nucleotide sequences within a tissue were counted, and the associated frequencies are summed. These frequencies were visualized using the TCR Enrichment Ana- lysis (TEA) webtool the code for which is archived at https://github.com/ eisascience/Wong-Gold-Lewinsohn/tree/v1.0.0 Isolation and TCR sequencing of T cells from lung granulomas. Diseased lung tissue (approximately 3 cm3) was isolated from surgically resected explants. Each sample was washed in multiple changes of Hank’s Balanced Salt Solution (HBSS), diced into smaller pieces (approximately 1 mm3), strained, resuspended in pre- warmed R10 supplemented with 0.5 mg/ml collagenase D (Roche) and 40 U/ml DNaseI (Roche), and transferred to GentleMACS C-tubes (Miltenyi Biotec) for mechanical digestion per the manufacturer’s instructions. The resulting suspension was incubated for 60 min at 37 °C, subjected to an additional mechanical digestion step, strained through a 70 μm ﬁlter, washed twice in HBSS, and stained prior to sorting CD4−T-cells using a FACSARIA ﬂow cytometer (BD Biosciences). Cells were gated as live (nearIR−, Invitrogen), single lymphocytes (determined on the basis of light scatter), then sorted as CD45+, CD3+, CD4−events directly into RLT buffer. Genomic DNA was extracted using a DNeasy Minikit (Qiagen) and high- throughput TCRα sequencing was performed using the ImmunoSEQ assay (Adaptive Biotechnologies Corp.)40. Data were analyzed using the ImmunoSEQ Analyser. Generation and characterization of T cell clones. Cells from BAL samples were stained with Aqua LIVE/DEAD (Invitrogen), MR1/5-OP-RU tetramer (0.3 nM, McCluskey Laboratory), α-CD4-FITC (clone OKT4; BioLegend), and α-CD8-APC- Cy7 (clone SK8; BioLegend). Live tetramer-binding cells were sorted by the basis of co-receptor expression using an Inﬂux ﬂow cytometer (BD Biosciences), rested overnight in RPMI 1640 supplemented with 10% heat-inactivated pooled human serum and 0.5 ng/ml rhIL-2, and then distributed in limiting dilution format with irradiated PBMCs (150 × 105/well) and irradiated B-lymphoblastoid cells (3 × 104/ well) in a 96-well round bottom plate. The cultures were stimulated with rhIL-2 (5 ng/ml), rhIL-12 (0.5 ng/ml), rhIL-7 (0.5 ng/ml), rhIL-15 (0.5 ng/ml) and α-CD3 (0.03 µg/ml). Clones were harvested after incubation for 20 days at 37 °C and assessed for clonality by ﬂow cytometry, TCR sequencing, and MR1-restricted function by ELISPOT. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 Nitrocellulose-backed multiscreen 96-well plates (Millipore) were coated overnight at 4 °C with a 10 µg/ml solution of α-IFNγ antibody (clone 1-D1K; Mabtech) in 0.1 M Na2CO3, 0.1 M NaHCO3, pH 9.6. The plate was washed three times with sterile PBS and blocked for 1 h at room temperature with RPMI 1640 containing 10% heat-inactivated pooled human serum. Uninfected, M. smegmatis mc2122-infected (multiplicity of infection = 3), or M. tuberculosis H37Rv-infected (multiplicity of infection = 30) wildtype or MR1-null32 A549 cells (1 × 104/well) and clonal T cells (1 × 104/well) were added and incubated overnight at 37 °C. The plates were then washed six times in PBS containing 0.05% Tween-20, incubated for 2 h at room temperature with a 1 µg/ml solution of α-IFNγ-biotin antibody (clone 7-B6-1; Mabtech) in PBS containing 0.5% bovine serum albumin and 0.05% Tween-20, washed again six times in PBS containing 0.05% Tween-20 followed by PBS alone, and developed using an AEC Vectastain Kit (Vector Laboratories). Spots were counted using an automated ELISPOT Reader System (Autoimmun Diagnostika GmbH). CDR3α sequence similarity. Similarity between CDR3α sequences was calculated as described previously21. This method allows similarities to be assigned between sequences of different length in an alignment-free manner. An implementation of the similarity matching between CDR3 sequences is publicly available at http://www.cbs.dtu.dk/services/MAIT_Match. The server takes as input a list of CDR3α sequences, and returns for each a score based on the maximal sequence similarity with a reference database of MAIT cell CDR3α sequences. A perfect match has a similarity score of 1, and a perfect mismatch a similarity score of 0. Collection, staining and stimulation of BAL lymphocytes. Bronchoscopies were performed by pulmonologists at IALCH. Patients received sedation and bronchodilators according to the local standard of care; Normal saline (200 mL) was lavaged into the lobe with the highest burden of pathology or, in patients with diffuse disease, the right middle lobe. Available BAL ﬂuid was combined in a 1:1 ratio with R10 (RPMI 1640 supplemented with 10% fetal bovine serum, L-gluta- mine, penicillin and streptomycin) and stored on ice. All samples were processed within 3 h of collection, BAL ﬂuid was ﬁltered through a 40 µm strainer (BD Pharmingen) and centrifuged. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 Rainbow Fluorescent Particles (BD Bioscience) and applications settings in FACSDiva7 were used to correct for day-to-day Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. Data availability The datasets generated during and/or analyzed during the current study are archived at https://github.com/eisascience/Wong-Gold-Lewinsohn or available from the corresponding author on reasonable request. pp g y y variations in instrument performance. Cells were gated as live (aqua viability dye negative) lymphocytes (determined on the basis of light scatter), and CD14++ cells were excluded prior to selecting CD3+ cells for analysis. Paired peripheral blood samples were collected where possible and freshly isolated PBMC were processed in parallel with matched BAL cells. Data were analyzed with FlowJo10.6 (Treestar). Background cytokine production was subtracted to calculate percentage of cells producing cytokine in response to stimulation. When available, paired cryopre- served BAL and PBMC cells were thawed and stained with some or all of the above antibodies and MR1/5-OP-RU or MR1/6-FP tetramers (courtesy of the McCluskey Laboratory). Cell suspensions were acquired and sorted on a FACSARIA ﬂow cytometer (BD Biosciences) into TRIzol (Invitrogen). Genomic DNA was extracted utilizing the phenol-chloroform method according to manufacturer protocol, using linear acrylamide (Invitrogen) as a carrier. High-throughput TCRα sequencing was performed using the ImmunoSEQ assay (Adaptive Biotechnologies Corp)40. ARTICLE ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 Resuspended BAL cells were aliquoted for staining with Aqua LIVE⁄DEAD (Invitrogen) and some or all of the following antibodies: α-CD3-PE-CF594 (BD Horizon, clone UCHT1), α-CD8-APC-H7 (BD Pharmingen, clone SK1), α-CD14-PerCP-Cy5.5 (BioLegend, clone HCD14), α- CD235a-PerCP-Cy5.5 (BioLegend, clone HIR2), α-TRAV1-2-APC (clone OF- 5A1212), α-CD161-PE-Cy7 (BioLegend, clone HP-3G10), α-CD26-PE TCR sequence analysis of CD8+ T cell clones isolated from BAL ﬂuid. For some clones, total RNA was extracted using an RNeasy Mini Kit (Qiagen). Unbiased ampliﬁcation of all expressed TRA and TRB gene products was then conducted using a template-switch anchored RT-PCR with chain-speciﬁc constant region primers41. Amplicons were sub-cloned, sampled, sequenced and analyzed as described previously42. Gene usage was assigned according to the IMGT nomen- clature. For other clones, genomic DNA was extracted using a DNeasy Mini Kit (Qiagen) and high-throughput TCRα and TCRβ sequencing was performed using the ImmunoSEQ assay (Adaptive Biotechnologies Corp)40. Data were analyzed using the ImmunoSEQ Analyser. (BioLegend, clone BA5b). All stains were performed at 4 °C. Cells were then ﬁxed with 4% paraformaldehyde. Functional studies were performed if sufﬁcient numbers of BAL lymphocytes were available. After depletion of macrophages via plastic adherence for 1 h, 1 × 106 lymphocytes were stimulated for 18 h at 37 °C with α-CD2/CD3/CD28-loaded Anti-Biotin MACSiBead Particles (Miltenyi Bio- tec) at a ratio of 2:1 in RPMI 1640 supplemented with 10% fetal bovine serum, L- glutamine, HEPES, penicillin, and streptomycin. Brefeldin A was added after the ﬁrst hour to inhibit protein transport from the endoplasmic reticulum. Statistics and reproducibility. Statistical analyses were performed using Prism 6 (GraphPad Software Inc). The non-parametric Mann–Whitney U test was used to assess differences between groups unless indicated otherwise. All statistical tests were two-sided unless indicated otherwise. P values < 0.05 were considered sig- niﬁcant for direct comparisons. In cases of multiple comparisons the Bonferonni correction was applied. Experiments were repeated with as many biologically indpendent samples as were available; when possible a minimum of two experi- mental replicates were performed. (Invitrogen) and the following antibodies: α CD235a PerCP Cy5.5, α CD14 PerCP-Cy5.5, α-CD8-APC-H7, α-TRAV1-2-APC, α-CD161-PE-Cy7. After a wash step, cells were ﬁxed with PERM/FIX Medium A (Invitrogen), permeabilized with PERM/FIX Medium B (Invitrogen), and stained with the following antibodies: α- CD3-PE-CF594, α-TNFa-PE (Beckman Coulter, clone IPM2), and α-IL-17-BV421 (BioLegend, clone BL168). Stained samples were acquired using a Fortessa ﬂow cytometer (BD Bioscience). Methods b Human subjects. Samples from Portland, Oregon, USA. Airway, lung, small intestine and associated lymph node tissues ineligible for transplantation were obtained from the Paciﬁc Northwest Transplant Bank under a protocol approved by the Institutional Review Board at Oregon Health & Science University. Limited clinical information was available for these individuals, who were generally con- sidered healthy prior to demise. For comparison with the organ samples, PBMCs were obtained by apheresis from healthy adult donors providing informed consent. Samples from Durban, South Africa. Explanted granulomatous lung tissue and associated lymph nodes were obtained under a protocol approved by the University of KwaZulu Natal Human Biomedical Research Ethics Commitee (UKZN BREC) Immunohistochemistry of airway tissues. Cryosections (10 µM) of frozen airway tissues were treated with acetone and air-dried prior to incubation with α-TRAV1- 2 antibody (clone-3C10; Biolegend) followed by goat α-mouse IgG1-Alexa Fluor 488 (1:1000), and then α-CD8 antibody (1:50; LSBio) followed by goat α-mouse IgG1-Alexa Fluor 568 (1:1000). Sections were washed and stained with DAPI. COMMUNICATIONS BIOLOGY \| (2019) 2:203 \| https://doi.org/10.1038/s42003-019-0442-2 \| www.nature.com/commsbio 10 COMMUNICATIONS BIOLOGY \| (2019) 2:203 \| https://doi.org/10.1038/s42003-019-0442-2 \| www.nature.com/commsbio References 29. Eberhard, J. M. et al. CD161+ MAIT cells are severely reduced in peripheral blood and lymph nodes of HIV-infected individuals independently of disease progression. 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MAIT cell function is modulated by PD-1 signaling in patients with active tuberculosis. Am. J. Respir. Crit. Care Med. https://doi.org/10.1164/ rccm.201401-0106OC (2014). 42. Price, D. A. et al. Acknowledgements 16. Gold, M. C. et al. MR1-restricted MAIT cells display ligand discrimination and pathogen selectivity through distinct T cell receptor usage. J. Exp. Med. https://doi.org/10.1084/jem.20140507 (2014). We would like to thank Hollis Shen of the AHRI Immunology Core for technical assistance, Kamini Gounder for HLA genotyping, James McCluskey and his laboratory for use of the MR1 tetramers, the Paciﬁc Northwest Transplant Bank for ongoing pro- vision of research tissue, the HIV Pathogenesis Programme Processing Laboratory staff, the AHRI Clinical Core, the staff of Inkosi Albert Luthuli Central Hospital, and the study participants. This work was funded in part by a Burroughs-Wellcome Fund/American Society of Tropical Medicine and Hygiene fellowship (EBW), a Fulbright Award (ZAS), the National Institutes of Health (grants T32 AI007387 and K08 AI118538 to E.B.W., grant R01AI078965 to M.C.G., grant R01AI048090 to D.M.L., grants R01AI37856 and R01AI97138 to W.R.B., grant R01AI106725 to S.M.B.), the National Institute of Allergy and Infectious Diseases Mucosal Immunology Studies Team (grant U01AI09577 to M.C.G. and D.M.L.), and Merit Review Awards # I01 BX001231 and I01 BX000533 from the United States Department of Veterans Affairs (VA) Biomedical Laboratory Research and Development, supported by use of the facilities and resources at the VA Portland Health Care System. T.N. received funding from the South African DST/NRF Research Chairs Initiative and the Victor Daitz Foundation. D.A.P. is a Wellcome Trust Senior Investigator. Collection of samples from the iThimba Cohort was supported by the Harvard University Center for AIDS Research (grant P30 AI060354). Research reported in this publication was supported by the Strategic Health Innovation Partnerships (SHIP) Unit of the South African Medical Research Council (SA MRC) with funds received from the South African Department of Science and Technology as part of a bilateral research collaboration agreement with the Government of India; and through a SA MRC Colla- borating Centre (ACT4TB/HIV).This work was also supported in part through the Sub- Saharan African Network for TB/HIV Research Excellence (SANTHE), a DELTAS Africa Initiative [grant # DEL-15-006]. The DELTAS Africa Initiative is an independent funding scheme of the African Academy of Sciences (AAS)’s Alliance for Accelerating Excellence in Science in Africa (AESA) and supported by the New Partnership for Africa’s Devel- opment Planning and Coordinating Agency (NEPAD Agency) with funding from the Wellcome Trust [grant # 107752/Z/15/Z] and the UK government. ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 References Avidity for antigen shapes clonal dominance in CD8+ T cell populations speciﬁc for persistent DNA viruses. J. Exp. Med. 202, 1349–1361 (2005). 15. Gold, M. C. & Lewinsohn, D. M. Co-dependents: MR1-restricted MAIT cells and their antimicrobial function. Nat. Rev. Microbiol. https://doi.org/10.1038/ nrmicro2918 (2012). Acknowledgements Open access pub- lication of this article has been made possible through support from the Victor Daitz Information Gateway, an initiative of the Victor Daitz Foundation and the University of KwaZulu-Natal. The views expressed in this publication are those of the authors and do not represent the views of the Unites States Department of Veterans Affairs, the United States Government, AAS, NEPAD Agency, Wellcome Trust or the UK government. 17. Sharma, P. K. et al. High expression of CD26 accurately identiﬁes human bacterial-reactive MR1-restricted MAIT cells. Immunology https://doi.org/ 10.1111/imm.12461 (2015). 18. Nunes-Alves, C. et al. Human and murine clonal CD8+ T cell expansions arise during tuberculosis because of TCR selection. PLoS Pathog. 11, e1004849 (2015). 19. Carlson, C. S. et al. Using synthetic templates to design an unbiased multiplex PCR assay. Nat. Commun. 4, 2680 (2013). y 20. Greenaway, H. Y. et al. NKT and MAIT invariant TCRalpha sequences can be produced efﬁciently by VJ gene recombination. Immunobiology https://doi. org/10.1016/j.imbio.2012.04.003 (2012). g gy and Infectious Diseases Mucosal Immunology Studies Team (grant U01AI09577 to M.C.G. and D.M.L.), and Merit Review Awards # I01 BX001231 and I01 BX000533 from the United States Department of Veterans Affairs (VA) Biomedical Laboratory Research and Development, supported by use of the facilities and resources at the VA Portland Health Care System. T.N. received funding from the South African DST/NRF Research Chairs Initiative and the Victor Daitz Foundation. D.A.P. is a Wellcome Trust Senior g j 21. Shen, W.-J. W., Hau-San, Xiao, Quan-Wu, Guo & Xin, Smale,Stephen Towards a mathematical foundation of immunology and amino acid chains. eprint arXiv 1205, 6031 (2012). p 22. Greenaway, H. Y. et al. NKT and MAIT invariant TCRalpha sequences can be produced efﬁciently by VJ gene recombination. Immunobiology 218, 213–224 (2013). Investigator. Collection of samples from the iThimba Cohort was supported by the Harvard University Center for AIDS Research (grant P30 AI060354). Research reported in this publication was supported by the Strategic Health Innovation Partnerships (SHIP) Unit of the South African Medical Research Council (SA MRC) with funds received from the South African Department of Science and Technology as part of a bilateral research collaboration agreement with the Government of India; and through a SA MRC Colla- borating Centre (ACT4TB/HIV).This work was also supported in part through the Sub- Saharan African Network for TB/HIV Research Excellence (SANTHE), a DELTAS Africa Initiative [grant # DEL-15-006]. Code availability Custom code for the MAIT Match tool is available at http://www.cbs.dtu.dk/services/ MAIT_Match/. Custom code for the visualization of frequencies of speciﬁc TCRα‘s in the TB granuloma tissues and the TCR Enrichment Analysis (TEA) webtool are archived at https://github.com/eisascience/Wong-Gold-Lewinsohn Received: 7 February 2019 Accepted: 24 April 2019 Received: 7 February 2019 Accepted: 24 April 2019 11 11 COMMUNICATIONS BIOLOGY \| (2019) 2:203 \| https://doi.org/10.1038/s42003-019-0442-2 \| www.nature.com/commsbio 1Africa Health Research Institute, KwaZulu-Natal, South Africa. 2Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA, USA. 3Harvard Medical School, Boston, MA, USA. 4Division of Infection and Immunity, University College London, London, UK. 5Department of Pulmonary & Critical Care Medicine, Oregon Health & Science University, Portland, OR, USA. 6VA Portland Health Care System, Portland, OR, USA. 7Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, OR, USA. 8Division of Bioinformatics and Computational Biology (BCB), Department of Medical Informatics and Clinical Epidemiology (DMICE), Oregon Health & Science University, Portland, OR, USA. 9Institute for Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark. 10Durban University of Technology, Durban, South Africa. 11Department of Pulmonology, Inkosi Albert Luthuli Hospital, Durban, South Africa. 12Department of Pulmonology & Critical Care, Nelson R. Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa. 13Department of Cardiothoracic Surgery, Nelson R. Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa. 14Centre for AIDS Programme of Research in South Africa (CAPRISA), Durban, South Africa. 15Institute of Infection & Immunity, Cardiff University School of Medicine, Cardiff, Wales, UK. 16Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA. 17Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, USA. 18Center for Biological Sequence Analysis, Department of Bio and Health Informatics, Technical University of Denmark, Lyngby, Denmark. 19Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, Buenos Aires, Argentina. 20HIV Pathogenesis Programme, Doris Duke Medical Research Institute, Nelson R. Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa. 21The Ragon Institute of MGH, MIT, and Harvard, Harvard Medical School, Cambridge, MA, USA. 22Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, MD, USA. 23Max Planck Institute for Infection Biology, Berlin, Germany. 24These authors contributed equally: Emily B. Wong, Marielle C. Gold. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-019-0442-2 Author contributions Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. E.B.W., M.C.G., S.M.B., A.L., T.N. and D.M.L. designed the experiments; E.B.W., M.C.G., B.Z.X., EW.M., S.K., Z.A.S., H.K., P.K.S., A.H.W., J.E.M., K.L., K.L.M. and M.N. per- formed the experiments; E.B.W., M.C.G., E.W.M., S.K., J.E.M., D.A.P., S.M.B., A.L., T.N., E.M. and D.M.L. analyzed the results. U.L., Z.R., P.B., A.A., L.N., R.M., M.S., V.O.K. and W.R.B. enrolled human subjects and performed procedures; E.B.W., M.C.G., D.A.P., E.M., T.N. and D.M.L. wrote the manuscript. All co-authors provided comments and approved the content. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. ARTICLE ARTICLE Additional information Supplementary information accompanies this paper at https://doi.org/10.1038/s42003- 019-0442-2. Competing interests: The authors declare no competing interests. Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ reprintsandpermissions/ © The Author(s) 2019 Emily B. Wong 1,2,3,4,24, Marielle C. Gold 5,6,7,24, Erin W. Meermeier 5, Bongiwe Z. Xulu1, Sharon Khuzwayo 1, Zuri A. Sullivan 1, Eisa Mahyari8, Zoe Rogers1, Hénrik Kløverpris1,4,9, Prabhat K. Sharma6, Aneta H. Worley6, Umesh Lalloo10, Prinita Baijnath10,11, Anish Ambaram11, Leon Naidoo11, Moosa Suleman11,12, Rajhmun Madansein13,14, James E. McLaren 15, Kristin Ladell 15, Kelly L. Miners15, David A. Price 15,16, Samuel M. Behar 17, Morten Nielsen 18,19, Victoria O. Kasprowicz1,20,21, Alasdair Leslie1,4, William R. Bishai22, Thumbi Ndung’u1,19,20,21,23 & David M. Lewinsohn 5,6,7 Emily B. Wong 1,2,3,4,24, Marielle C. Gold 5,6,7,24, Erin W. Meermeier 5, Bongiwe Z. Xulu1, Sharon Khuzwayo 1, Zuri A. Sullivan 1, Eisa Mahyari8, Zoe Rogers1, Hénrik Kløverpris1,4,9, Prabhat K. Sharma6, Aneta H. Worley6, Umesh Lalloo10, Prinita Baijnath10,11, Anish Ambaram11, Leon Naidoo11, Moosa Suleman11,12, Rajhmun Madansein13,14, James E. McLaren 15, Kristin Ladell 15, Kelly L. Miners15, David A. Price 15,16, Samuel M. Behar 17, Morten Nielsen 18,19, Victoria O. Kasprowicz1,20,21, Alasdair Leslie1,4, William R. Bishai22, Thumbi Ndung’u1,19,20,21,23 & David M. Lewinsohn 5,6,7 1Africa Health Research Institute, KwaZulu-Natal, South Africa. 2Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA, USA. 3Harvard Medical School, Boston, MA, USA. 4Division of Infection and Immunity, University College London, London, UK. 5Department of Pulmonary & Critical Care Medicine, Oregon Health & Science University, Portland, OR, USA. 6VA Portland Health Care System, Portland, OR, USA. 7Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, OR, USA. 8Division of Bioinformatics and Computational Biology (BCB), Department of Medical Informatics and Clinical Epidemiology (DMICE), Oregon Health & Science University, Portland, OR, USA. 9Institute for Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark. 10Durban University of Technology, Durban, South Africa. 11Department of Pulmonology, Inkosi Albert Luthuli Hospital, Durban, South Africa. 12Department of Pulmonology & Critical Care, Nelson R. Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa. 13Department of Cardiothoracic Surgery, Nelson R. Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa. 14Centre for AIDS Programme of Research in South Africa (CAPRISA), Durban, South Africa. 15Institute of Infection & Immunity, Cardiff University School of Medicine, Cardiff, Wales, UK. Acknowledgements The DELTAS Africa Initiative is an independent funding scheme of the African Academy of Sciences (AAS)’s Alliance for Accelerating Excellence in Science in Africa (AESA) and supported by the New Partnership for Africa’s Devel- opment Planning and Coordinating Agency (NEPAD Agency) with funding from the Wellcome Trust [grant # 107752/Z/15/Z] and the UK government. Open access pub- lication of this article has been made possible through support from the Victor Daitz Information Gateway, an initiative of the Victor Daitz Foundation and the University of KwaZulu-Natal. The views expressed in this publication are those of the authors and do not represent the views of the Unites States Department of Veterans Affairs, the United States Government, AAS, NEPAD Agency, Wellcome Trust or the UK government. 23. McWilliam, H. E. et al. The intracellular pathway for the presentation of vitamin B-related antigens by the antigen-presenting molecule MR1. Nat. Immunol. 17, 531–537 (2016). 24. Dusseaux, M. et al. Human MAIT cells are xenobiotic-resistant, tissue- targeted, CD161hi IL-17-secreting T cells. Blood 117, 1250–1259 (2011). 25. Gibbs, A. et al. MAIT cells reside in the female genital mucosa and are biased towards IL-17 and IL-22 production in response to bacterial stimulation. Mucosal Immunol. https://doi.org/10.1038/mi.2016.30 (2016). g 26. Sharma, P. K. et al. High expression of CD26 accurately identiﬁes human bacteria-reactive MR1-restricted MAIT cells. Immunology https://doi.org/ 10.1111/imm.12461 (2015). 27. Tobin, D. M. et al. Host genotype-speciﬁc therapies can optimize the inﬂammatory response to mycobacterial infections. Cell 148, 434–446 (2012). 28. Leeansyah, E. et al. Activation, exhaustion and persistent decline of the anti- microbial MR1-restricted MAIT cell population in chronic HIV-1 infection. Blood https://doi.org/10.1182/blood-2012-07-445429 (2012). 12 COMMUNICATIONS BIOLOGY \| (2019) 2:203 \| https://doi.org/10.1038/s42003-019-0442-2 \| www.nature.com/commsbio Additional information 16Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA. 17Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, USA. 18Center for Biological Sequence Analysis, Department of Bio and Health Informatics, Technical University of Denmark, Lyngby, Denmark. 19Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, Buenos Aires, Argentina. 20HIV Pathogenesis Programme, Doris Duke Medical Research Institute, Nelson R. Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa. 21The Ragon Institute of MGH, MIT, and Harvard, Harvard Medical School, Cambridge, MA, USA. 22Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, MD, USA. 23Max Planck Institute for Infection Biology, Berlin, Germany. 24These authors contributed equally: Emily B. Wong, Marielle C. Gold. 13 COMMUNICATIONS BIOLOGY \| (2019) 2:203 \| https://doi.org/10.1038/s42003-019-0442-2 \| www.nature.com/commsbio
https://openalex.org/W2294293971	https://mediatum.ub.tum.de/doc/1353668/document.pdf	English	null	More Consistently Altered Connectivity Patterns for Cerebellum and Medial Temporal Lobes than for Amygdala and Striatum in Schizophrenia	Frontiers in human neuroscience	2,016	cc-by	8,824	ORIGINAL RESEARCH published: 17 February 2016 doi: 10.3389/fnhum.2016.00055 More Consistently Altered Connectivity Patterns for Cerebellum and Medial Temporal Lobes than for Amygdala and Striatum in Schizophrenia Henning Peters 1,2,3, Junming Shao 4,5,6, Martin Scherr 1, Dirk Schwerthöffer 1, Claus Zimmer 7, Hans Förstl 1, Josef Bäuml 1, Afra Wohlschläger 2,7, Valentin Riedl 2,7,8, Kathrin Koch 2,7† and Christian Sorg 1,2,7† 1 Department of Psychiatry, Technische Universität München, München, Germany, 2 TUM-Neuroimaging Center, Klinikum Rechts der Isar, Technische Universität München, München, Germany, 3 Klinik für Psychiatrie und Psychotherapie, Klinikum der Universität München, München, Germany, 4 School of Computer Science and Engineering, University of Electronic Science and Technology of China, Chengdu, China, 5 Big Data Research Center, University of Electronic Science and Technology of China, Chengdu, China, 6 Center for Information in BioMedicine, University of Electronic Science and Technology of China, Chengdu, China, 7 Department of Neuroradiology, Technische Universität München, München, Germany, 8 Department of Nuclear Medicine, Technische Universität München, München, Germany Background: Brain architecture can be divided into a cortico-thalamic system and modulatory “subcortical-cerebellar” systems containing key structures such as striatum, medial temporal lobes (MTLs), amygdala, and cerebellum. Subcortical-cerebellar systems are known to be altered in schizophrenia. In particular, intrinsic functional brain connectivity (iFC) between these systems has been consistently demonstrated in patients. While altered connectivity is known for each subcortical-cerebellar system separately, it is unknown whether subcortical-cerebellar systems’ connectivity patterns with the cortico-thalamic system are comparably altered across systems, i.e., if separate subcortical-cerebellar systems’ connectivity patterns are consistent across patients. Edited by: Rachael D. Seidler, University of Michigan, USA Reviewed by: Stephan F. Taylor, University of Michigan, USA Jessica A. Bernard, Texas A&M University, USA Correspondence: Kathrin Koch kathrin.koch@tum.de †These authors have contributed equally to this work. Received: 16 November 2015 A t d 05 F b 2016 Reviewed by: Stephan F. Taylor, University of Michigan, USA Jessica A. Bernard, Texas A&M University, USA *Correspondence: Kathrin Koch kathrin.koch@tum.de Methods: To investigate this question, 18 patients with schizophrenia (3 unmedicated, 15 medicated with atypical antipsychotics) and 18 healthy controls were assessed by resting-state functional magnetic resonance imaging (fMRI). Independent component analysis of fMRI data revealed cortical intrinsic brain networks (NWs) with time courses representing proxies for cortico-thalamic system activity. Subcortical-cerebellar systems’ activity was represented by fMRI-based time courses of selected regions-of- interest (ROIs; i.e., striatum, MTL, amygdala, cerebellum). Correlation analysis among ROI- and NWs-time courses yielded individual connectivity matrices [i.e., connectivity between NW and ROIs (allROIs-NW, separateROI-NW), only NWs (NWs-NWs), and only ROIs (allROIs-allROIs)] as main outcome measures, which were classiﬁed by support-vector-machine-based (SVM) leave-one-out cross-validation. Differences in classiﬁcation accuracy were statistically evaluated for consistency across subjects and systems. †These authors have contributed equally to this work. Received: 16 November 2015 Accepted: 05 February 2016 Published: 17 February 2016 INTRODUCTION microcircuit alterations, are consistent features of schizophrenia and are linked with emotional and cognitive deficits, respectively (Andreasen and Pierson, 2008; Benes, 2010; Anticevic et al., 2012; Mier et al., 2014; Millan et al., 2014; Vai et al., 2015). In more detail, amygdala alterations are heterogeneous and appear to differ depending on the clinical syndrome and disorder state (Rasetti et al., 2009). For example, amygdala hyperactivity has been shown to relate to paranoid ideation in schizophrenia, suggesting symptom-based subgroups (Pinkham et al., 2015). Findings across subjects at high-risk, early and chronic states of schizophrenia, suggest differentially affected amygdala iFC from increased to reduced levels, also reflecting symptom severity (Anticevic et al., 2014; Mukherjee et al., 2014). Considering altered cerebellar functional connectivity, mainly reduced connectivity has been reported consistently across different tasks and disorder states (Lungu et al., 2013). Interestingly, the cerebellum has been identified as one of the key regions of hypoconnectivity with the thalamus related to conversion of subjects at ultra high risk to psychosis (Anticevic et al., 2015). Widespread brain dysconnectivity is an essential element in the pathophysiology of schizophrenia (Stephan et al., 2009). The cortico-thalamic system is particularly affected by aberrant functional and structural connectivity (Flynn et al., 2003; Begré and Koenig, 2008; Lynall et al., 2010; van den Heuvel et al., 2010; Welsh et al., 2010; Woodward et al., 2012; Palaniyappan et al., 2013; Wagner et al., 2013, 2015; Manoliu et al., 2014). For example, the coherence of ongoing, slowly fluctuating neural activity (<0.1 Hz), which reflects a basic form of brain organization called intrinsic functional connectivity (iFC; Fox and Raichle, 2007), is consistently changed in patients relative to healthy controls for both cortico-thalamic (Welsh et al., 2010; Woodward et al., 2012) and cortico-cortical connectivity (Lynall et al., 2010; Palaniyappan et al., 2013; Manoliu et al., 2014; van den Heuvel and Fornito, 2014). One should note that, rather than overall reduced or increased connectivity, alterations reflect rather complex reorganization of network interaction. Beyond the cortico-thalamic system, several other non-cortical/non-thalamic brain systems are involved in schizophrenia. For example, the striatum shows increased pre-synaptic dopamine concentrations that are crucial for schizophrenia’s psychotic symptoms (Howes et al., 2012) and increased activity is linked with symptom dimensions, for putamen with positive symptoms during psychosis, for ventral striatum with negative symptoms during remission (Sorg et al., 2013). Citation: Peters H, Shao J, Scherr M, Schwerthöffer D, Zimmer C, Förstl H, Bäuml J, Wohlschläger A, Riedl V, Koch K and Sorg C (2016) More Consistently Altered Connectivity Patterns for Cerebellum and Medial Temporal Lobes than for Amygdala and Striatum in Schizophrenia. Front. Hum. Neurosci. 10:55. doi: 10.3389/fnhum.2016.00055 February 2016 \| Volume 10 \| Article 55 Frontiers in Human Neuroscience \| www.frontiersin.org 1 Peters et al. Subcortical-Cerebellar System Connectivity in Schizophrenia Results: Correlation matrices based on allROIs-NWs yielded 91% classiﬁcation accuracy, which was signiﬁcantly superior to allROIs-allROIs and NWs-NWs (56 and 74%, respectively). Considering separate subcortical-cerebellar systems, cerebellum- NWs and MTL-NWs reached highest accuracy values with 91 and 85%, respectively, while those of striatum-NW and amygdala-NW were signiﬁcantly lower with about 65% classiﬁcation accuracy. Conclusion: Results provide initial evidence for differential consistency of altered intrinsic connectivity patterns between subcortical-cerebellar systems and the cortico- thalamic system. Data suggest that differential dysconnectivity patterns between subcortical-cerebellar and cortical systems might reﬂect different disease states or patient subgroups. Keywords: fMRI, support vector machine, schizophrenia, functional connectivity (FC), multivariate pattern analysis, subcortical Frontiers in Human Neuroscience \| www.frontiersin.org INTRODUCTION Moreover, substantial alterations within the medial temporal lobes (MTLs) include altered microstructure, transmitters, perfusion, and activity, particularly in the hippocampus (Tamminga et al., 2010). In more detail, structural deficits were recently attributed to hippocampal subfields in large samples of schizophrenia patients (Mathew et al., 2014; Arnold et al., 2015; Haukvik et al., 2015) and were associated with decreased default-mode-network suppression in healthy subjects at genetic risk (Seidman et al., 2014). Patients’ memory deficits, particularly with regard to relational information (vs. item-specific), have been linked with diminished hippocampal activation (Ragland et al., 2015). Furthermore, abnormalities in amygdala and cerebellum, ranging from molecular to Intriguingly, all these brain systems—striatum, MTL, amygdala, and cerebellum—are massively connected with the cortico-thalamic system (Swanson, 2003). They are assumed to play key modulatory roles in cortico-thalamic processes (Buzsaki, 2006) and beyond structural aberrations (Bois et al., 2015; van Erp et al., 2015), several studies demonstrated their aberrant iFC with the cortico-thalamic system in patients with schizophrenia (Collin et al., 2011; Anticevic et al., 2012; Fornito et al., 2013; Kraguljac et al., 2014; Peters et al., in press). We use the term ‘‘subcortical-cerebellar system’’ to refer to these systems. We are aware that it is a matter of debate to separate these systems from the cortico-thalamic system; for example, the striatum is massively integrated in cortico-basal ganglia- thalamo-cortical loops. However, differences in anatomy and connectivity, physiology and function, as well as their distinct role in schizophrenia pathophysiology suggest that treating these systems separately from thalamo-cortical connectivity is plausible (Buzsaki, 2006; Andreasen and Pierson, 2008; Benes, 2010; Tamminga et al., 2010; Howes et al., 2012). While previous studies investigated each ‘‘subcortical-cerebellar’’ system and its connectivity separately, it is unknown how the whole pattern of February 2016 \| Volume 10 \| Article 55 Frontiers in Human Neuroscience \| www.frontiersin.org 2 Subcortical-Cerebellar System Connectivity in Schizophrenia Peters et al. by specific iFC patterns (Fox and Raichle, 2007). We used intrinsic networks, which cover preferentially the cerebral cortex, as proxy for cortico-thalamic sub-systems (Figure 1). Corresponding time courses, reflecting networks’ activity (NWs) were used for further analysis. (iii) Concerning subcortical- cerebellar systems, we focused on striatum, amygdala, MTL, and cerebellum (Figure 2). For each system, we chose typical sub-regions, which have been used previously to study systems’ iFC (details in the method part). For these regions-of-interest, we extracted representative rs-fMRI signal time courses (ROIs). INTRODUCTION Displayed are regions of interest (ROI) based on coordinates derived from previous studies in case of striatum, medial temporal lobe and cerebellum (Kahn et al., 2008; Etkin et al., 2009; Krienen and Buckner, 2009; Peters et al., in press). Deﬁnition of amygdala ROI was derived from Anatomy toolbox for SPM (http://www.fz-juelich.de/inb/inb-3/spm_anatomy_toolbox). TABLE 1 \| Demographic and psychometric data. TABLE 1 \| Demographic and psychometric data. SP (n = 18) HC (n = 18) SP vs. HC Measure Mean (SD) Mean (SD) T-score p-value Age 35.33 (12.49) 34.10 (13.4) 0.318 0.75 Sex (m/f) 9/9 9/9 PANSS-Total 76.44 (18.45) 30.10 (0.66) 11.23 <0.001∗ PANSS-Positive 18.06 (5.74) 6.95 (0.23) 8.57 <0.001∗ PANSS-Negative 19.94 (8.11) 6.90 (0.44) 7.08 <0.001∗ GAF 41.50 (11.55) 99.50 (1.12) −22.46 <0.001∗ CPZ 466.72 (440.49) Two-sample t-tests for age and psychometric tests; ∗signiﬁcant for p < 0.05. Abbreviations: SP, patients with schizophrenia during acute psychosis; HC, healthy control group; PANSS, Positive and Negative Syndrome Scale; GAF, Global Assessment of Functioning Scale; CPZ, chlorpromazine equivalent dose. We hypothesized that: 1. Connectivity matrices based on allROIs-NWs reflect more consistent connectivity patterns within groups of patients and healthy controls than allROIs- allROIs and NWs-NWs connectivity matrices. Therefore, they classify subjects more accurately. 2. Separate-ROI- NWs connectivity matrices describe differentially consistent connectivity patterns. Therefore, separate ROIs classify subjects with different accuracy. INTRODUCTION (iv) Then, we calculated iFC connectivity matrices (i.e., Pearson correlation of pairs of signal time courses of NWs or ROIs, respectively) for combinations of allROIs-NWs, NWs-NWs, allROIs-allROIs as well as separateROI-NWs for each subject. (v) Finally, we used multivariate pattern classification (i.e., support vector machine) and leave-one-out cross validation to estimate group differences and particularly inter-individual consistency of iFC patterns for different subcortical-cerebellar systems. connectivity changes between ‘‘subcortical-cerebellar’’ systems and the cortico-thalamic system is configured in schizophrenia, particularly whether abnormal connectivity is consistent across patients for different systems. This might be an important question, since graded patterns of aberrant connectivity across subcortical-cerebellar systems might vary across disease states and patient subgroups. To obtain initial evidence for this question, the current study investigates connectivity patterns in the cortico-thalamic system across mentioned subcortical- cerebellar systems, and—most importantly—between these two types of systems in psychotic patients with schizophrenia to evaluate the inter-individual consistency of potential changes across systems. To reach the study’s objective, the following steps have been performed (for an overview, see Figure S1): (i) We assessed slowly fluctuating ongoing brain activity (Leopold and Maier, 2012) in patients with schizophrenia and healthy controls with resting- state functional MRI (rs-fMRI). (ii) Ongoing brain activity is organized in intrinsic brain networks (NWs) characterized FIGURE 1 \| Cortical networks. Displayed are spatial maps of cortical networks (NWs) derived from independent component analysis (ICA) of patients’ and healthy controls’ resting state fMRI data, representing the cortico-thalamic sub-systems. The 22 NWs shown were selected following an automated spatial multiple-regression on reference-templates provided by Allen et al. (2011). FIGURE 1 \| Cortical networks. Displayed are spatial maps of cortical networks (NWs) derived from independent component analysis (ICA) of patients’ and healthy controls’ resting state fMRI data, representing the cortico-thalamic sub-systems. The 22 NWs shown were selected following an automated spatial multiple-regression on reference-templates provided by Allen et al. (2011). February 2016 \| Volume 10 \| Article 55 Frontiers in Human Neuroscience \| www.frontiersin.org 3 3 Peters et al. Subcortical-Cerebellar System Connectivity in Schizophrenia FIGURE 2 \| Subcortical-cerebellar systems. Displayed are regions of interest (ROI) based on coordinates derived from previous studies in case of striatum, medial temporal lobe and cerebellum (Kahn et al., 2008; Etkin et al., 2009; Krienen and Buckner, 2009; Peters et al., in press). Deﬁnition of amygdala ROI was derived from Anatomy toolbox for SPM (http://www.fz-juelich.de/inb/inb-3/spm_anatomy_toolbox). FIGURE 2 \| Subcortical-cerebellar systems. Frontiers in Human Neuroscience \| www.frontiersin.org DATA ACQUISITION All participants underwent 10 min of rs-fMRI with the instruction to keep their eyes closed and not to fall asleep. We verified that subjects stayed awake by interrogating via intercom immediately after the rs-fMRI scan. Before and after scanning, a medical examination of patients validated their stable condition and investigated whether they had feelings of odd situations during the scanning. No patient dropped out during the scanning session. Using in-house scripts employing SPM 8 and matlab3 routines, for each subject and ROI, fMRI-time courses were extracted, Butterworth bandpass-filtered for the frequency range from 0.009 to 0.08 Hz, and reduced to ROI-representative time courses by singular value decomposition. To account for partial volume and movement effects, linear effects of global gray matter (GM), white matter (WM), cerebrospinal fluid (CSF) fMRI- signal, and six movement parameters were regressed out. We included the global GM signal as a nuisance regressor since it is thought to reflect a combination of physiological processes (such as cardiac and respiratory fluctuations) and scanner drift. To extract the nuisance covariate time series for GM, WM and CSF, each individual’s high-resolution T1-weighted structural image was segmented. Mean images of the study sample’s T1- segmentation were used to create ROIs for the extraction of GM, WM, and CSF nuisance signals. MRI was carried out on a 3 T whole body scanner (Achieva, Philips Healthcare). FMRI was based on gradient echo EPI sequence (TE = 35 ms, TR = 2000 ms, flip angle = 82◦, FoV = 220 × 220 mm2, matrix = 80 × 80, 32 slices, slice thickness = 4 mm, and 0 mm interslice gap; 300 volumes). T1-weighted anatomical MRI was based on magnetization-prepared rapid acquisition gradient echo sequence (TE = 4 ms, TR = 9 ms, TI = 100 ms, flip angle = 5◦, FoV = 240 × 240 mm2, matrix = 240 × 240, 170 slices, voxel size = 1 × 1 × 1 mm3). MATERIALS AND METHODS Eighteen healthy controls and Eighteen patients participated in the study (Table 1). All participants had been investigated in a previous study (Manoliu et al., 2014), which addressed cortical iFC changes for three selected intrinsic networks i.e., default mode, salience, and central executive network; here, we focus on a larger number of cortical networks and their iFC with subcortical-cerebellar systems. Written informed consent in accordance with the Human Research Committee guidelines of the Klinikum Rechts der Isar, Technische Universität München was obtained from all participants. Patients were recruited from the Department of Psychiatry, Klinikum Rechts der Isar, TU München, controls by word-of-mouth advertising. Participants’ examination included medical history, psychiatric interview, psychometric assessment, and additionally blood tests for patients. Psychiatric diagnoses relied on the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV). To assess psychiatric diagnoses, the Structured Clinical Interview for DSM-IV (SCID-I) was used (Spitzer et al., 1992). The global level of social, occupational, and psychological functioning was measured with the Global Assessment of Functioning Scale (GAF; Spitzer et al., 1992). For rating severity of clinical symptoms on the day of scanning, the Positive and Negative Syndrome Scale (PANSS) was applied (Kay et al., 1987). Clinical- psychometric assessment was completed by psychiatrists DS and MS who have been professionally trained for SCID and PANSS- based interviews with inter-rater reliability of more than 95%. Inclusion criteria for the study were diagnosis of schizophrenia and age between 18 and 60 years. Exclusion criteria were current or past neurological or internal systemic disorder, current depressive or manic episode, substance abuse (except for nicotine), schizoaffective disorder, and cerebral pathology in MRI. All patients included were diagnosed with paranoid schizophrenia during acute psychosis as indicated by clinical Frontiers in Human Neuroscience \| www.frontiersin.org Frontiers in Human Neuroscience \| www.frontiersin.org February 2016 \| Volume 10 \| Article 55 4 Subcortical-Cerebellar System Connectivity in Schizophrenia Peters et al. exacerbation and increased positive symptom scores on the PANSS (Table 1). Seven out of eighteen had significant hallucinations (PANSS P3 ≥3), 15 delusions (P1 ≥3). The mean duration of illness was 7.15 years (SD = 6.89 years), the mean number of hospital stays was 2.98 (SD = 2.48). Preprocessing For each participant, the first three rs-fMRI scans were discarded due to magnetization effects. SPM8 (Wellcome Department of Cognitive Neurology, London) was used for motion correction, spatial normalization into the stereotactic space of the Montreal Neurological Institute (MNI) and spatial smoothing with an 8 mm × 8 mm × 8 mm Gaussian kernel. To ensure data quality, particularly concerning motion-induced artifacts, temporal signal-to-noise ratio (tSNR) and point-to-point head motion were estimated for each subject (Murphy et al., 2007; Van Dijk et al., 2012). Point-to-point motion was defined as the absolute displacement of each brain volume compared to its previous volume. Moreover, root mean square (RMS) of the translational head movement parameters was calculated for each subject. Excessive head motion (cumulative motion translation >3 mm and mean point-to-point translation or rotation >0.15 mm or 0.1◦) was applied as exclusion criterion. None of the 1http://www.fz-juelich.de/inb/inb-3/spm_anatomy_toolbox 2http://marsbar.sourceforge.net 3www.mathworks.com MATERIALS AND METHODS Concerning medication, three patients were free of any antipsychotic medication, the others received mono- or dual therapy with atypical antipsychotic medication including Amisulpride (n = 2), Olanzapine (n = 11), Clozapine (n = 4), Quetiapine (n = 2), Ziprasidone (n = 1), Risperidone (n = 5), Aripiprazole (n = 2), Paliperidone (n = 3; cp. Table S2 for individual medication protocols and dosage; see Table S1 for mean chlorpromazine equivalent dose). All healthy subjects were free of psychotropic medication and any neurological and psychiatric disorder, current and in history. participants had to be excluded. Two-sample t-tests yielded no significant differences between groups regarding mean point-to- point translation or rotation of any direction (p > 0.18), RMS (p > 0.25), or tSNR (p > 0.35). Subcortical-Cerebellar Systems: ROIs and Preprocessing For each subcortical-cerebellar system, representative ROIs were defined based on previous studies, which investigated systems’ cortical iFC (Kahn et al., 2008; Etkin et al., 2009; Krienen and Buckner, 2009; Peters et al., in press; Figure 2). For the amygdala, left and right basolateral amygdala ROI were derived from the Anatomy toolbox for SPM1 following Etkin et al. (2009) and converted to corresponding ROIs via Marsbar2. For other subcortical-cerebellar systems, center coordinates in striatum, cerebellum, and MTL, respectively, were derived from the literature and spherical ROIs (6 mm radius) were created via Marsbar: left and right ventral and dorsal striatum following Peters et al. (in press) with MNI coordinates [XYZ]: (±12, 9, −9) and (±24, 12, 0), left and right Crus 1 and 2 following Krienen and Buckner (2009) with MNI coordinates [XYZ]: (±12.0, −80.0, −24.0) and (±22.0, −86.0, −40.0), left and right hippocampus and parahippocampus ROIs following Kahn et al. (2008) with MNI coordinates [XYZ]: (±24.0, −18.0, −18.0) and (±26.0, −40.0, −12.0). Frontiers in Human Neuroscience \| www.frontiersin.org RESULTS To generate individual feature vectors as input parameter for pattern classification via SVM, respective Pearson-correlations of NWs’ and ROIs’ time courses and subsequent Fisher’s r-to-z transformation were performed for each subject (allROIs-NWs) and represented as typical correlation matrices. Correlation matrices for NWs-NWs and allROIs- allROIs connectivity were analogously created. Besides a global analysis including all ROIs and NWs as input for classification, also separateROIs-NWs correlation matrices were analyzed, including only one of the four subcortical- cerebellar system-ROIs at a time (i.e., cerebellum, amygdala, striatum, or MTL), thus facilitating comparison of classification accuracy obtained by each of the subcortical-cerebellar systems’ component. Typical Cortical Intrinsic Networks have been Identiﬁed in Patients and Controls ICA analysis of rs-fMRI data from patients and controls identified 22 intrinsic networks, which cover mainly the cerebral cortex (Figure 1; for more details, Figures S2–6). Networks were consistent across groups, and matched with previously defined networks of an identical methodological approach in 603 healthy controls (Allen et al., 2011). Typical Cortical Intrinsic Networks have been Identiﬁed in Patients and Controls ICA analysis of rs-fMRI data from patients and controls identified 22 intrinsic networks, which cover mainly the cerebral cortex (Figure 1; for more details, Figures S2–6). Networks were consistent across groups, and matched with previously defined networks of an identical methodological approach in 603 healthy controls (Allen et al., 2011). 4http://icatb.sourceforge.net 5http://www.cs.waikato.ac.nz/ml/weka/index.html Cortico-Thalamic Networks: NW Identiﬁcation and Selection Further reasons to follow Allen’s approach were both greater comparability of results across studies and most importantly, reduction of subjective bias for network selection by using Allen’s templates to identify networks-of-interest. Preprocessed data were decomposed into 75 spatially independent components within a group-ICA framework based on the infomax-algorithm as implemented in the GIFT-software4. FMRI data were concatenated and reduced by two-step principal component analysis, followed by independent component estimation with the infomax-algorithm. We subsequently ran 40 ICA (ICASSO) to ensure stability of the estimated components. This resulted in a set of average group components, which are then back- reconstructed into single subject space. Each back-reconstructed component consists of a spatial z-map reflecting the component’s functional connectivity pattern across space and an associated time course reflecting component’s activity across time. To select ICs which covered mainly the cortico-thalamic system, we chose templates as provided by Allen et al. (2011), i.e., t-maps of 28 components that reflect canonical intrinsic networks. We chose components mainly covering the cerebral cortex (22 of 28 maps, see Supplementary Material) to reflect networks-of-interest in an automated and objective way, and performed multiple spatial regression analyses of our 75 independent components’ spatial maps on these templates. Components of highest correlation coefficient with the templates were selected, resulting in 22 ICs of interest. In the end, this approach yielded a component’s z-map and time course for each subject and cortical intrinsic network, which reflect network’s coherent activity. Time courses of NW were used as proxies for cortico-thalamic system activity for further analysis. Cortico-Thalamic Networks: NW Identiﬁcation and Selection NWs were identified by ICA of pre-processed fMRI data within an identical framework as defined by Allen et al. (2011) and as applied previously (Manoliu et al., 2014). Briefly, selection of the optimal ICA model-order to analyze rs-fMRI data is subject of ongoing debate. However, it has been demonstrated that a model-order around 70 components may represent an optimal level to detect between-group differences and to avoid false positive results. Therefore, we followed the approach of Frontiers in Human Neuroscience \| www.frontiersin.org Frontiers in Human Neuroscience \| www.frontiersin.org February 2016 \| Volume 10 \| Article 55 5 Subcortical-Cerebellar System Connectivity in Schizophrenia Peters et al. both classes (i.e., groups). Among all possible hyperplanes, that one with the maximum margin between classes is selected and subsequently applied to classify test instances. In order to evaluate classification effectiveness, SVM procedure has been realized within a leave-one-out validation framework, in which in each round of classification one subject is used as test instance while the others represent training instances. The specific algorithm implemented in WEKA applies sequential minimal optimization and a linear kernel (Platt, 1999). Measures-of-interest for SVM-based classification were classification accuracy, sensitivity and specificity, which together reflect the power of the particular feature (i.e., NWs- NWs, allROIs-allROIs, allROIs-NWs, and separateROIs-NMs) to discriminate groups. These measures reflect how consistent patients/controls are correctly classified due to the corresponding iFC pattern. We intended to examine different subcortical- cerebellar systems for such consistency of connectivity pattern changes. In addition, statistical significance of differences in classification measures was determined by reading out the individual classification results (i.e., correct or misclassified) and pairwise identification of diverging classification accuracy for pairs of features between NWs-NWs, allROIs-allROIs, allROIs- NWs, and separateROIs-NWs (Japkowicz and Shah, 2011). In more detail, let (X1,Y1), (X2,Y2), . . ., (X35,Y35) indicate the individual classification results, where Xi = 0 or Yi = 0 (misclassified), Xi = 1 or Yi = 1 (correct). We counted the number of pairs Xi > Yi. Let W be the number of pairs (Xi > Yi), N is the number of pairs that Xi and Yi are different, then W follows a binomial distribution W ∼B(N, 0.5). The exact p-value was obtained by checking binomial distribution. Allen et al. (2011), who used ICA model order of 75 components. Striatum-NWs and Amygdala-NWs h d d l f h l h Striatum-NWs and Amygdala-NWs When separating individual patients from healthy controls via SVM for different subcortical-cerebellar systems separately, classification accuracies were significantly higher for iFC patterns of cerebellum-NWs (91%, sensitivity 82%, specificity 100%) and MTL-NWs (85%, sensitivity 77%, specificity 94%) than for striatum-NWs (65%, sensitivity 65%, specificity 65%) and amygdala-NW (68%, sensitivity 71%, specificity 65%; Table 3 and S3, Figure 3). Superior Classiﬁcation Accuracy for allROIs-NWs than allROIs-allROIs or NWs-NWs The resulting correlation matrices of the two groups (feature vectors) were used for pattern recognition based classification via the WEKA toolbox5. Specifically, a support-vector machine based algorithm (SVM) was applied together with leave-one-out cross validation. The basic idea of SVM procedures (Vapnik, 1999) is to construct a separating hyperplane between multi- dimensional training instances (i.e., correlation matrices) of When separating individual patients from healthy controls via SVM classification accuracy for iFC patterns between all subcortical-cerebellar systems’ ROIs and cortical NWs (allROIs-NWs) was 91% with high sensitivity and specificity levels of about 90% (Table 2). Classification accuracies for iFC patterns among subcortical-cerebellar systems allROIs-allROIs and cortical networks NWs-NWs, were significantly lower with 56 and 73%, respectively (Tables 2, 4). February 2016 \| Volume 10 \| Article 55 Frontiers in Human Neuroscience \| www.frontiersin.org 6 Subcortical-Cerebellar System Connectivity in Schizophrenia Peters et al. TABLE 3 \| Classiﬁcation results based on intrinsic functional connectivity (iFC) between single subcortical-cerebellar systems and cortical networks. Connectivity matrix Classiﬁcation Sensitivity Speciﬁcity accuracy [%] Cerebellum-NWs 91.3 0.82 1.00 MTL-NWs 85.3 0.77 0.94 Striatum-NWs 64.7 0.65 0.65 Amygdala-NWs 67.6 0.71 0.65 Classiﬁcation results based on support vector machine algorithm and leave-one- out cross validation. Input to the classiﬁer were connectivity matrices based on Pearson correlations between time courses of region-of-interest (ROI) of subcortical-cerebellar systems and cortical intrinsic networks of Figures S2–6. TABLE 3 \| Classiﬁcation results based on intrinsic functional connectivity (iFC) between single subcortical-cerebellar systems and cortical networks. TABLE 2 \| Classiﬁcation results based on intrinsic functional connectivity (iFC) among subcortical-cerebellar systems and cortical networks, respectively. Connectivity matrix Classiﬁcation Sensitivity Speciﬁcity accuracy [%] allROI-allROI 55.9 0.59 0.53 NW-NW 73.5 0.71 0.77 allROI-NW 91.2 0.88 0.94 Classiﬁcation results based on support vector machine algorithm and leave-one- out cross validation. Input to the classiﬁer were connectivity matrices based on Pearson correlations among time courses of region-of-interest (ROI) of subcortical- cerebellar systems and cortical intrinsic networks (NW) of Figures S2–6. TABLE 2 \| Classiﬁcation results based on intrinsic functional connectivity (iFC) among subcortical-cerebellar systems and cortical networks, respectively. Classiﬁcation results based on support vector machine algorithm and leave-one- out cross validation. Input to the classiﬁer were connectivity matrices based on Pearson correlations among time courses of region-of-interest (ROI) of subcortical- cerebellar systems and cortical intrinsic networks (NW) of Figures S2–6. Superior Classiﬁcation Accuracy for MTL-NWs and Cerebellum-NWs than networks (NWs) in patients with schizophrenia. (i) Taking all subcortical-cerebellar systems of interest together, i.e., MTL, cerebellum, striatum, and amygdala (allROIs-NWs), iFC separated patients from controls with an accuracy of about 90%, which was significantly superior in comparison to allROIs-allROIs and NWs-NWs connectivity. This result highlights the role of iFC between subcortical- cerebellar systems and cortical networks in contrast to both cortico-cortical and subcortical-cerebellar-subcortical- cerebellar systems’ connectivity in schizophrenia. (ii) Across subcortical-cerebellar systems, MTL-NWs and cerebellum- NWs connectivity showed highest classification accuracies of about 90%, which were significantly higher in comparison to iFC patterns of striatum-NWs and amygdala-NWs with accuracies of about 65%. This finding provides first evidence for differential across-system consistency of changes networks (NWs) in patients with schizophrenia. (i) Taking all subcortical-cerebellar systems of interest together, i.e., MTL, cerebellum, striatum, and amygdala (allROIs-NWs), iFC separated patients from controls with an accuracy of about 90%, which was significantly superior in comparison to allROIs-allROIs and NWs-NWs connectivity. This result highlights the role of iFC between subcortical- cerebellar systems and cortical networks in contrast to both cortico-cortical and subcortical-cerebellar-subcortical- cerebellar systems’ connectivity in schizophrenia. (ii) Across subcortical-cerebellar systems, MTL-NWs and cerebellum- NWs connectivity showed highest classification accuracies of about 90%, which were significantly higher in comparison to iFC patterns of striatum-NWs and amygdala-NWs with accuracies of about 65%. This finding provides first evidence for differential across-system consistency of changes Striatum-NWs and Amygdala-NWs iFC Between Subcortical-Cerebellar Systems and Cortical Networks is more Consistently Changed than iFC within Modulatory Systems and Cortical Networks, Respectively In line with the studies from Collin et al. (2011) and Duan et al. (2015), which suggest longer lasting MTL- and cerebellum-iFC changes, we found highly consistent changes of both MTL- and cerebellum-cortex iFC in patients. This finding suggests that aberrant cortex-cerebellum iFC and cortex-MTL iFC together are stable features in schizophrenia. iFC Between Subcortical-Cerebellar Systems and Cortical Networks is more Consistently Changed than iFC within Modulatory Systems and Cortical Networks, Respectively Networks, Respectively Changes in iFC patterns between all subcortical-cerebellar and cortical networks are highly consistent in psychotic patients with schizophrenia indicated by classification accuracy, sensitivity, and specificity of about 90% (Table 2). Interestingly, such iFC changes are more consistent than those among only subcortical-cerebellar (allROIs-allROIs) or only cortical networks (NWs-NWs), respectively, highlighting the prominent role of subcortical-cerebellar system connectivity into the cortico-thalamic system in schizophrenia (Tables 2, 5). This result is in line with several previous studies, which demonstrated separately impaired iFC between a selected subcortical-cerebellar system and cortical regions (Collin et al., 2011; Anticevic et al., 2012; Fornito et al., 2013; Kraguljac et al., 2014; Wagner et al., 2015; Peters et al., in press). For instance, in a study by Wagner et al. (2015) an abnormal effective connectivity was observed between thalamus, anterior cingulate and dorsolateral prefrontal cortex in patients with schizophrenia compared to healthy controls. Moreover, patients have been found to show a decreased functional connectivity between the putamen with right anterior insula and dorsal prefrontal cortex and the ventral striatum with left anterior insula compared to healthy control subjects (Peters et al., in press). The present result goes beyond these studies by demonstrating that the whole pattern of iFC between several subcortical-cerebellar systems and cortical systems is more consistently aberrant than iFC among subcortical-cerebellar systems or among cortical systems alone. TABLE 5 \| Statistical evaluation of differences in classiﬁcation accuracy for different connectivity matrices focused on single subcortical-cerebellar systems. CB MTL AY ST CB – 0.2188 0.0097∗ 0.0095∗ MTL – 0.0537 0.0269∗ AY – 0.2256 Distinct connectivity matrices based on Pearson correlations among time courses of region-of-interest of subcortical-cerebellar systems and cortical intrinsic networks of Figures S2–6 have been classiﬁed by support vector machine with distinct classiﬁcation accuracy results (see Table 3). Classiﬁcation accuracies were compared among each other via statistical testing (pairwise binominal testing, details in methods). CB-network and MTL-network connectivity yielded signiﬁcantly highest classiﬁcation accuracy (∗indicates signiﬁcance at threshold p = 0.05, ∗∗p = 0.01). Abbreviations: CB, cerebellum; MTL, media temporal lobe; AY, amygdala; ST, striatum. TABLE 5 \| Statistical evaluation of differences in classiﬁcation accuracy for different connectivity matrices focused on single subcortical-cerebellar systems. consistently decreased cerebellar iFC into widespread parts of the cortex, in patients and their unaffected siblings, suggesting impaired cerebellum iFC as putative risk endophenotype for schizophrenia and therefore, as stable feature in the disease. DISCUSSION The current study focused on iFC between different subcortical-cerebellar systems (ROIs) and cortical intrinsic FIGURE 3 \| ROI-network connectivity. Connectivity matrices between cortical networks (NWs, cp. Figure 1) and different ROI, i.e., cerebellum, medial temporal lobe, amygdala and striatum (cp. Figure 2), served as input features for support-vector-machine-based classiﬁcation of schizophrenia patients and healthy subjects. Displayed classiﬁcation accuracies [%] indicate differential discriminatory power of separate ROI-NWs-systems and the degree of inter-subject consistency of ROI-speciﬁc connectivity pattern changes in schizophrenia. FIGURE 3 \| ROI-network connectivity. Connectivity matrices between cortical networks (NWs, cp. Figure 1) and different ROI, i.e., cerebellum, medial temporal lobe, amygdala and striatum (cp. Figure 2), served as input features for support-vector-machine-based classiﬁcation of schizophrenia patients and healthy subjects. Displayed classiﬁcation accuracies [%] indicate differential discriminatory power of separate ROI-NWs-systems and the degree of inter-subject consistency of ROI-speciﬁc connectivity pattern changes in schizophrenia. February 2016 \| Volume 10 \| Article 55 7 Frontiers in Human Neuroscience \| www.frontiersin.org Peters et al. Subcortical-Cerebellar System Connectivity in Schizophrenia TABLE 4 \| Statistical evaluation of differences in classiﬁcation accuracy for different connectivity matrices. allROI-NW NW-NW allROI-allROI allROI-NW – 0.0439∗ 0.0002∗∗ NW-NW – 0.0439∗ Distinct connectivity matrices based on Pearson correlations among time courses of regions-of-interest (ROI) of subcortical-cerebellar systems (Figure 1) and cortical intrinsic networks (NW) of Figures S2–6 have been classiﬁed by support vector machine with distinct classiﬁcation accuracy results (see Table 2). Classiﬁcation accuracies were compared among each other via statistical testing (pairwise binominal testing, details in methods). allROI-NW connectivity yielded signiﬁcantly highest classiﬁcation accuracy (∗indicates signiﬁcance at threshold p = 0.05, ∗∗p = 0.01). Abbreviations: ROI, Regions of interest; NW, networks. TABLE 4 \| Statistical evaluation of differences in classiﬁcation accuracy for different connectivity matrices. in intrinsic connectivity between different subcortical- cerebellar systems and the cortico-thalamic system. Data suggest that the whole pattern of cortical-subcortical- cerebellar system dysconnectivity might be differential for patients, maybe indicating different patient subgroups or disease states. Frontiers in Human Neuroscience \| www.frontiersin.org Across Subcortical-Cerebellar Systems, iFC Changes with Cortical Networks are more Consistent for MTLs and Cerebellum than Striatum and Amygdala In contrast, iFC between striatum and amygdala, respectively, and cortical networks separated patients from controls with significantly lower accuracy levels of about 65% (Table 3 and S3, Figure 3). Aberrant amygdala and striatum iFC with the cortex is in line with several previous studies. Anticevic et al. (2012) showed decreased iFC between amygdala and the orbitofrontal cortex, which was only present in patients of early and chronic disease stages but not in unaffected high-risk persons. This finding suggests that iFC between cortex and amygdala is modulated by disease state. Concerning striatum, previous studies demonstrated a complex pattern of in- and decreased iFC with the cortex along the ventromedial to dorsolateral axis of the striatum; in particular, Intrinsic connectivity changes between cerebellum and MTL respectively, and cortical networks were highly consistent in patients with accuracies of about 90% (Table 3, Figure 3). This result is well in line with previous findings reporting aberrant seed-based iFC of hippocampus and cerebellum, respectively, with cortical areas (Collin et al., 2011; Kraguljac et al., 2014). In a longitudinal study over 2 years in more than 60 patients with schizophrenia, Duan et al. (2015) demonstrated lasting iFC decrease between hippocampus and both, temporal and parietal areas, suggesting aberrant hippocampus-cortex iFC as a permanent feature in schizophrenia. Collin et al. (2011) showed Frontiers in Human Neuroscience \| www.frontiersin.org February 2016 \| Volume 10 \| Article 55 Frontiers in Human Neuroscience \| www.frontiersin.org 8 Subcortical-Cerebellar System Connectivity in Schizophrenia Peters et al. with schizophrenia (Sambataro et al., 2010). Therefore, we cannot exclude that observed changes in iFC in comparison to healthy controls are confounded by medication effects. However, one should note that the main focus of the study was differential iFC changes across subcortical-cerebellar systems in patients i.e., differences in changes of patients instead of differences in patients relative to controls, suggesting that—due to consistent confound across patients—findings might be more robust against medication influences. Nevertheless, studies in non-medicated patients would be favorable. (ii) Sample size of the study is small, limiting the power of our study results to some degree. Therefore and due to confounding medication effects, we have to categorize our study as a preliminary study about differential iFC changes across subcortical-cerebellar systems in schizophrenia. (iii) One might ask why we used SVM instead of canonical two-sample t-tests to compare iFC across subjects. AUTHOR CONTRIBUTIONS CS and HP designed the study; JB, MS, DS and HF recruited participants and acquired data; VR, CZ and AW acquired data; JS and HP analyzed data; CS, KK, VR, AW and HP interpreted data; CS, KK and HP drafted the article; all authors critically revised and approved the final version of the article. CONCLUSION Results provide preliminary evidence for more consistent iFC changes with cortical networks for MTL and cerebellum than for striatum and amygdala in schizophrenia. Differential iFC changes might reflect distinct disease states or patient subgroups. Differential Patterns of Aberrant iFC Between Subcortical-Cerebellar Systems and Cortical Networks as Candidates to Separate Sub-Groups of Patients and/or Disease States Widespread brain dysconnectivity is central to schizophrenia (Stephan et al., 2009). Several models proposed specific patterns of dysconnectivity being associated with specific syndromes, symptoms, states, and subgroups of patients in the disease (Aleman and Kahn, 2005; Andreasen and Pierson, 2008; Stephan et al., 2009; Tamminga et al., 2010; Williamson and Allman, 2012). Due to both their essential role in modulating the cortico-thalamic system (Buzsaki, 2006) and the overwhelming evidence of their multi-level changes in schizophrenia (Andreasen and Pierson, 2008; Benes, 2010; Tamminga et al., 2010; Howes et al., 2012), striatum, cerebellum, MTL, and amygdala are promising candidates to specify schizophrenia’s dysconnectivity. The current study provides initial evidence that these main modulatory systems of the cortico-thalamic system and their iFC with cortical networks might specify distinct dysconnectivity patterns across patients despite limitations such as medication effects in particular as laid out further below. Future studies are necessary to identify how patterns of subcortical-cerebellar systems’ dysconnectivity vary across disease states, unaffected at-risk persons, and patients, and whether specific patterns may define patient subgroups. Across Subcortical-Cerebellar Systems, iFC Changes with Cortical Networks are more Consistent for MTLs and Cerebellum than Striatum and Amygdala Typical two-sample t-tests for seed-based iFC maps would provide information about spatially specific group differences in cortical iFC for distinct subcortical-cerebellar systems; but such group difference maps are difficult to compare across subjects due to both, varying spatial extent of cortical changes and most importantly, the lack of direct measures about how consistent iFC changes are across systems and subjects. Combining measures of classification and their statistical comparison via binominal testing yields exactly such information. (iv) Why did we select intrinsic networks as proxies for cortico-thalamic sub-systems instead of brain-atlas-based anatomically defined regions? Concerning brain activity, we focused on slowly fluctuating activity as measured by rs-fMRI and correspondingly on iFC of such ongoing activity. Overwhelming evidence indicates that iFC is organized by intrinsic networks particularly in the human cortex and exceeds anatomically defined cortex parcellations (Fox and Raichle, 2007). this pattern of change seems to be modulated by the disease state and to be intimately linked with psychotic symptoms (Fornito et al., 2013; Manoliu et al., 2013; Peters et al., in press). However, psychotic symptoms seem to be not necessarily linked with pathophysiological striatum changes. For example, Demjaha et al. (2012) demonstrated in psychotic non-responders of anti-dopaminergic treatment, that presynaptic dopamine synthesis levels are unchanged in comparison to healthy controls, suggesting striatal dopamine synthesis being independent from psychosis. In line with the studies from Anticevic and Demjaha, which suggest inconsistent amygdala and striatum changes in patients with schizophrenia, we found that changes of both amygdala- and striatum-cortex iFC were of moderate accuracy and sensitivity, and of significant lower accuracy than iFC changes of cerebellum and MTL. Considering that cerebellum and MTL iFC changes appear as rather permanent features in schizophrenia, this finding suggests that aberrant cortex-amygdala iFC and cortex-striatum iFC are less consistent in schizophrenia and may reflect different disease states or heterogeneity across patients. Differential Patterns of Aberrant iFC Between Subcortical-Cerebellar Systems and Cortical Networks as Candidates to Separate Sub-Groups of Patients and/or Disease States REFERENCES risk phenotype for psychosis. JAMA Psychiatry 70, 1143–1151. doi: 10. 1001/jamapsychiatry.2013.1976 risk phenotype for psychosis. 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Antipsychotic medication has been demonstrated to alter functional connectivity in patients This work was supported by the German Federal Ministry of Education and Research (BMBF 01EV0710 to AW) and the Kommission für Klinische Forschung of the Klinikum Rechts February 2016 \| Volume 10 \| Article 55 Frontiers in Human Neuroscience \| www.frontiersin.org 9 Peters et al. Subcortical-Cerebellar System Connectivity in Schizophrenia SUPPLEMENTARY MATERIAL der Isar der Technischen Universität München (KKF 8765162 to CS). This work was partially supported by the National Natural Science Foundation of China (Grant Nos. 61403062 and 61433014 to JS) and Fundamental Research Funds for the Central Universities (Grant No. ZYGX2014J053 to JS). The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fnhum. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). REFERENCES The use, distribution and 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. Spitzer, R. L., Williams, J. B., Gibbon, M., and First, M. B. (1992). The Structured Clinical Interview for DSM-III-R (SCID). I: history, rationale and description. Arch. Gen. Psychiatry 49, 624–629. doi: 10.1001/archpsyc.1992. 01820080032005 Stephan, K. E., Friston, K. J., and Frith, C. D. (2009). Dysconnection in schizophrenia: from abnormal synaptic plasticity to failures of self-monitoring. Schizophr. Bull. 35, 509–527. doi: 10.1093/schbul/sbn176 February 2016 \| Volume 10 \| Article 55 Frontiers in Human Neuroscience \| www.frontiersin.org 11
https://openalex.org/W4392653245	https://www.nature.com/articles/s41598-024-56669-z.pdf	English	null	Increasing silage maize yield and nitrogen use efficiency as a result of combined rabbit manure and mineral nitrogen fertilization	Scientific reports	2,024	cc-by	13,219	Andrzej Wysokinski * & Monika Kożuchowska Combined application of organic and mineral fertilizers is crucial to obtaining high crop yields, increasing the utilization of nutrients by plants, and limiting their dispersion, thus protecting the environment, which underscores the importance of sustainable and minimally invasive agriculture. The aim of the field experiment was to determine the effect of application of rabbit manure (RM) and mineral nitrogen (Nmin) on the dry matter (DM) yield of maize and on nitrogen content, uptake, and use efficiency (NUE). RM application was tested at levels of 0, 20, 40 and 60 t·ha−1, and Nmin application at 0, 50, 100 and 150 kg·ha−1. Significant differences were noted in yield and in the content and uptake of nitrogen depending on both experimental factors. Increasing the application of RM and Nmin led to an increase in the yield of harvested maize and in the content and uptake of nitrogen. In terms of DM yield and nitrogen uptake (yield of crude protein), the most beneficial fertilizer variant was 60 t·ha−1 RM applied together with 100 kg·ha−1 Nmin. The highest NUE value was obtained following application of 20 t·ha−1 RM together with 150 kg·ha−1 Nmin. To meet the high and continually growing demand for food, agriculture is required to continually increase its production power. High crop yields can be obtained by providing them with access to a large amount of nutrients. This has led to the appearance of areas that have been excessively fertilized with mineral fertilizers, resulting in environmental pollution from agricultural sources. Therefore, there is a need for more efficient plant utilization of fertilizer nutrients in order to protect the environment and at the same time ensure food security1,2. The 4R principle should be applied: (1) the right nutrient source, (2) the right application rate, (3) the right application time, and (4) the right place of application. This will ensure adequate utilization of soil-applied nutrients by l optimizing productivity.h p g p y The use of organic fertilizers for crops reduces the amount of nutrients introduced to the soil with inorganic fertilizers. Closing the nutrient cycle on a farm is important for protection of the natural environment and also can reduce fertilizer costs. Manure contains macro and micronutrients for plants, and land application in crop production recycles those manure nutrients. www.nature.com/scientificreports www.nature.com/scientificreports University of Siedlce, Faculty of Agricultural Sciences, Konarskiego 2 Str., 08110 Siedlce, Poland. email: andrzej. wysokinski@uws.edu.pl OPEN Andrzej Wysokinski & Monika Kożuchowska Scientific Reports \| (2024) 14:5856 Experimental designhi The field experiment was carried out in the years 2018–2019 at the Experimental Agricultural Station of the University of Siedlce (GPS, N: 52° 03′ 42.01″, E:22° 33′ 09.01″, 168 m above sea level, eastern Poland). Before set- ting up the experiment, 10 soil cores were taken from a 24 × 60 m area, from a 0.30 m layer, from which one soil sample was made. The soil consisted of the following fractions: 2.0–0.05 mm = 72.45%; 0.05–0.02 mm = 15.86%; 0.02–0.002 mm = 10.11%; < 0.002 mm = 1.56%. The soil pH measured in KCl 1 mol·dm−1 was 5.5. The content of total nitrogen (Ntot) and organic carbon (Corg) amounted 0.79 and 8.40 g·kg−1, respectively. The content of plant available phosphorus (Pav), potassium (Kav) and magnesium (Mgav) were 66, 87 and 53 mg·kg−1 of soil, respec- tively. Rainfall and air temperature recordings were obtained from the University’s own weather station (Table 1).hhi The experiment was two-factorial, set up in a randomized block design in triplicate. The first factor was varied application rates of rabbit manure (RM): 0, 20, 40 and 60 t·ha−1. The second factor was varied application rates of mineral nitrogen (Nmin): 0, 50, 100 and 150 kg·ha−1. The area of each plot was 9 m2 (3 × 3 m). Supplementary fer- tilization with phosphorus and potassium was used in the treatments to obtain a nitrogen:phosphorus:potassium ratio of no less than 1:0.35:1. In the treatment without nitrogen application, phosphorus and potassium were applied in amounts corresponding to the average application rate in all fertilizer treatments. Mineral nitrogen was applied in the form of ammonium nitrate (34.0% N), phosphorus in the form of triple superphosphate (19.4% P), and potassium in the form of potassium chloride (49.8% K). All fertilizers were incorporated to the soil once in the first year (2018), prior to the experiment, 10 days before sowing the test plant. The effect on the succeeding crop was examined the following year. No fertilizer was applied for crop in 2019, and the experiment took place on the exact same plot and plot layout in both years. In both growing seasons the test plant was the ‘Tapas’ cultivar of maize (Zea mays L.), harvested for silage. www.nature.com/scientificreports/ Rabbit manure can be used directly to fertilize crops10,12,13, as an ingredient in plant growth substrates14,15, as an adsorbent16, for biochar production17,18, and even in anaerobic fermentation to produce methane19.h Organic fertilizers are known to improve the physical, chemical, and biological properties of soils. These fertilizers, including rabbit manure, increase the content of organic matter15, pH, cation exchange capacity20, organic carbon content in soil, total fungi, and bacteria21. The use of manure as fertilizer increases the yield and quality of crops, e.g. the content of protein and minerals15,21,22.if Rabbit manure used in combination with chemical fertilizers has a particularly beneficial effect on crop yield and quality10,22,23. Combined application of nitrogen from RM (organic) and from mineral/inorganic nitrogen (from chemical fertilizers) increases the efficiency of nitrogen utilization from these fertilizers. In a pot experi- ment, combined application of rabbit manure and mineral nitrogen compared to the only nitrogen mineral treatment was shown to reduce the percentage of nitrogen lost from sandy and clayey soils while increasing the amount of mineral nitrogen and nitrogen present in the form of microbial biomass24. According to Wu and coauthors24, the data cannot be extrapolated to field conditions, and future research should verify the results in field conditions. i In Poland, in the years preceding the experiment, the production of silage maize systematically increased: 2010 year—395,000 ha; 2013 year—462,000 ha; 2015 year—555,000 ha; 2017 year—596,000 ha6. Including manures in the fertilization of silage maize can have a major impact on the success of its cultivation, affecting not only the yield, but also reducing the costs of cultivation.hif y y g The aim of the field experiment was to determine the effect of rabbit manure and supplementary application of nitrogen on the yield of silage maize, the content and uptake of nitrogen, and nitrogen use efficiency by this plant. Andrzej Wysokinski * & Monika Kożuchowska The use of nutrients in animal manure can to a large extent (for example 37.3% N, 87.6% P2O5 and 65.9% K2O in China) replace chemical fertilizers3. p Some countries (France, Spain, China, and Argentina) have large rabbit (Oryctolagus cuniculus) breeding industries4,5. In some parts of Poland the development of large-scale rabbit farming is observed as well. Accord- ing to the Statistical Yearbook of Agriculture6, between 2010 and 2020 the number of rabbits sent to industrial slaughter in Poland increased from 290,000 to 370,000. One fattening unit rabbit produces on average 10–12 kg of manure in one cycle (about 150 g per day as the sum of faeces and urine). The population of 370,000 slaughter rabbits will therefore produce approximately 4000 tons of manure, which must be managed in an environmen- tally-safe manner. Rabbit manure is a fertilizer rich in organic matter and nutrients for plants and has a low or medium C/N ratio7–10. The average efficiency of ingested nitrogen in rabbit fattening is about 40% and drops sharply with age11. Thus about 60% of ingested nitrogen ends up in faeces. Calvet et al.11 report that urine and faeces contributed to overall nitrogen excretion in approximately the same proportions. \| https://doi.org/10.1038/s41598-024-56669-z Scientific Reports \| (2024) 14:5856 www.nature.com/scientificreports/ Crop samplings, measurements and calculations Maize plants were harvested from entire plots and then weighed. Four representative plants were collected from each plot for laboratory analyses. They were first chopped into pieces 1–2 cm in length and dried to obtain a constant mass at 105 °C for determination of dry matter (DM), and then they were ground in laboratory mills. Nitrogen content was measured by a standard Kjeldahl method following wet mineralization in concentrated H2SO4 26. Nitrogen uptake by maize (Nup): Nup = Y · Nc, where Y is the dry mass of maize, Nc is the total nitrogen content (concentration) in maize’s dry mass. Nitrogen use efficiency from total nitrogen doses (NUEtot): NUEtot = Nup_N −Nup_N0 /Napplied × 100%, where Nup_N is the nitrogen uptake by maize fertilized with nitrogen, Nup_N0 is the nitrogen uptake by maize nonfertilized with nitrogen, Napplied is the total amount of nitrogen applied into soil with RM and Nmin. Nitrogen use efficiency from total nitrogen doses as the sum from 2 years (NUEtot_2): where Nup_N is the nitrogen uptake by maize fertilized with nitrogen, Nup_N0 is the nitrogen uptake by maize nonfertilized with nitrogen, Napplied is the total amount of nitrogen applied into soil with RM and Nmin. Nitrogen use efficiency from total nitrogen doses as the sum from 2 years (NUEtot 2): g applied g pp Nitrogen use efficiency from total nitrogen doses as the sum from 2 years (N NUEtot_2 = Nup_N_2 −Nup_N0_2 /Napplied × 100%, where Nup_N_2 is the sum for 2 years nitrogen uptake by maize fertilized with nitrogen, Nup_N0_2 is the sum for 2 years nitrogen uptake by maize nonfertilized with nitrogen, Napplied is the total amount of nitrogen applied into soil with RM and Nmin. N ffi f l bl d (NUE ) where Nup_N_2 is the sum for 2 years nitrogen uptake by maize fertilized with nitrogen, Nup_N0_2 is the sum for 2 years nitrogen uptake by maize nonfertilized with nitrogen, Napplied is the total amount of nitrogen applied into soil with RM and Nmin.fi Nitrogen use efficiency from available nitrogen doses (NUEav): NUEav = Nup_N −Nup_N0 /Nav × 100%, where Nup_N is the nitrogen uptake by maize fertilized with different doses of nitrogen, (in 1st, in 2nd, and as sum for 2 years, respectively), Nup_N0 is the nitrogen uptake by maize nonfertilized with nitrogen. Experimental designhi Seeds were sown in the last 10 days of April at the rate of 100,000 per hectare, and the above-ground parts were harvested at an average height of 20 cm in the first 10 days of September. Winter triticale (Triticosecale Wittm. ex A. Camus) was grown in the field as a forecrop for maize. Its grain and straw were harvested in the summer of 2017. Rabbit manure contained 28.4% dry mat- ter, total carbon 370 g·kg−1 DM, total nitrogen 20.6 g·kg−1 DM, carbon/nitrogen ratio 18.0, N-NH4 + 4.22 g·kg−1 Table 1. Rainfall and air temperatures in 2018 and 2019 (weather station of the University in Siedlce). Month Average monthly temperatures in experimental years, °C Total monthly rainfall in experimental years, mm 2018 2019 2018 2019 April 13.1 9.8 34.5 5.9 May 17.0 13.3 27.3 59.8 June 18.3 17.9 31.5 35.9 July, VII 20.4 18.5 22.4 29.7 August 20.6 19.9 24.5 43.9 September 15.9 14.2 27.4 17.4 Average/sum April–September 17.6 15.6 167.6 192.6 Average/sum May–August 19.1 17.4 105.7 169.3 Table 1. Rainfall and air temperatures in 2018 and 2019 (weather station of the University in Siedlce). https://doi.org/10.1038/s41598-024-56669-z Scientific Reports \| (2024) 14:5856 \| www.nature.com/scientificreports/ DM, N-NO3 − 0.07 g·kg−1 DM, total phosphorus 7.06 gP·kg−1 DM, and total potassium 13.4 gK·kg−1 DM. The total amounts of nitrogen introduced to the soil and the plant available nitrogen in the treatments are shown in Table 2. According to legal regulations in Poland25, the total amount of nitrogen introduced to the soil with manure is converted to nitrogen available for plants (nitrogen which can actually be taken up by the plant). The amount of this form of nitrogen is calculated by multiplying the total amount of nitrogen applied by appropri- ate factors (multipliers), established for the next 2 years after manure application. For rabbit manure applied in spring they are 0.35 in the first year and 0.15 in the second year. Nitrogen from mineral fertilizers is assumed to be available for plants only in the first year after their application. Crop samplings, measurements and calculations (in 1st, in 2nd, and as sum for 2 years, respectively), Nav is the amount of theoretically available pool of nitrogen, (from RM and Nmin in 1st year, only from RM in 2nd year, and as the sum of nitrogen available in 1st and 2nd years, respectively). Yield increase after application of RM (Yi_RM) and Nmin (Yi_Nmin) separately: where Nup_N is the nitrogen uptake by maize fertilized with different doses of nitrogen, (in 1st, in 2nd, and as sum for 2 years, respectively), Nup_N0 is the nitrogen uptake by maize nonfertilized with nitrogen. (in 1st, in 2nd, and as sum for 2 years, respectively), Nav is the amount of theoretically available pool of nitrogen, (from RM and Nmin in 1st year, only from RM in 2nd year, and as the sum of nitrogen available in 1st and 2nd years, respectively). Yield increase after application of RM (Yi_RM) and Nmin (Yi_Nmin) separately: Yi_RM = YRM −Y0, Yi_Nmin = YNmin −Y0, Table 2. Amounts of total nitrogen (Ntot) and nitrogen theoretically available for plants (Nav) in experimental treatments, kg·ha−1. Treatments Nitrogen forms Nmin kg·ha−1 0 50 100 150 RM (t·ha−1) 0 Ntot 0 50 100 150 Nav in 2018 0 50 100 150 Nav in 2019 0 0 0 0 20 Ntot 117 167 217 267 Nav in 2018 41 91 141 191 Nav in 2019 17 17 17 17 40 Ntot 233 283 333 383 Nav in 2018 82 132 182 232 Nav in 2019 35 35 35 35 60 Ntot 350 400 450 500 Nav in 2018 123 173 223 273 Nav in 2019 53 53 53 53 3 4:5856 \| https://doi.org/10.1038/s41598-024-56669-z Table 2. Amounts of total nitrogen (Ntot) and nitrogen theoretically available for plants (Nav) in experimental treatments, kg·ha−1. www.nature.com/scientificreports/ where YRM is the yield of maize after only RM application, YNmin is the yield of maize after only Nmin application, Y0 is the yield of maize without fertilization (control object). P t f i ld i ft li ti f RM (Y ) d N (Y ) t l where YRM is the yield of maize after only RM application, YNmin is the yield of maize after only Nmin application, Y0 is the yield of maize without fertilization (control object).t 0 y ( j ) Percent of yield increase after application of RM (Yi_RM) and Nmin (Yi_Nmin) separately: %Yi_RM = Yi_RM/Y0 × 100%, %Yi_RM = Yi_RM/Y0 × 100%, %Yi_RM = Yi_RM/Y0 × 100%, %Yi_Nmin = Yi_Nmin/Y0 × 100%, where Yi_RM is the yield increase of maize after only RM application, Yi_Nmin is the yield increase of maize after only Nmin application, Y0 is the yield of maize without fertilization (control object). Yield increase after combined application of RM and Nmin (interaction effect, Yi_RM_Nmin): where Yi_RM is the yield increase of maize after only RM application, Yi_Nmin is the yield increase of maize after only Nmin application, Y0 is the yield of maize without fertilization (control object). Yi ld i ft bi d li ti f RM d N (i t ti ff t Y ) Yi_RM_Nmin = YRM_Nmin −Y0 −Yi_RM −Yi_Nmin, where YRM_Nmin is the yield of maize after combined application of RM and Nmin, Y0 is the yield of maize without fertilization (control object), Yi_RM is the increase of maize yield after only RM application, Yi_Nmin is the increase of maize yield after only Nmin application.tf where YRM_Nmin is the yield of maize after combined application of RM and Nmin, Y0 is the yield of maize without fertilization (control object), Yi_RM is the increase of maize yield after only RM application, Yi_Nmin is the increase of maize yield after only Nmin application.tf yt y min pp Percent of yield increase after combined application of RM and Nmin (interaction effect, %Yi_RM_Nmin): %Yi_RM_Nmin = Yi_RM_Nmin/ Yi_RM + Yi_Nmin × 100%, %Yi_RM_Nmin = Yi_RM_Nmin/ Yi_RM + Yi_Nmin × 100%, where Yi_RM_Nmin is the yield increase after combined application of RM and Nmin, Yi_RM is the increase of maize ield after only RM application, Yi_Nmin is the increase of maize yield after only Nmin application. Statistical analysish y The results were subjected to analysis of variance (ANOVA) with the Fisher–Snedecor distribution at a signifi- cance level of α = 0.05. Least significant difference (LSD) values at a significance level of α = 0.05 were calculated by the Tukey test. The Statistica 13.1.336.0 PL statistics package (StatSoft Inc., Tulsa, OK, USA) was used for the calculations.h Three-factor analysis of variance for data of yield, nitrogen content, uptake and NUEtot were perform according to the following model: yijlp = m + ai + bj + cl + abij + acil + bcjl + abcijl + eijl, where yijlp is the value of the examined characteristic, m is the population average, ai is the effect of differenti- ated fertilization with RM, bj is the effect of differentiated fertilization with Nmin, cl is the effect of year (Y), abij is the effect of the interaction of RM x Nmin, acil is the effect of the interaction of RM × Y, bcjl is the effect of the interaction of Nmin × Y, abcijl is the effect of the interaction of RM × Nmin × Y, eijl is the random error (numbers). Two-factor analysis of variance for data of total NUEtot as sum for 2 years and NUEav calculated for nitrogen where yijlp is the value of the examined characteristic, m is the population average, ai is the effect of differenti- ated fertilization with RM, bj is the effect of differentiated fertilization with Nmin, cl is the effect of year (Y), abij is the effect of the interaction of RM x Nmin, acil is the effect of the interaction of RM × Y, bcjl is the effect of the interaction of Nmin × Y, abcijl is the effect of the interaction of RM × Nmin × Y, eijl is the random error (numbers). interaction of Nmin × Y, abcijl is the effect of the interaction of RM × Nmin × Y, eijl is the random error (numbers). Statistical analysish Two-factor analysis of variance for data of total NUEtot as sum for 2 years and NUEav calculated for nitrogen theoretically available in subsequent years of study were performed according to the following model: jf j Two-factor analysis of variance for data of total NUEtot as sum for 2 years and NUEav calculated for nitro theoretically available in subsequent years of study were performed according to the following model: yijp = m + ai + bj + abij + eij, where yijp is the value of the examined characteristic, m is the population average, ai is the effect of differentiated fertilization with RM, bj is the effect of differentiated fertilization with Nmin, abij is the effect of the interaction of RM × Nmin, eij is the random error (numbers). where yijp is the value of the examined characteristic, m is the population average, ai is the effect of differentiated fertilization with RM, bj is the effect of differentiated fertilization with Nmin, abij is the effect of the interaction of RM × Nmin, eij is the random error (numbers). j Relationships between selected traits were evaluated by simple correlation analysis (α = 0.05) by the Statistica software. Ethical approvalh pp The authors hereby declare that all methods were carried out in accordance with relevant guidelines. Crop samplings, measurements and calculations Treatments Nitrogen forms Nmin kg·ha−1 0 50 100 150 RM (t·ha−1) 0 Ntot 0 50 100 150 Nav in 2018 0 50 100 150 Nav in 2019 0 0 0 0 20 Ntot 117 167 217 267 Nav in 2018 41 91 141 191 Nav in 2019 17 17 17 17 40 Ntot 233 283 333 383 Nav in 2018 82 132 182 232 Nav in 2019 35 35 35 35 60 Ntot 350 400 450 500 Nav in 2018 123 173 223 273 Nav in 2019 53 53 53 53 Treatments Nitrogen forms Nmin kg·ha−1 0 50 100 150 RM (t·ha−1) 0 Ntot 0 50 100 150 Nav in 2018 0 50 100 150 Nav in 2019 0 0 0 0 20 Ntot 117 167 217 267 Nav in 2018 41 91 141 191 Nav in 2019 17 17 17 17 40 Ntot 233 283 333 383 Nav in 2018 82 132 182 232 Nav in 2019 35 35 35 35 60 Ntot 350 400 450 500 Nav in 2018 123 173 223 273 Nav in 2019 53 53 53 53 Table 2. Amounts of total nitrogen (Ntot) and nitrogen theoretically available for plants (Nav) in experimental treatments, kg·ha−1. https://doi.org/10.1038/s41598-024-56669-z Scientific Reports \| (2024) 14:5856 \| www.nature.com/scientificreports/ Resultsh 1 Yi_RM, effect of using only RM. 2 Yi_Nmin, effect of using only Nmin. 3 %Yi_RM, effect of using only RM. 4 %Yi_Nmin, effect of using only Nmin. 5 Yi_RM_Nmin, effect of using combined application of RM and Nmin (interaction effect). 6 %Yi_RM_Nmin, % effect of using combined application of RM and Nmin (interaction effect). Table 4. Effect of the interaction of RM and Nmin in determining maize yield (DM t·ha−1) and % increase in yield. 1 Yi_RM, effect of using only RM. 2 Yi_Nmin, effect of using only Nmin. 3 %Yi_RM, effect of using only RM. 4 %Yi_Nmin, effect of using only Nmin. 5 Yi_RM_Nmin, effect of using combined application of RM and Nmin (interaction effect). 6 %Yi_RM_Nmin, % effect of using combined application of RM and Nmin (interaction effect). (20.1–30.0%) RM 60 t + Nmin 100 kg; 3 (10.1–20.0%) none; 4 (0.1–10.0%) RM 20 t, 40 t and 60 t, all + Nmin 50 kg, RM 40 t + Nmin 100 kg, RM 20 t and 60 t, both + Nmin 150 kg; 5 (≤ 0.0%) RM 20 t + Nmin 100 kg. The positive effects of the interaction may be due on the one hand to the increase in the mineralization rate of organic fertilizers induced by chemical nitrogen fertilizers, and on the other hand to the reduction in the dissipation of mineral nitrogen in the environment caused by organic fertilizer.hil (20.1–30.0%) RM 60 t + Nmin 100 kg; 3 (10.1–20.0%) none; 4 (0.1–10.0%) RM 20 t, 40 t and 60 t, all + Nmin 50 kg, RM 40 t + Nmin 100 kg, RM 20 t and 60 t, both + Nmin 150 kg; 5 (≤ 0.0%) RM 20 t + Nmin 100 kg. The positive effects of the interaction may be due on the one hand to the increase in the mineralization rate of organic fertilizers induced by chemical nitrogen fertilizers, and on the other hand to the reduction in the dissipation of mineral nitrogen in the environment caused by organic fertilizer.hil The content of nitrogen in the entire dry weight of maize was significantly influenced by the RM application rates, mineral nitrogen application rates, and years of the study (Table 5). Resultsh Maize yield [DM t·ha−1] as a result of different RM and Nmin application rates and years, mean values ± standard deviation (SD). a,b,c,d—means for investigated factors with different lowercase letters are significantly different; A,B,C—means for interactions with different capital letters in the columns are significantly different. Table 3. Maize yield [DM t·ha−1] as a result of different RM and Nmin application rates and years, mean values ± standard deviation (SD). a,b,c,d—means for investigated factors with different lowercase letters are significantly different; A,B,C—means for interactions with different capital letters in the columns are significantly different. Table 3. Maize yield [DM t·ha−1] as a result of different RM and Nmin application rates and years, mean values ± standard deviation (SD). a,b,c,d—means for investigated factors with different lowercase letters are significantly different; A,B,C—means for interactions with different capital letters in the columns are significantly different. Table 4. Effect of the interaction of RM and Nmin in determining maize yield (DM t·ha−1) and % increase in yield. 1 Yi_RM, effect of using only RM. 2 Yi_Nmin, effect of using only Nmin. 3 %Yi_RM, effect of using only RM. 4 %Yi_Nmin, effect of using only Nmin. 5 Yi_RM_Nmin, effect of using combined application of RM and Nmin (interaction effect). 6 %Yi_RM_Nmin, % effect of using combined application of RM and Nmin (interaction effect). Treatments Nmin kg·ha−1 Averages 0 50 100 150 t·ha−1 % t·ha−1 % t·ha−1 % t·ha−1 % t·ha−1 % RM t·ha−1 0 – – 0.92 8.54 1.72 16.04 2.42 22.64 1.72 15.74 20 2.11 19.83 0.15 3.36 − 0.35 − 7.96 0.35 7.96 < 0.15 1.16 40 3.11 29.23 0.45 10.06 0.35 6.36 1.75 30.96 0.85 15.76 60 4.71 44.33 0.15 1.86 1.45 21.96 0.25 2.86 0.65 8.86 Averages 3.31 31.13 0.25 5.06 0.55 6.86 0.75 13.96 0.55 8.56 Treatments Nmin kg·ha−1 Averages 0 50 100 150 t·ha−1 % t·ha−1 % t·ha−1 % t·ha−1 % t·ha−1 % RM t·ha−1 0 – – 0.92 8.54 1.72 16.04 2.42 22.64 1.72 15.74 20 2.11 19.83 0.15 3.36 − 0.35 − 7.96 0.35 7.96 < 0.15 1.16 40 3.11 29.23 0.45 10.06 0.35 6.36 1.75 30.96 0.85 15.76 60 4.71 44.33 0.15 1.86 1.45 21.96 0.25 2.86 0.65 8.86 Averages 3.31 31.13 0.25 5.06 0.55 6.86 0.75 13.96 0.55 8.56 Table 4. Effect of the interaction of RM and Nmin in determining maize yield (DM t·ha−1) and % increase in yield. Resultsh The dry matter yield of silage maize differed between RM application rates, mineral nitrogen application rates, and years of the study (Table 3). On average for the 2 years of the study, application of increasing amounts of RM caused an increase in the dry weight of harvested maize. In the first year, the difference in yield following application of 40 and 60 t·ha−1 RM was not significant. In the second year, a significant increase in the amount of silage maize was obtained following application of 40 and 60 t·ha−1 RM, while the dose of 20 t·ha−1 RM did not significantly affect the yield. As in the first year, the differences between these rates were also not significant. O f h f h d l f l (N ) b k h 1 d d ififi On average for the 2 years of the study, increasing application of mineral nitrogen (Nmin) by 50 kg·ha−1 did not result in an increase in the dry weight of harvested maize between adjacent application rates (Table 3). In comparison with the control without Nmin application, the yield was higher following application of 100 and 150 kg Nmin·ha−1, and in comparison with application of 50 kg Nmin·ha−1, the yield was higher following applica- tion of 150 kg Nmin·ha−1. Analysis of interactions indicates that in the first year the yield increased significantly at application of up to 100 kg Nmin·ha−1, while application of 150 kg Nmin·ha−1 did not result in a significant increase in yield. In the second year, maize yield was not significantly dependent on the amount of Nmin applied in the first year. On average for the 2 years of the study, the yield in the second year was lower than in the first year.hf i y g y y y yi y The actual effect of the interaction of RM and Nmin in determining maize yield, in comparison with the sum of the effects of RM and Nmin applied separately, is shown in Table 4. Based on the data the effect of the interac- tion of RM and Nmin can be ranked in the following ranges of percentages: 1 (> 30.0%) RM 40 t + Nmin 150 kg; 2 https://doi.org/10.1038/s41598-024-56669-z Scientific Reports \| (2024) 14:5856 \| www.nature.com/scientificreports/ Table 3. Maize yield [DM t·ha−1] as a result of different RM and Nmin application rates and years, mean values ± standard deviation (SD). Resultsh a,b,c,d—means for investigated factors with different lowercase letters are significantly different; A,B,C—means for interactions with different capital letters in the columns are significantly different. Treatments Years LSD0.05, pRM × Years, Nmin × Years Averages LSD0.05, p RM, Nmin 2018 2019 RM t·ha−1 0 11.3 ± 1.6 A 12.4 ± 1.4 A 1.7 p < 0.0001 11.8 ± 1.6 a 1.2 p < 0.0001 20 14.0 ± 1.7 B 13.9 ± 0.6 AB 14.0 ± 1.3 b 40 16.9 ± 3.6 C 14.1 ± 1.1 BC 15.5 ± 3.0 c 60 18.3 ± 3.1 C 15.7 ± 1.7 C 17.0 ± 2.8 d Nmin kg·ha−1 0 12.5 ± 2.5 A 13.6 ± 1.7 A 1.7 p = 0.0002 13.1 ± 2.2 a 1.2 p < 0.0001 50 14.3 ± 2.8 B 13.9 ± 1.8 A 14.1 ± 2.3 ab 100 16.1 ± 4.1 C 14.1 ± 1.8 A 15.1 ± 3.2 bc 150 17.5 ± 3.8 C 14.5 ± 1.7 A 16.0 ± 3.3 c Averages 15.1 ± 3.8 b 14.0 ± 1.7 a LSD0.05, p for years 0.7, p = 0.0016 Table 3. Maize yield [DM t·ha−1] as a result of different RM and Nmin application rates and years, mean values ± standard deviation (SD). a,b,c,d—means for investigated factors with different lowercase letters are significantly different; A,B,C—means for interactions with different capital letters in the columns are significantly different. Treatments Years LSD0.05, pRM × Years, Nmin × Years Averages LSD0.05, p RM, Nmin 2018 2019 RM t·ha−1 0 11.3 ± 1.6 A 12.4 ± 1.4 A 1.7 p < 0.0001 11.8 ± 1.6 a 1.2 p < 0.0001 20 14.0 ± 1.7 B 13.9 ± 0.6 AB 14.0 ± 1.3 b 40 16.9 ± 3.6 C 14.1 ± 1.1 BC 15.5 ± 3.0 c 60 18.3 ± 3.1 C 15.7 ± 1.7 C 17.0 ± 2.8 d Nmin kg·ha−1 0 12.5 ± 2.5 A 13.6 ± 1.7 A 1.7 p = 0.0002 13.1 ± 2.2 a 1.2 p < 0.0001 50 14.3 ± 2.8 B 13.9 ± 1.8 A 14.1 ± 2.3 ab 100 16.1 ± 4.1 C 14.1 ± 1.8 A 15.1 ± 3.2 bc 150 17.5 ± 3.8 C 14.5 ± 1.7 A 16.0 ± 3.3 c Averages 15.1 ± 3.8 b 14.0 ± 1.7 a LSD0.05, p for years 0.7, p = 0.0016 Table 3. Resultsh Treatments Years LSD0.05, p RM × Years, Nmin × Years Averages LSD0.05, p RM, Nmin 2018 2019 RM t·ha−1 0 123.9 ± 29.9 83.3 ± 11.2 p = 0.0506 103.6 ± 30.3 a 17.0 p < 0.0001 20 166.8 ± 39.5 119.6 ± 24.3 143.2 ± 40.1 b 40 215.9 ± 50.0 140.9 ± 14.9 178.4 ± 52.6 c 60 230.5 ± 41.0 172.1 ± 27.5 201.3 ± 45.3 d Nmin kg·ha−1 0 141.1 ± 44.6 A 114.4 ± 29.8 A 24.0 p = 0.0005 127.7 ± 39.5 a 17.0 p < 0.0001 50 170.1 ± 43.2 B 125.4 ± 36.2 AB 147.7 ± 45.2 b 100 204.6 ± 60.1 C 129.2 ± 40.8 AB 166.9 ± 63.3 c 150 221.4 ± 51.9 C 146.9 ± 42.2 B 184.1 ± 59.9 d Averages 184.3 ± 58.0 b 129.0 ± 38.2 a LSD0.05, p for years 9.1, p < 0.0001 Table 6. Nitrogen uptake [kg N·ha−1] by maize as a result of different RM application rates and Nmin and l ±SD b d f i ti t d f t ith diff t l l tt i ifi tl Table 6. Nitrogen uptake [kg N·ha−1] by maize as a result of different RM application rates and Nmin and years, mean values ± SD. a,b,c,d—means for investigated factors with different lowercase letters are significa different; A B C means for interactions with different capital letters in the columns are significantly differe Table 6. Nitrogen uptake [kg N·ha−1] by maize as a result of different RM application rates and Nmin and years, mean values ± SD. a,b,c,d—means for investigated factors with different lowercase letters are significantly different; A,B,C—means for interactions with different capital letters in the columns are significantly different. Table 6. Nitrogen uptake [kg N·ha−1] by maize as a result of different RM application rates and Nmin and years, mean values ± SD. a,b,c,d—means for investigated factors with different lowercase letters are significantly different; A,B,C—means for interactions with different capital letters in the columns are significantly different. of Nmin. On average for the years of the study, nitrogen uptake by maize in the first year was 42.9% greater than in the second year.hi The total nitrogen uptake as a sum from 2 years of research was significantly dependent on RM application rates or mineral nitrogen application rates (Table 7). This parameter increased with the RM application rate. Resultsh a,b,c—means for investigated factors with different lowercase letters are significantly different; A,B,C—means for interactions with different capital letters in the columns are significantly differen Treatments Years LSD0.05, p RM × Years, Nmin × Years Averages LSD0.05 RM, Nmin 2018 2019 RM t·ha−1 0 10.87 ± 1.22 A 6.72 ± 0.48 A 1.15 p = 0.0015 8.79 ± 2.31 a 0.81 p < 0.0001 20 11.77 ± 1.59 AB 8.61 ± 1.70 B 10.19 ± 2.28 b 40 12.70 ± 0.69 B 9.99 ± 0.91 C 11.35 ± 1.60 c 60 12.63 ± 0.69 B 11.00 ± 1.71 C 11.81 ± 1.52 c Nmin kg·ha−1 0 11.04 ± 1.61 8.35 ± 1.59 p = 0.3941 9.69 ± 2.08 a 0.81 p < 0.0001 50 11.81 ± 1.21 8.90 ± 1.97 10.35 ± 2.18 ab 100 12.56 ± 0.92 9.03 ± 2.19 10.80 ± 2.44 bc 150 12.57 ± 0.88 10.04 ± 2.31 11.30 ± 2.15 c Averages 11.99 ± 1.32 b 9.08 ± 2.06 a LSD0.05, p for years 0.43, p < 0.0001 Table 5. Nitrogen content [g N·kg−1 DM] in maize as a result of different RM and Nmin application rates and years, mean values ± SD. a,b,c—means for investigated factors with different lowercase letters are significantly different; A,B,C—means for interactions with different capital letters in the columns are significantly different. Table 6. Nitrogen uptake [kg N·ha−1] by maize as a result of different RM application rates and Nmin and years, mean values ± SD. a,b,c,d—means for investigated factors with different lowercase letters are significantly different; A,B,C—means for interactions with different capital letters in the columns are significantly different. Resultsh On average for the 2 years and in the second year of the study, the nitrogen content in maize increased following the application of 20 and 40 t·ha−1 RM, while the application of 60 t·ha−1 RM did not cause a significant increase in this parameter. Analysis of interactions shows that in the first year following the application of RM significantly more nitrogen was obtained in maize following the application of 40 and 60 t·ha−1 RM than when RM was not applied. 1 g On average for the 2 years of the study, increasing application of mineral nitrogen (Nmin) by 50 kg·ha−1 did not significantly increase the content of nitrogen in the maize between adjacent rates of application. In comparison to the control without Nmin application, maize contained more nitrogen following application of 100 and 150 kg Nmin·ha−1. In comparison to the application of 50 kg Nmin·ha−1, more nitrogen was noted in maize following application of 150 kg Nmin·ha−1. On average for the years of the study, the nitrogen content in maize harvested in the first year was higher than in the second year.i i Nitrogen uptake by maize was significantly dependent on RM application rates, mineral nitrogen applica- tion rates, and the years of the study (Table 6). On average for the 2 years of the study, nitrogen uptake by maize increased with the RM application rate. Following application of 20, 40 and 60 t·ha−1 RM, nitrogen uptake was 38.2%, 72.2%, and 94.3% higher, respectively, than without RM application. On average for the 2 years of the study, each increase in the application of mineral nitrogen (Nmin) by 50 kg·ha−1 caused a significant increase in nitrogen uptake by maize. Following application of 50, 100 and 150 kg Nmin·ha−1, nitrogen uptake was 15.7%, 30.7%, and 44.2% higher, respectively, than without application Scientific Reports \| (2024) 14:5856 \| https://doi.org/10.1038/s41598-024-56669-z www.nature.com/scientificreports/ Table 5. Nitrogen content [g N·kg−1 DM] in maize as a result of different RM and Nmin application rates and years, mean values ± SD. www.nature.com/scientificreports/ Treatments Years p RM × Years, Nmin × Years Averages p RM, Nmin 2018 2019 RM t·ha−1 20 43.8 ± 7.8 22.1 ± 9.9 p = 0.1095 33.0 ± 14.1 p = 0.2706 40 43.9 ± 9.7 20.3 ± 3.8 32.1 ± 14.0 60 35.1 ± 8.6 22.5 ± 5.8 28.8 ± 9.7 Nmin kg·ha−1 50 36.4 ± 6.1 22.5 ± 7.2 p = 0.1933 29.5 ± 9.7 p = 0.3826 100 43.1 ± 9.2 19.3 ± 6.6 31.2 ± 14.5 150 43.3 ± 11.4 23.1 ± 6.7 33.2 ± 13.8 Averages 40.9 ± 9.4 b 21.6 ± 6.8 a LSD0.05, p for years 4.4, p < 0.0001 Table 8. Nitrogen use efficiency [%] from total nitrogen application (Ntot) as a result of different RM application rates and years, mean values ± SD. a,b—means for investigated factors with different lowercase letters are significantly different. Table 8. Nitrogen use efficiency [%] from total nitrogen application (Ntot) as a result of different RM application rates and years, mean values ± SD. a,b—means for investigated factors with different lowercase letters are significantly different. Table 8. Nitrogen use efficiency [%] from total nitrogen application (Ntot) as a result of different RM application rates and years, mean values ± SD. a,b—means for investigated factors with different lowercase letters are significantly different. Table 9. Total nitrogen use efficiency (sum from 2 years) [%] from total nitrogen application (Ntot_2) as a result of different RM and Nmin application rates, mean values ± SD. Treatments Nmin kg·ha−1 Averages p for RM doses 0 50 100 150 RM t·ha−1 20 50.3 ± 6.5 60.2 ± 4.7 61.6 ± 12.4 75.9 ± 11.5 62.0 ± 12.4 p = 0.3396 40 55.7 ± 11.9 60.8 ± 7.5 62.9 ± 11.2 68.9 ± 16.4 62.1 ± 11.5 60 52.1 ± 1.6 55.7 ± 9.9 62.6 ± 19.4 54.4 ± 5.6 56.2 ± 10.5 Averages 52.7 ± 7.2 58.9 ± 7.1 62.4 ± 12.8 66.4 ± 14.1 Interaction RM/Nmin p = 0.6158 p for Nmin doses p = 0.0829 Table 9. Total nitrogen use efficiency (sum from 2 years) [%] from total nitrogen application (Ntot_2) as a result of different RM and Nmin application rates, mean values ± SD. Resultsh The total nitrogen uptake from 2 years did not increase significantly after each increase in the mineral nitrogen dose by 50 kg·ha−1. Significant differences in this parameter were obtained between the objects differing in the application of 100 kg Nmin·ha−1 into the soil.hfii The nitrogen use efficiency of maize was not significantly dependent on RM application rates or mineral nitrogen application rates (Table 8). The value of this parameter was significantly dependent on the year of the study. The nitrogen use efficiency of maize obtained in the first year following application of fertilizer was twice as high as in the second year.f g y Table 9 shows the effect of application of mineral nitrogen on NUE from RM as the sum from 2 years. NUE was shown to increase with each successive increase in application of mineral nitrogen, but the differences were not confirmed statistically. The total NUE from the 2 years of the study following application of 20 and 40 t·ha−1 RM was nearly identical, while following application of 60 t·ha−1 RM it was somewhat lower, but the significance of the differences was not confirmed statistically in this case as well. All NUE values were above 50%. An NUE value above 70% was obtained only after the application of 20 t·ha−1 RM and 150 kg·ha−1 Nmin. NUE values in the range of 60.1–70% were obtained following application of 20 t·ha−1 RM with supplementary application of Nmin at 50 and 100 kg·ha−1; 40 t·ha−1 RM with supplementary application of Nmin at all rates; and 60 t·ha−1 RM with supplementary application of Nmin at 100 kg·ha−1. NUE values in the range of 50–60% were Scientific Reports \| (2024) 14:5856 \| https://doi.org/10.1038/s41598-024-56669-z www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 7. Total nitrogen uptake (sum from 2018 and 2019) [kg N·ha−1] as a result of different RM and Nmin application rates, mean values ± SD. a,b,c—means for investigated factors with different lowercase letters are significantly different. Treatments Nmin kg·ha−1 Averages LSD0.05, p for RM doses 0 50 100 150 RM t·ha−1 20 221.4 ± 7.6 263.1 ± 7.9 296.2 ± 26.9 365.2 ± 30.6 286.4 ± 57.8 a 39.9 p < 0.0001 40 292.8 ± 27.7 334.9 ± 21.2 372.6 ± 37.5 426.9 ± 62.9 356.8 ± 62.1b 60 345.1 ± 5.7 385.7 ± 39.8 444.6 ± 87.4 434.9 ± 28.1 402.6 ± 59.8 c Averages 286.4 ± 55.8 a 327.9 ± 58.1 ab 371.1 ± 81.1bc 409.0 ± 50.1c Interaction RM/Nmin p = 0.7204 LSD0.05, p for Nmin doses 50.9 p < 0.0001 Table 7. Total nitrogen uptake (sum from 2018 and 2019) [kg N·ha−1] as a result of different RM and Nmin application rates, mean values ± SD. a,b,c—means for investigated factors with different lowercase letters are significantly different. Treatments Years p RM × Years, Nmin × Years Averages p RM, Nmin 2018 2019 RM t·ha−1 20 43.8 ± 7.8 22.1 ± 9.9 p = 0.1095 33.0 ± 14.1 p = 0.2706 40 43.9 ± 9.7 20.3 ± 3.8 32.1 ± 14.0 60 35.1 ± 8.6 22.5 ± 5.8 28.8 ± 9.7 Nmin kg·ha−1 50 36.4 ± 6.1 22.5 ± 7.2 p = 0.1933 29.5 ± 9.7 p = 0.3826 100 43.1 ± 9.2 19.3 ± 6.6 31.2 ± 14.5 150 43.3 ± 11.4 23.1 ± 6.7 33.2 ± 13.8 Averages 40.9 ± 9.4 b 21.6 ± 6.8 a LSD0.05, p for years 4.4, p < 0.0001 Table 8. Nitrogen use efficiency [%] from total nitrogen application (Ntot) as a result of different RM application rates and years, mean values ± SD. a,b—means for investigated factors with different lowercase letters are significantly different. www.nature.com/scientificreports/ Level of RM × Nmin interaction for yield data, nitrog Treatments Nmin kg·ha−1 0 50 100 150 RM t·ha−1 0 Yield 10.6 ± 1.9 11.5 ± 1.2 12.3 ± 1.0 13.0 ± 1.4 N content 7.88 ± 1.59 8.77 ± 2.52 9.09 ± 2.50 9.43 ± 2.79 N uptake 81.3 ± 7.7 99.2 ± 23.8 111.0 ± 28.9 123.1 ± 40.1 20 Yield 12.7 ± 1.0 13.7 ± 0.4 14.1 ± 0.6 15.4 ± 1.3 N content 8.84 ± 1.56 9.65 ± 1.87 10.49 ± 2.64 11.78 ± 2.31 N uptake 110.7 ± 11.8 131.5 ± 22.8 148.1 ± 38.2 182.6 ± 44.3 NUEtot – 30.1 ± 12.1 30.8 ± 15.9 38.0 ± 15.3 40 Yield 13.7 ± 0.7 15.0 ± 1.3 15.7 ± 2.3 17.8 ± 4.7 N content 10.72 ± 1.74 11.14 ± 1.64 11.67 ± 2.03 11.85 ± 0.96 N uptake 146.4 ± 24.6 167.4 ± 33.1 186.3 ± 56.2 213.5 ± 70.4 NUEtot – 30.4 ± 10.4 31.5 ± 15.8 34.5 ± 17.4 60 Yield 15.3 ± 1.6 16.3 ± 2.4 18.4 ± 4.0 17.9 ± 2.0 N content 11.33 ± 1.62 11.84 ± 1.52 11.93 ± 1.97 12.16 ± 1.19 N uptake 172.6 ± 24.8 192.6 ± 34.0 222.3 ± 68.2 217.4 ± 33.3 NUEtot – 27.9 ± 7.7 31.3 ± 14.5 27.2 ± 6.0 p value for interaction RM × Nmin, and RM × Nmin × Year Yield RM × Nmin = 0.7483, RM × Nmin × Year = 0.3647 N content RM × Nmin = 0.4938, RM × Nmin × Year = 0.3799 N uptake RM × Nmin = 0.5563, RM × Nmin × Year = 0.7202 NUEtot RM × Nmin = 0.5214, RM × Nmin × Year = 0.7663 Table 10. Level of RM × Nmin interaction for yield data, nitrogen content and uptake, and NUEtot value, mean values ± SD. Table 10. Level of RM × Nmin interaction for yield data, nitrogen content and uptake, and NUEtot value, me values ± SD. Table 11. Nitrogen use efficiency [%] from nitrogen theoretically available for plants (presented in Table 2) in each year of the study as a result of different RM and Nmin application rates, mean values ± SD. a,b—means for investigated factors with different lowercase letters are significantly different. www.nature.com/scientificreports/ Treatments Nmin kg·ha−1 Averages p for RM doses 0 50 100 150 RM t·ha−1 20 50.3 ± 6.5 60.2 ± 4.7 61.6 ± 12.4 75.9 ± 11.5 62.0 ± 12.4 p = 0.3396 40 55.7 ± 11.9 60.8 ± 7.5 62.9 ± 11.2 68.9 ± 16.4 62.1 ± 11.5 60 52.1 ± 1.6 55.7 ± 9.9 62.6 ± 19.4 54.4 ± 5.6 56.2 ± 10.5 Averages 52.7 ± 7.2 58.9 ± 7.1 62.4 ± 12.8 66.4 ± 14.1 Interaction RM/Nmin p = 0.6158 p for Nmin doses p = 0.0829 Table 9. Total nitrogen use efficiency (sum from 2 years) [%] from total nitrogen application (Ntot_2) as a result of different RM and Nmin application rates, mean values ± SD. obtained following application of 60 t·ha−1 RM and Nmin at 50 and 150 kg·ha−1 and following application of all levels of RM without Nmin.if obtained following application of 60 t·ha−1 RM and Nmin at 50 and 150 kg·ha−1 and following application of all levels of RM without Nmin. fi f d b d ff d h f ld d No significant interactions were found between different RM and Nmin rates in the case of yield, content nitrogen uptake (as an average and sum for 2-years) by maize.h g g y y The mean values of the examined property for the two-way interactions RM × Nmin presented in Table 10 were not significant—all p-values > 0.05. i NUEav values calculated separately for each year for nitrogen theoretically available for plants were not sig- nificantly dependent on the level of RM application in either year or on the amount of Nmin applied in the first year (Table 11). In the second year of the study, application of 0, 50 and 100 kg·ha−1 Nmin together with RM did not significantly influence NUEav from the pool of theoretically available nitrogen applied in the form of RM. The highest value for this parameter was obtained following the application of Nmin at 150 kg·ha−1 together with RM. NUEav values calculated for N theoretically available for plants in total for the 2 years of the study were not significantly dependent on either the RM or Nmin application rate. https://doi.org/10.1038/s41598-024-56669-z Scientific Reports \| (2024) 14:5856 \| www.nature.com/scientificreports/ Table 10. Level of RM × Nmin interaction for yield data, nitrogen content and uptake, and NUEtot value, mean values ± SD. www.nature.com/scientificreports/ Treatments Nmin kg·ha−1 0 50 100 150 RM t·ha−1 0 Yield 10.6 ± 1.9 11.5 ± 1.2 12.3 ± 1.0 13.0 ± 1.4 N content 7.88 ± 1.59 8.77 ± 2.52 9.09 ± 2.50 9.43 ± 2.79 N uptake 81.3 ± 7.7 99.2 ± 23.8 111.0 ± 28.9 123.1 ± 40.1 20 Yield 12.7 ± 1.0 13.7 ± 0.4 14.1 ± 0.6 15.4 ± 1.3 N content 8.84 ± 1.56 9.65 ± 1.87 10.49 ± 2.64 11.78 ± 2.31 N uptake 110.7 ± 11.8 131.5 ± 22.8 148.1 ± 38.2 182.6 ± 44.3 NUEtot – 30.1 ± 12.1 30.8 ± 15.9 38.0 ± 15.3 40 Yield 13.7 ± 0.7 15.0 ± 1.3 15.7 ± 2.3 17.8 ± 4.7 N content 10.72 ± 1.74 11.14 ± 1.64 11.67 ± 2.03 11.85 ± 0.96 N uptake 146.4 ± 24.6 167.4 ± 33.1 186.3 ± 56.2 213.5 ± 70.4 NUEtot – 30.4 ± 10.4 31.5 ± 15.8 34.5 ± 17.4 60 Yield 15.3 ± 1.6 16.3 ± 2.4 18.4 ± 4.0 17.9 ± 2.0 N content 11.33 ± 1.62 11.84 ± 1.52 11.93 ± 1.97 12.16 ± 1.19 N uptake 172.6 ± 24.8 192.6 ± 34.0 222.3 ± 68.2 217.4 ± 33.3 NUEtot – 27.9 ± 7.7 31.3 ± 14.5 27.2 ± 6.0 p value for interaction RM × Nmin, and RM × Nmin × Year Yield RM × Nmin = 0.7483, RM × Nmin × Year = 0.3647 N content RM × Nmin = 0.4938, RM × Nmin × Year = 0.3799 N uptake RM × Nmin = 0.5563, RM × Nmin × Year = 0.7202 NUEtot RM × Nmin = 0.5214, RM × Nmin × Year = 0.7663 Table 10. Table 11. Nitrogen use efficiency [%] from nitrogen theoretically available for plants (presented in Table 2) in each year of the study as a result of different RM and Nmin application rates, mean values ± SD. a,b—means for investigated factors with different lowercase letters are significantly different. Discussion Animal excrements (faeces and urine) are traditionally used to fertilize soil and plants. They owe their value as fertilizer to high content of organic matter and nutrients for plants. One of the most important nutrients for plants contained in these fertilizers is nitrogen8,27. Organic forms of nitrogen applied to the soil gradually become available for plants28. Mineralization of organic compounds from these fertilizers, spread out over time, causes the gradual release of nitrogen in available forms, which enables a regular supply of this macronutrient to plants. In the case of chemical fertilizers, the entire pool of nitrogen is usually available for plants immediately after their application, but decreases significantly in the later stage of their growth29. This is the result of nitrogen being taken up by plants and dissipated in the environment. The combined application of these fertilizers limits the dissipation of nitrogen from mineral fertilizers in the environment30. This may be the result of biological sorption of mineral nitrogen (its immobilization process). Fertilization based on the combined application of organic fertilizers and nitrogen mineral fertilizers can ensure an optimal supply of nitrogen to plants31 and may result in higher crop yields30,32. The present study showed that the biomass yield of maize increased in the first year after application of RM up to 40 t·ha−1 and Nmin up to 100 kg·ha−1. Further increases to 60 t·ha−1 RM and 150 kg·ha−1 Nmin did not lead to a significant increase in the yield of maize. In that second year of the study, an increase in yield in comparison to the treatment without RM application was obtained following application at the two highest rates (40 and 60 t·ha−1). In that year, varied application of Nmin did not significantly differentiate the yields of the test plant, which is consistent with the idea of the rapid but short-term fertilizing effect of chemical nitrogen fertilizers. The dry matter yield per kg of applied nitrogen decreases as the rate of application increases. Nitrogen applied in small amounts is then the major growth-limiting factor. Increasing the supply of nitrogen reduces productivity of applied N, because the yield is determined by factors other than the limiting nutrient33,34.i p y pp y y g An important element of the experiment was the determination of the most beneficial proportion of rabbit manure and mineral nitrogen application for the yield of the test plant, nitrogen content and uptake, and NUE. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Correlation analysis revealed significant relationships between total and available nitrogen doses, and: silage maize yield, nitrogen content, nitrogen uptake (Table 12). Nitrogen use efficiency in 2019 and NUE as sum from 2018 and 2019 were significantly correlated with total nitrogen doses. Table 12. Linear correlation coefficients between Ntot and Nav rates and selected properties of silage maize, values marked with * are important (α< 0.05). www.nature.com/scientificreports/ Years of study Treatments RM, t·ha−1 Averages LSD0.05, p for Nmin doses 20 40 60 2018 Nmin kg·ha−1 p = 0.0552 0 87.3 ± 4.6 97.6 ± 8.8 88.2 ± 3.2 91.0 ± 5.5 50 67.4 ± 8.9 83.9 ± 3.4 77.4 ± 5.9 76.2 ± 6.1 100 68.2 ± 5.4 81.0 ± 11.3 82.1 ± 12.9 77.1 ± 9.2 150 70.2 ± 0.7 79.9 ± 13.0 57.4 ± 5.0 69.1 ± 11.4 Averages 73.3 ± 9.2 85.6 ± 10.0 76.3 ± 7.7 Interaction RM/Nmin p = 0.8180 p for RM doses p = 0.1688 2019 Nmin kg·ha−1 99.3 p = 0.0055 0 135.2 ± 21.1 143.3 ± 20.9 139.8 ± 19.6 139.4 ± 18.1a 50 230.4 ± 121.8 175.7 ± 43.9 168.3 ± 32.5 191.5 ± 72.9ab 100 220.0 ± 90.1 178.7 ± 26.6 186.7 ± 84.3 195.1 ± 65.9ab 150 403.1 ± 178.6 225.6 ± 40.8 218.3 ± 37.4 282.3 ± 130.2b Averages 247.2 ± 142.8 180.8 ± 42.4 178.3 ± 51.9 Interaction RM/Nmin p = 0.3841 p for RM doses p = 0.0614 For N available in both years Nmin kg·ha−1 p = 0.4688 0 101.4 ± 13.1 111.3 ± 23.7 103.7 ± 3.2 105.4 ± 14.4 50 93.0 ± 7.3 103.2 ± 12.7 98.7 ± 17.6 98.3 ± 12.3 100 84.5 ± 17.0 96.8 ± 17.3 102.2 ± 21.7 94.5 ± 21.4 150 97.4 ± 14.7 99.0 ± 23.6 83.5 ± 8.6 93.3 ± 16.3 Averages 94.1 ± 13.2 102.6 ± 17.9 97.0 ± 18.0 Interaction RM/Nmin p = 0.8513 p for RM doses p = 0.4978 Table 11. Nitrogen use efficiency [%] from nitrogen theoretically available for plants (presented in Table 2) in each year of the study as a result of different RM and Nmin application rates, mean values ± SD. a,b—means for investigated factors with different lowercase letters are significantly different. Table 11. Nitrogen use efficiency [%] from nitrogen theoretically available for plants (presented in Table 2) in each year of the study as a result of different RM and Nmin application rates, mean values ± SD. a,b—means for investigated factors with different lowercase letters are significantly different. https://doi.org/10.1038/s41598-024-56669-z Discussion The study showed that the most favourable proportion in terms of the biomass yield of maize was 60 t·ha−1 RM and 100 kg·ha−1 Nmin, which introduced a total of 450 kg·ha−1 nitrogen into the soil, while the amount of nitrogen theoretically available for plants in the first and second year was 223 and 53 kg·ha−1, respectively. Other research- ers reported that for cultivation of forage maize in semi-arid areas, the optimal application rate of nitrogen in chemical fertilizer, and thus nitrogen available for plants, was 210 kg·ha−1, but that was the highest rate tested in the study34. Correlation analysis (p < 0.05) shows that the silage maize yields were significantly dependent on the application rates of nitrogen: total (r = + 0.862) and theoretically available for plants (r = + 0.849). Increas- ing maize yields caused by increasing nitrogen application rates is a common phenomenon and consistent with researchers’ expectations35,36. Good supply of nitrogen to plants promotes faster photosynthesis by increasing capture of sunlight by crops and the efficiency of conversion to biomass, thus improving biomass production37. Photosynthesis activity is associated with nitrogen content in leaves38. The amount of dry matter of silage maize increases with the level of nitrogen in the leaves33. Combined application of organic and chemical nitrogen fer- tilizers has proven very effective in crops of other plants. In potato, the ideal combination for optimizing yield has been shown to be, on average, 31 t·ha−1 of organic manure and 187.5 kg·ha−1 of nitrogen fertilizer39. Similar fertilization systems based on partial replacement of mineral nutrients for plants with nutrients applied in the form of organic fertilizers, including rabbit manure, have resulted in similar or even higher maize yields than inorganic fertilizer applied at the same nitrogen:phosphorus:potassium rate40. g pp g p p p Increasing the amount of nitrogen introduced to the soil caused an increase in its content in the biomass of maize as well as its uptake by the plant. Converting nitrogen content to crude protein content (CP) (× 6.25) and nitrogen uptake to crude protein yield (CPY) shows that application of RM at 20–60 t·ha−1 resulted in CP from 7.4 to 7.9% in the biomass of maize grown in the first year after fertilizer application, and CPY from 1042 to Table 12. Linear correlation coefficients between Ntot and Nav rates and selected properties of silage maize, values marked with * are important (α< 0.05). www.nature.com/scientificreports/ 1441 kg·ha−1. In the second year following application of RM at 20–60 t·ha−1, CP ranged from 5.4 to 6.9%, and CPY from 748 to 1076 kg·ha−1. The values obtained in the first year after fertilizer application correspond to the typical protein content in maize silage41,42. Analysis of the interaction of RM and Nmin application shows that the highest average CP content from the 2 years (7.6%) was obtained following the combined application of RM and Nmin at the highest rates, i.e. 60 t·ha−1 RM and 150 kg·ha−1 Nmin, which introduced a total of 500 kg·ha−1 N to the soil, including 273 and 53 kg·ha−1 of nitrogen theoretically available for plants in the first and second year, respec- tively. However, the highest average crude protein yield from the 2 years (1389 kg·ha−1) was obtained following the combined application of 60 t·ha−1 RM and 100 kg·ha−1 Nmin. On this treatment into soil was applied 50 kg less total and theoretically available nitrogen than in the case of the highest nitrogen application. Ma and coauthors34 tested nitrogen application rates from 70 to 210 kg·ha−1 in chemical fertilizer and obtained 5.2% to 7.0% protein content in forage maize. In the present study, the average crude protein content in the maize biomass was slightly higher. Literature data indicate that it is possible to obtain even higher CP content in forage maize43. Following application of Nmin alone at 50–150 kg·ha−1, CP content ranged from 5.5 to 5.9%, while following application of RM alone at 20–60 t·ha−1, CP content ranged from 5.5 to 7.1%. After combined application of RM and Nmin in all combinations tested, CP ranged from 6.0 to 7.6%. Correlation analysis (p < 0.05) indicates that the nitrogen content and, consequently, the crude protein content in the first and second year after fertilizer application was significantly correlated with the total amount of nitrogen applied (r = + 0.671 and r = + 0.829, respectively), as well as with the amount of nitrogen theoretically available for plants (r = + 0.696 and r = + 0.715, respectively). Nitrogen uptake and, consequently, crude protein yield in the first and second year after fertilization was also significantly correlated with the total nitrogen applied (r = + 0.872 and r = + 0.899, respectively), as well as with the amount of nitrogen theoretically available for plants (r = + 0.870 and r = + 0.770, respectively). www.nature.com/scientificreports/ This means that as the level of nitrogen application increased (total and theoretically available for plants), uptake of nitrogen and its accumulation in the maize biomass increased. This translates directly to the NUE values, which were generally somewhat smaller following application of higher levels of nitrogen, but did not differ significantly depending on the levels of RM and Nmin application. However, literature data indicate that the NUE value significantly decreases as N application increases34,44. NUE values ranged from 35.1 to 43.9% in the first year following application of RM and Nmin, and from 19.3 to 23.1% in the second year. These are higher values than in other studies, in which NUE from urea applied at 70–210 kg·ha−1 nitrogen was within the range of 24.4–31.7%34. Other researchers obtained a significant decrease in nitrogen recovery efficiency from various levels of nitrogen application after replacing inorganic nitrogen with organic nitrogen (applied with manure) at a level of at least 50%45. In that study, the highest value of this parameter was obtained following application of inorganic and organic nitrogen in a 0.75/0.25 ratio (about 40–50% in maize cultivation). Another study found that the optimal level of replacement of synthetic nitrogen with organic nitrogen, leading to an increase in NUE, was 44%46. The NUE values above 60% obtained in the present study following application of RM at 20 t·ha−1 (117 kg·ha−1 total nitrogen, 41 kg·ha−1 theoretically available nitrogen) and 40 t·ha−1 (233 kg·ha−1 total N, 82 kg·ha−1 theoretically available nitrogen) with supplementary application of Nmin at 50–150 kg·ha−1 and following application of 60 t·ha−1 RM (350 kg·ha−1 total nitrogen, 123 kg·ha−1 theoretically available nitrogen) with 100 kg·ha−1 Nmin indicate an appropriate level of nitrogen use by the plants and are in line with current sustainable development strategies for the agricultural sector. Fertilization regimes based on balanced synthetic nitrogen and manure are ideal for sustainable nitrogen management47, increasing NUE48. Calculation of NUE for the pool of nitrogen theoretically available for plants (NUEav) sheds new light on NUE values. The NUEav values obtained in the first year, most often in the range of 60–80%, are indicative of incomplete use of the pool of nitrogen theoretically available for plants. This may have been due to unfavourable moisture conditions during the growing season. Conclusion Given the dry matter yield of maize, nitrogen uptake (crude protein yield), nitrogen use efficiency from applied nitrogen, and the effect of the interaction of organic and mineral fertilizer, fertilization of maize for silage with rabbit manure at up to 40 t·ha−1 and supplementary application of mineral nitrogen in amounts up to 150 kg·ha−1 is justified. When application of rabbit manure is increased to 60 t·ha−1, application of Nmin should be decreased so as not to exceed 100 kg·ha−1. These fertilizer combinations introduce up to 232 kg·ha−1 of nitrogen theoretically available for plants to the soil in the first growing season, and the NUEav from this pool of nitrogen is about 80%. The values of all parameters tested (yield, nitrogen content and uptake, NUEtot and NUEav) indicate the need for additional application of nitrogen for crops grown in the second year following application of RM, but after taking into account the small amount of available nitrogen remaining after application of RM in the first year. www.nature.com/scientificreports/ The NUEav values for the pool of nitrogen theoretically available for plants in the second year, exceeding 100% in each case, and in some cases even 200%, are indicative of greater nitrogen availability for plants than was predicted. They may also indicate that the pool of available nitrogen unused in the first year was used in the second year. NUEav values for the pool of nitrogen theoretically available for plants in total for both years of the study was most often over 90%, and in five cases exceeded 100% (on average 98%), indicating total or nearly total utilization of this pool of nitrogen. It also indicates that the factors chosen to convert the total amount of nitrogen introduced to the soil with manure to nitrogen available for plants were well-selected25. Discussion Specification Ntot dose Nav dose Yield in 2018 0.875* 0.824* Yield in 2019 0.688* 0.568* Yield, as the sum 2018 + 2019 0.862* 0.849* N content in 2018 0.671* 0.696* N content in 2019 0.829* 0.715* N uptake in 2018 0.872* 0.870* N uptake in 2019 0.899* 0.770* N uptake, as the sum 2018 + 2019 0.927* 0.872* NUEtot in 2018 0.027 – NUEtot in 2019 0.570* – NUEtot as the sum 2018 + 2019 0.308* – Table 12. Linear correlation coefficients between Ntot and Nav rates and selected properties of silage maize, values marked with * are important (α< 0.05). Specification Ntot dose Nav dose Yield in 2018 0.875* 0.824* Yield in 2019 0.688* 0.568* Yield, as the sum 2018 + 2019 0.862* 0.849* N content in 2018 0.671* 0.696* N content in 2019 0.829* 0.715* N uptake in 2018 0.872* 0.870* N uptake in 2019 0.899* 0.770* N uptake, as the sum 2018 + 2019 0.927* 0.872* NUEtot in 2018 0.027 – NUEtot in 2019 0.570* – NUEtot as the sum 2018 + 2019 0.308* – Specification Ntot dose Nav dose Yield in 2018 0.875* 0.824* Yield in 2019 0.688* 0.568* Yield, as the sum 2018 + 2019 0.862* 0.849* N content in 2018 0.671* 0.696* N content in 2019 0.829* 0.715* N uptake in 2018 0.872* 0.870* N uptake in 2019 0.899* 0.770* N uptake, as the sum 2018 + 2019 0.927* 0.872* NUEtot in 2018 0.027 – NUEtot in 2019 0.570* – NUEtot as the sum 2018 + 2019 0.308* – Table 12. Linear correlation coefficients between Ntot and Nav rates and selected properties of silage maize, values marked with * are important (α< 0.05). Table 12. Linear correlation coefficients between Ntot and Nav rates and selected properties of silage maize, values marked with * are important (α< 0.05). https://doi.org/10.1038/s41598-024-56669-z Scientific Reports \| (2024) 14:5856 \| www.nature.com/scientificreports/ Data availability Th d d y The datasets generated and analysed during the current study are not publicly available due to they are the authors’ own data, but are available from the corresponding author on reasonable request. Received: 14 December 2023; Accepted: 8 March 2024 Received: 14 December 2023; Accepted: 8 March 2024 https://doi.org/10.1038/s41598-024-56669-z Scientific Reports \| (2024) 14:5856 \| www.nature.com/scientificreports/ References References 1. Penuelas, J., Coello, F. & Sardans, J. A better use of fertilizers is needed for global food security and environmental sustainability. Agric. Food Secur. 12, 5. https://doi.org/10.1186/s40066-023-00409-5 (2023).fi 1. Penuelas, J., Coello, F. & Sardans, J. 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W4299652829.txt	https://zenodo.org/record/54238/files/paper137.pdf	en		Analysis and improvement of correlation-based watermarking methods for digital images	Zenodo (CERN European Organization for Nuclear Research)	2,002	cc-by	2,882	Analysis and Improvement of Correlation-Based Watermarking Methods for Digital Images Akio MIYAZAKI and Akihiro OKAMOTO Graduate School of Information Science and Electrical Engineering, Kyushu University 6-10-1, Hakozaki, Fukuoka, 812-8581 JAPAN ABSTRACT This paper aims to design robust correlation-based watermarking methods for images. We rst present a model of watermark embedding and extracting processes and carry out their analyses. Then we examine the robustness of the watermarking method against image processing and clarify the reason why detection errors occur in the watermark extracting process. Based on the result, we improve the watermark extracting process and design robust watermarking systems. The improvement is accomplished using the deconvolution technique. Numerical experiments using the DCT-based watermarking system show good performance as expected by us. 1 Introduction With the rapid spread of computer networks and the further development of multimedia technologies, the copyright protection of digital contents such as audio, image and video, has been one of the most serious problems because digital copies can be made identical to the original. The digital watermark technology is now drawing attention as a new method of protecting copyrights of digital contents. A digital watermark is realized by embedding information data, e.g., owner, distributor, or recipient identi ers, transaction dates, serial number, etc., directly into digital contents with an imperceptible form for human audio/visual systems, and should satisfy the following requirements: The embedded watermark should not spoil the quality of the original contents and should not be perceptible. It should be dicult for an attacker to remove the watermark and should be robust to signal processing and geometric distortions. A signi cant number of watermarking methods have been recently reported[1],[2]. Most of these methods embed the watermark into the spectral coecients of images by using signal transformation such as discrete cosine transformation (DCT) or discrete wavelet transformation (DWT) because embedding in the frequency domain is more tolerant to attacks and image processing than embedding in the spatial domain. However, theoretical limits to their robustness against attacks and image processing have not been given, and the watermarking methods that are robust to almost all image processing have not been reported yet. On the contrary, watermark-removal software such as StirMark[3] has succeeded in washing the watermark away for most of the known watermarking systems. Such a situation is quite discouraging but will foster new research in this eld such as the analysis of the performance of watermarking systems and the development of watermarking systems with the desired robustness. Therefore, as the rst stage in the development of watermarking technology, it is important to analyze the watermark embedding and extracting processes and apply the result to the design of robust watermarking systems. In this paper, we concentrate on the watermarking of still images and deal with the correlation-baed watermarking method[2] in which a watermark code (pseudorandom sequence) is perceptually weighted and added to the spectral coecient, and is detected by computing the correlation between the watermarked coecient and the watermark code to be checked for the presence. We rst analyze the watermark embedding and extracting processes and then investigate the robustness of the watermarking system against attacks and image processing. It is clari ed how distortion of the watermark occurs by image processing and the reason why the distortion causes detection errors in the watermark extracting procedure. Based on the result, we furthermore carry out an improvement of the watermark extracting process by designing a deconvolution lter that attempts to undo the e ect of watermark distortion. The design is achieved using the deconvolution technique with adaptive algorithms. Numerical experiments with the DCT-based watermarking system reveal the desired performance is achieved. 2 Analysis of Correlation-Based Watermarking Methods In correlation-based watermarking methods, we rst prepare a set of watermarks W = fwl ; 1 l Lg where wl = [wl (k)] (B-dimensional vector) and the elements wl (k)'s are random sequences of real number drawn from the Gaussian distribution with zero-mean and variance = 1: The watermark embedding and extracting processes are as follows. In the watermark embedding process, an (original) image s = [s(n)] (N -dimensional vector) , where s(n) denotes a pixel quantized to 256 levels (represented by 8 bits), is rst converted into a spectral coecient c = [c(n)] by using signal transformation such as DCT or DWT as c = T s; (1) where T is an N 2 N matrix created from the transfrom kernels. Next, we select B spectral coecients c(i ); c(i ); 11 1 ; c(iB ) and one watermark w = [w(k)] from c and W , respectively, and embed w(k) into c(ik ) in the form of c0 (ik ) = c(ik ) + 1k w(k); (2) where 1k is a positive number called embedded intensity and is usually determined by exploiting the properties of the frequency model for the human visual system (HVS) so that one can make watermarks that have higher energy perceptually invisible. Then, the watermarked coecient d = [d(n)] is dened as c0 (ik ) ; n = ik (1 k B ) d(n) = (3) c(n) ; otherwise : By de ning the N 2 B matrix EM = [eM (m; n)] where eM (m; n) = 1k for (m; n) = (ik ; k); = 0, otherwise, and 1 k B; d can be written as d = c + EM w : (4) By the inverse transform T 01 of d; the watermarked image x = [x(n)] is obtained as x = T 01 d : (5) It is necessary to determine EM so that images may not be degraded through watermark embedding . From EM ; we construct the B 2 N matrix EX = [eX (m; n)] where eX (m; n) = 1 for (m; n) = (k; ik ); = 0, otherwise, and 1 k B: The set of parameters K = fEX ; W g is saved and used as key data in watermark detection. In the watermark extracting process, we transform the watermarked image z = [z(n)] into the spectral coecient d0 = [d0(n)] by the transformation T as d0 = Tz; (6) 2 1 1 2 2 1 An image is usually denoted by an 2 array = [ ( )] In this paper, for simplicity of description, putting = 2 we map = [ ( )] into a vector = [ ( )] of size by row ordering. 2 We can discuss the relationship between embedded intensity 1k (1 ) and the watermarked image quality when the orthogonal transform is used. Since 1 ( k ) = ( k ) 0 ( k ) = 1k ( ) is the random variable with zero-mean and variance 12k , the MSE ofPtheB original and watermarked images is given by n = Nx1Ny k=1 12k where we put the size of images as x 2 y . M M s s m; n N s s m; n s s n M ; N B k B T c i 0 c i c i w k P N N : and obtain the watermarked coecients u as u = EX d0 : (7) Then, we calculate the correlation rW (l) (1 l L) between u and all wl 2 W as rW (l) =< u; wl >= B X k=1 u(k)wl (k ): (8) Then we search for w3 that maximizes rW (l)'s, that is, w3 = arg wmax rW (l) (9) l 2W and determine that there exists w3 in the watermarked image. 3 Robust Watermarking Systems We consider the robustness of the watermarking system against image processing. Let f be an image operator that represents a certain image processing, and let z = f (x) = [ f (x); f (x); 1 1 1 ; fN (x) ]t ; (10) where x is a watermarked image. Then, from Eqs. (4) and (5), we have x = s + T 01 EM w: (11) Since watermarked images are not degraded through watermark embedding, ksk k T 01 EM w k; k 1 k being the norm of vectors, z = f (s + T 01 EM w) ' f (s) + F (s)(T 01 EM w) (12) is obtained, where fm = fm (x); xn = x(n) and 1 2 i.e., @fm @xn : (13) xs Hence, from Eqs. (6) and (7), distortion of the watermarked coecient u occurs as u = EX T f (s) + EX T F (s)T 01 EM w; (14) and detection errors arise in the watermark extracting procedure. It is noted that in the case of linear transformation, z = Fx; Eq. (14) can be written as u = EX T Fs + EX T F T 01 EM w: (15) We can see from Eq. (14) that the distortion of the watermarked coecients u depends on not only the image operator f but also the original image s; and the watermark w is convolved with the lter, incorporating the e ect of distortion, whose impulse response is described by the elements of the B 2 B matrix EX T F (s)T 01EM ; F (s) = [fm;n(s)] = = and furthermore the bias EX T f (s) corrupts the watermark w: This implies that we can design a deconvolution lter that attempts to undo the e ects of the convolution lter and the bias as follows: From Eq. (14), putting H = (EX TF (s)T 01 EM )0 and r = 0HEX Tf (s); we have the improved watermark extracting procedure 9 (i) u = EX d0 > > > = (ii) v = Hu + r 0 (16) (iii) rW (l) =< v; wl >0 > > w3 = arg wmax rW (l) : > ; l 2W 1 4 Improvement of Watermark Extracting Process using Deconvolution Technique In correlation-based watermarking methods, the parameter EM is decided in consideration of the quality of watermarked images and the robustness of watermarks against basic signal processing like JPEG compression and ltering. Hence there exist some image processing techniques that destroy the watermark in image processing and attacking tools as Photoshop and StirMark. Moreover, even if the watermark is robust against a certain image processing tool at present, it may be destroyed by a new version of this tool in the future. In such a situation, a present solution is considered to be the redesign of the watermarking system to revise the watermark detector for the image processing f to which the system is not robust. Using the result is Section 3, we can carry out the revision of the watermark detector provided that we can access the original image s and get a set of input-output pairs fx(k); z(k)g from z = f (x): Then the revised watermark detector may be designed by application of the adaptive signal processing technique because the deconvolution lter can be represented as a system described by a matrix equation and training data, a set of pairs of watermark fw(k)g and watermarked coecient fu(k )g, can be obtained from a set of fx(k); z(k)g of f : That is, by training the system, it will automatically produce an adequate deconvolution lter in accordance with f and s: The design procedure is shown brie y as follows: We rst construct the deconvolution lter ( (ii) in Eq. (16) ) by 3 i.e., v(k ) = B X l=1 h(k; l)u(l) + r(k ) (1 k B) : (17) Then we train the system in order to undo the e ects of the convolution lter and the bias in Eq. (14). The objective of the training process is that the weights h(k; l)'s and r(k)'s are set to the optimum values by minimizing 3 The use of the revised watermark detector is as follows: If we cannot extract the waternark with the ordinary watermark detector (Eqs. (6){(9)), then we utilize the revised watermark detector (Eq. (16)), with which the watermark extraction may be carried out on a trial-and-error basis for the image processings to which the system is not robust. the square of error e(k) = w(k) 0 v(k) between various training watermarks fw(k)g and the outputs fv(k)g of the system. As is well known, a method for setting the weights to these values is the LMS algorithm[4], which is an iterative gradient algorithm. Training take place during many trials or runs until the weights converge to the optimum values, that is, the system (Eq. (17)) learns the characteristic of the deconvolution lter. 5 Numerical Experiments In this section, we focus on the DCT-based watermarking method, in which the watermark is embedded into DCT coecients of an image, properly selected, and try to improve the watermark extracting process using the design technique described in Section 4. The improvement will be veri ed from numerical experiments using the image LENNA with the size of 128 2 128 pixels (Figure 1 (a)). (a) (b) Figure 1 : (a) Original image s (LENNA). (b) Watermarked image x. The watermark embedding procedure given in this experiment is based on Refs.[5] and [6]. The parameters 1k ; B; L, and EM are set as follows: 1k = jc(ik )j ( = 0:8), B = 324, L = 1000, and c(i ), c(i ), 1 1 1, c(i ), c(i ) are equal, respectively, to c(50; 50), c(50; 51), 1 1 1, c(67; 66), c(67; 67) in the 128 2 128 DCT coecient array. (This selection of DCT coecients is denoted by EM ). Figure 1 (b) shows the watermarked image, whose MSE and PSNR are 3.18 and 43.1 [dB], respectively. Figure 2 shows the response rW (l) (1 l L) of the watermark detector D to the watermarked image into which we embed the 500th watermark in L randomly generated watermarks. It can be seen that one spike clearly indicates the presence of the 500th watermark embedded in this image. Next we examine the robustness of the above watermarked image against JPEG compression with standard quality and `StirMark' random geometric distortions from StirMark 3.1. The results are illustrated in Figures 3{5. We can see from Figures 3 and 4 that the correct (500th) watermark is detected from the JPEG compression image of standard quality, but the watermark detection from the `StirMark' attacked image ends in failure. Thus we design the deconvolution lter in the 1 99 324 2 case of `StirMark' random geometric distortions. Training data are 300 pairs of watermark fw(k)g and watermarked coecient fu(k)g, which are di erent from 1000 watermarks used in testing. The initial value of h(k; l)'s and r(k)'s are, respectively, h (k; l) = 1 for k = l; = 0 for k 6= l; and r (k) = 0: We show the result of the watermark detection with the deconvolution lter in Figure 5. As the result, the response rW0 (l) has the peak whose location corresponds to that of the correct (500th) watermark. Hence the watermark extracting process is improved as expected. (0) (0) 6 Concluding Remarks Using the proposed technique, we can revise the watermark detector of the DCT-based watermarking system against other image operator f ; e.g., ltering, sharpening, scaling, and so on. The design method can also be applied to the watermarking system using another signal transformation method such as the DWT-based watermarking method. We believe that based on the results we can develop and improve the watermarking technology. These results will be reported in forthcoming papers. In the case that the image operator f is not known or a set of input-output pairs fx; zg of f is not obtained, the design problem can be formulated as a kind of blind deconvolution problem, that is, the problem of nding H; r; and v in Eq. (16) from processed or attacked watermarked image(s) z: It seems that this problem is dicult because the distortion (convolution) model of Eq. (14) is generally a shift-variant system[7]. The authors would like to make an attempt to solve this problem. : Response of the watermark detector to the watermarked image. Figure 2 : Response of the watermark detector to the JPEG compression image of standard quality Figure 3 References [1] B. Macq (Guest Editor), Special Issue on Identi cation and Protection of Multimedia Information, Proc. IEEE, vol.87, no.7, July 1999. [2] G. C. Langelaar, I. Setyawan, and R. L. Lagendijk, \Watermarking Digital Image and Video Data," IEEE Signal Processing Magazine, vol.17, no.5, pp.20-46, Sep. 2000. [3] StirMark: http:// www.cl.cam.ac.uk/ ~fapp2/ watermarking/ stirmark [4] B. Widrow and S. D. Stearns, Adaptive Signal Processing, Prentice-Hall, Englewood Cli s, New Jersey, 1985. [5] A. Piva, M. Barni, F. Bartoline, and V. Cappellini, \DCT-Based Watermark Recovering without Resorting to the Uncoruppted Original Image," Proc. IEEE Int. Conf. Image Processing, vol.1, pp.520-523, Oct. 1997. [6] H. Inoue, A. Miyazaki, and T. Katsura, \An Image Watermarking Method based on the Wavelet Transform," Proc. IEEE Int. Conf. Image Processing, vol.1, pp.296300, Oct 1999. [7] R. Liu and L. Tong (Guest Editor), Special Issue on Blind System Identi cation and Estimation, Proc. IEEE, vol.86, no.10, Oct. 1998. : Response of the watermark detector to the `StirMark' attacked image. Figure 4 : Response of the watermark detector using the deconvolution lter to the `StirMark' attacked image. Figure 5
https://openalex.org/W3024190241	https://periodicos2.uesb.br/index.php/geo/article/download/6150/4756, https://www.redalyc.org/journal/5743/574363075002/574363075002.pdf	es		Ilegalidad de la tenencia y desigualdad en la distribución de la tierra en Ecuador como condiciones de vulnerabilidad	Geopauta	2,020	cc-by	5,538	Volume 4, nº. 1 2020 ISSN: 2594-5033 http://periodicos2.uesb.br/index.php/geo DOI: https://doi.org/10.22481/rg.v4i1.6150 Ilegalidad de la tenencia y desigualdad en la distribución de la tierra en Ecuador como condiciones de vulnerabilidad Desigualdade na distribuição e ilegalidade na posse da terra no Equador como determinantes da vulnerabilidade. Inequality in Distribution and Illegality in Land Tenure in Ecuador as Vulnerability Determiners. Julio Ramiro León Paz 1 http://orcid.org/0000-0002-6278-7852 Amanda Rivera 2 http://orcid.org/0000-0002-9162-7698 1 PósGrado em Geografia - Pontificia Universidad Católica del Ecuador – Quito, Equador y Universidad Andina Simón Bolívar – Quito, Equador email: ramiroleonpaz@gmail.com 2 Ingeniera ambiental-Universidad Andina Simón Bolívar –Quito, Equador– email . escenario77@gmail.com Resumen El artículo aborda la inequidad en la repartición y la ilegalidad en la tenencia de la tierra en el Ecuador y como estos factores son condicionantes de vulnerabilidad, tomando en cuenta dos casos de estudio específicos como son los cantones de Pedernales en la provincia de Manabí y de Rioverde en la provincia de Esmeraldas. Se utilizó una metodología de trabajo empírica con levantamiento de campo insitu e investigativa con la aplicación del Modelo PAR (Presión y Liberación de los Desastres) y el coeficiente de GINI, para la tenencia de la tierra, también se observa que las presiones dinámicas del territorio afectan las población económicamente menos favorecida, concluye que la gestión adecuada de la tierra es una acción que permitirá minimizar el riesgo existente y reducir el riesgo a futuro. Palabras clave: Inequidad. Vulnerabilidad. Reducción de Riesgos. Resumo Este artigo trata da desigualdade na distribuição e ilegalidade da posse da terra no Equador e como esses fatores são fatores determinantes da vulnerabilidade, levando em consideração dois estudos de caso específicos, como os cantões de Pedernales, na província. Manabí e Rioverde, na província de Esmeraldas. Utilizou-se uma metodologia empírica de trabalho com uma pesquisa de campo in situ e investigativa, com a aplicação do Modelo PAR (Pressão e Liberação Desastres) e o coeficiente GINI, para a posse da terra, também é observado que as pressões dinâmicas do território afetam a população economicamente menos favorecida em uma porcentagem maior, conclui-se que o manejo adequado da terra é uma ação o que minimiza o risco existente e reduz o risco futuro. Palavras-chave: Desigualdade. Vulnerabilidade. Redução de risco. Abstract This article deals with inequality in the distribution and illegality of land tenure in Ecuador and how these factors are determinants of vulnerability, taking into account two specific case studies, such as the cantons of Pedernales, in the province. Manabí and Rioverde, in the province of Esmeraldas. An empirical methodology of work was used with an in situ and investigative field research, with the application of the PAR Model (Pressure and Disaster Release) and the GINI coefficient, for land tenure, it is also observed that the dynamic pressures of the territory affect the economically less favored population in a higher percentage, it is concluded that the proper management of the land is an action that minimizes the existing risk and reduces the future risk. V. 4, n. 1, 2020 http://periodicos2.uesb.br/index.php/geo Este é um artigo de acesso aberto sob a licença Creative Commons da CC BY 34 Ilegalidad de la tenencia y desigualdad en la distribución de la tierra en Ecuador como condiciones de vulnerabilidad PAZ,R.L. RIVERA, A. Keywords: Inequity. Vulnerability. Reduction of Riesgos. Recebido em: 20/01/2020 Aceito para publicação em: 30/03/2020 “Vale, pero millones de veces más la vida de un solo ser humano que todas las propiedades del hombre más rico de la tierra” Ernesto “Che” Guevara Introducción La inequidad en el proceso de repartición de la Tierra en el Ecuador se ha venido dando a lo largo de la historia, principalmente con el aparecimiento de los booms económicos relacionados a la agro exportación y a la extracción del petróleo, lo que dio como resultado el apoderamiento de grandes extensiones de terreno por parte de las familias más acaudaladas del país, ocasionando el asentamiento irregular alrededor de las haciendas de pobladores que trabajaban dentro de las mismas; el principal objetivo de este artículo es dar a conocer como la inequidad en la repartición de la tierra y la ilegalidad de posesión de la misma, son factores subyacentes que se convierten en condicionantes de vulnerabilidad para la población aumentando el riesgo de desastres en el país. El artículo planteado se basa en el análisis de la inequidad en la repartición de la tierra y la ilegalidad en la tenencia de la misma como condicionantes de vulnerabilidad, por lo que se analizara el porcentaje de legalidad de las tierras en los cantones de Rioverde y Pedernales y el por qué hay niveles tan bajos de legalización, además de observar cómo afecta al desarrollo social la inequitativa repartición de recurso tierra, dichos resultados servirán para poder evidenciar la problemática y motivar a que los gobiernos cuenten con información sobre la propiedad de la tierra, que ayuden a aumentar el porcentaje de legalidad en la tenencia de la tierra, una equitativa repartición de los recursos y establecer medidas de reducción de riesgos, respuesta y recuperación post desastre por lo que se ha hecho un trabajo de levantamiento de datos en campo e investigativo en gabinete para así poder establecer la correlación de estas dos variables en el aumento de la vulnerabilidad. V. 4, n.1, 2020 http://periodicos2.uesb.br/index.php/geo Este é um artigo de acesso aberto sob a licença Creative Commons da CC BY 35 Ilegalidad de la tenencia y desigualdad en la distribución de la tierra en Ecuador como condiciones de vulnerabilidad PAZ,R.L. RIVERA, A. El Ecuador es uno de los países más pequeños de América del Sur, sin embargo tiene uno de los índices más altos de inequidad en el acceso de la tierra con un coeficiente de Gini de 0.81, de acuerdo a lo descrito anteriormente se observa que la ilegalidad en la tenencia de la tierra es un problema más crítico aún ya que en poblaciones que fueron afectadas por el Terremoto del 16 de abril del 2016, como el caso de Pedernales en la provincia de Manabí tan solo el 35% de los predios afectados tenían escritura, el mismo caso se da en el cantón Rioverde en la provincia de Esmeraldas donde tan solo el 23% de los predios están legalizados. Ahora bien, si se entiende que la vulnerabilidad es un proceso eminentemente social por lo que hace referencia a las características de una población o sistema, que los hace susceptibles a los efectos dañinos de una amenaza y lo relacionamos con lo descrito en párrafos anteriores podremos visualizar que la falta de acceso al recurso, en este caso la tierra y la necesidad de recibir servicios como educación, salud o conectividad han hecho que la población menos favorecida se ubique en lugares con un mayor grado de exposición ante las amenazas naturales preexistentes mediante invasiones y asentamientos ilegales. Ecuador en la última década se caracterizó por la construcción de grandes obras de infraestructura en ejes como salud y educación, teniendo la particularidad que se ubicaban en los centros poblados principales, por lo que se eliminaron los servicios de este tipo en las comunidades vecinales casi obligando de manera involuntaria a un traslado de la población buscando estos beneficios para sus familias generando condiciones de vulnerabilidad social ante las amenazas naturales existentes. La inequidad e ilegalidad de la tierra en nuestro país es un condicionante de vulnerabilidad debido a que expone a los elementos ante posibles eventos peligrosos que de un simple proceso natural termina siendo un desastre. Una mejor distribución del recurso tierra es una acción para reducir el riesgo presente y minimizar el riesgo futuro. Inequidad: marco teórico La Inequidad se defina como la diferencia existente entre los grupos sociales que conforman una sociedad, la desigualdad de oportunidades para poder acceder a recursos y servicios como vivienda, educación o salud y como resultado de esto la ausencia de un desarrollo para poder tener una vida digna. Los polos de desarrollo son zonas geográficas relativamente reducidas en las que se estimula la localización de actividades industriales para que impulsen la actividad V. 4, n.1, 2020 http://periodicos2.uesb.br/index.php/geo Este é um artigo de acesso aberto sob a licença Creative Commons da CC BY 36 Ilegalidad de la tenencia y desigualdad en la distribución de la tierra en Ecuador como condiciones de vulnerabilidad PAZ,R.L. RIVERA, A. económica en un área geográfica de mayor amplitud. Aunque con algunos matices diferenciadores, también se denominan polos de crecimiento y polos de promoción industrial, ahora bien tomando en cuenta la evolución urbana del Ecuador se localiza dos Polos de desarrollo históricamente bien definidos tanto en la sierra como en la costa, que son Quito y Guayaquil, los mismos que han sido ejes en el desarrollo del resto de ciudades cercanas a ellas. La Unidad de Producción Agropecuaria -UPA se refiere a una extensión de tierra de 500 m² o más, dedicada total o parcialmente a la producción agropecuaria, considerada como una unidad económica. Según el EIRDi - 2009 la vulnerabilidad se define como las características y circunstancias de una comunidad, sistema o bien, que los hacen susceptibles a los efectos dañinos de una amenaza, hay que tomar en cuenta que tanto la exposición como la vulnerabilidad son resultados de determinadas acciones humanas o procesos sociales. El UNISDRii-2009 define los desastres como una grave interrupción en el funcionamiento de una comunidad o sociedad que ocasiona una situación generalizada de impactos y pérdidas humanas, materiales, económicas y ambientales; que exceden la capacidad de la comunidad o la sociedad afectada para hacer frente a la situación mediante el uso de sus propios recursos. Se entiende por amenaza como un fenómeno, sustancia, actividad humana o condición peligrosa que puedan ocasionar la muerte, lesiones u otros impactos a la salud, al igual que daños a la propiedad, la perdida de medios de sustento y de servicios, trastornos sociales y económicos o daños ambientales (EIRD- 2009). El riesgo de desastre se define como las perdidas probables que ocasionaría un desastre, en términos de vidas humanas, las condiciones de salud, los medios y los servicios, que pueden ocurrir en una comunidad o sociedad en particular en un periodo especifico de tiempo en el futuro (EIRD- 2009). La gestión de riesgo de desastre definida en forma genérica se refiere a un proceso social cuyo fin último es la prevención, la reducción y el control permanente de los factores de riesgo de desastre en la sociedad, en consonancia con, e integrada al logro de pautas de desarrollo humano, económico, ambiental y territorial, sostenibles. El coeficiente de gini es un indicador utilizado para medir el nivel de desigualdad en el acceso al recurso existente entre los habitantes de una región, es decir el coeficiente demuestra hasta qué punto la distribución de ingreso entre individuos u hogares o el acceso a los principales recursos dentro de una economía se aleja de la V. 4, n.1, 2020 http://periodicos2.uesb.br/index.php/geo Este é um artigo de acesso aberto sob a licença Creative Commons da CC BY 37 Ilegalidad de la tenencia y desigualdad en la distribución de la tierra en Ecuador como condiciones de vulnerabilidad PAZ,R.L. RIVERA, A. distribución equitativa, cuando el resultado se acerca más a 1 quiere decir que existe una mayor desigualdad en la repartición de los recursos. El modelo par, modelo de presión y liberación de los desastres, explica la forma como los factores subyacentes y causas de fondo incorporadas en la vida diaria dan origen a presiones dinámicas que afectan grupos particulares y conducen a condiciones específicamente inseguras; tomando en cuenta que dos dinámicas generan el riesgo de desastre y en su caso extremo el desastre, la amenaza y la vulnerabilidad creciente. La tenencia de la tierra en el Ecuador a lo largo de la historia. En nuestro país los procesos de apoderamiento de la tierra inicia en la época de la colonización española, a medida que pasaron los años el sistema de apropiación arbitraria de las tierras se intensificó principalmente en la sierra cuando se obligó a los indígenas a retirarse de los valles llevándolos a ubicar en las zonas de montaña, el resultado de la conformación y ubicación en el territorio al final del período colonial, muestra una red de ciudades establecidas en la sierra, demostrando una tendencia hacia las desigualdades en la tenencia de la tierra tanto en sus dimensiones como en la calidad de la misma. Para inicios del siglo XX el avance del modelo agroexportador y la intensificación de la producción de banano, café y cacao en la costa ecuatoriana genera que los grupos económicos más fuertes acaparen el recurso tierra dejando sin acceso a los menos favorecidos, además existe un cambio significativo en la relación de la población con el territorio, debido a que estos procesos de crecimiento económico desenfrenado, dan como resultado la formación de nuevos asentamientos cercanos a las zonas de producción convirtiendo así el paisaje en ciudades flotantes que dependen de la cosecha, y que con el paso del tiempo se convierten en ciudades establecidas pero sin servicios y principalmente asentados en zonas de riesgo. La tenencia de la tierra en el país siempre ha estado ligada a los grandes booms económicos que han dado a lo largo de la historia, a mediados de siglo pasado con la extracción del petróleo se dio un poblamiento en la zona limítrofe del país y un cambio en el uso de suelo en la zona causando un deterioro ambiental ya que se transformó grandes cantidades de selva amazónica en pastizales para ganadería. La Inequidad del acceso al recurso tierra en Ecuador En el Ecuador un 98.3% del recurso tierra se encuentra en manos privadas pero con una desigualdad total de repartición, según datos del Censo Agropecuario del año V. 4, n.1, 2020 http://periodicos2.uesb.br/index.php/geo Este é um artigo de acesso aberto sob a licença Creative Commons da CC BY 38 Ilegalidad de la tenencia y desigualdad en la distribución de la tierra en Ecuador como condiciones de vulnerabilidad PAZ,R.L. RIVERA, A. 2000, las UPAiii menores a 5 hectáreas representan al 63,96% de las UPAS totales pero acceden solamente al 6,53% de la superficie agrícola del Ecuador, lo que quiere decir que cada una tiene un promedio de 1,4 ha, mientras que las propiedades entre 50 y 100 ha representan el 3,97 ha de las UPA y un 18.33% de la superficie agrícola, mientras que las propiedades superiores a 500 ha constituyen menos del 1% de las UPA pero controlan alrededor del 16% de la superficie agrícola, promediando un tamaño de 1400 ha, lo que hace una diferencia directa de 1000 a 1 entre los propietarios de grandes y pequeñas extensiones de terreno. La inequidad en la tenencia de tierra en el Ecuador es una de las más altas a nivel de América Latina tomando en cuenta que somos un país relativamente pequeño en comparación con otros de la región, el coeficiente de Gini utilizado en este caso para medir la desigualdad en el acceso al recurso tierra señala um 0.81 lo que es un resultado alarmante, el 99.9% de las UPA tienen extensiones inferiores a 640 ha, es decir tan solo el 0.01% tiene extensiones superiores a la mencionada anteriormente. Mapa 1- Índice de Gini a Nivel Provincial – Equador Fuente: Atlas de la Tenencia de la Tierra en Ecuador Elaborado por: SIPAE 2010-2011 La inequidad en nuestro país es tan marcada que la mitad de las UPA tiene una extensión que no permite el desarrollo adecuado de los campesinos, el aumento en la concentración de la tierra en condiciones de presión a los propietarios de pequeñas extensiones de terreno está generando que la población se quede sin tierra literalmente, V. 4, n.1, 2020 http://periodicos2.uesb.br/index.php/geo Este é um artigo de acesso aberto sob a licença Creative Commons da CC BY 39 Ilegalidad de la tenencia y desigualdad en la distribución de la tierra en Ecuador como condiciones de vulnerabilidad PAZ,R.L. RIVERA, A. los datos existentes en el III Censo Agropecuario establecen que existen 165.000 UPA que no llegan a 0.5 ha. Esta situación de inequidad se presenta en el país a pesar de que en este existieron dos Reformas Agrarias entre los años 60 y 70, las mismas tenían el objetivo de frenar el sistema latifundista que había despojado a los indígenas de sus tierras, el principal problema fue que los territorios entregados eran de mala calidad para la agricultura o estaban ubicados en zonas de riesgo, hay que tomar en cuenta que los propietarios de minifundios en muchos casos no tenían acceso al agua de riego por lo que existía una baja productividad por parcela, además de la falta de recursos para la inversión de tecnología para poder mejorar el rendimiento de sus tierras. La Inequidad en la repartición de le tierra como condicionante de vulnerabilidad. Tomando en cuenta que la vulnerabilidad es un proceso eminentemente social, y que un país como el nuestro está rodeado de amenazas naturales de todo tipo, el que un evento peligroso se considere o no riesgo, dependerá directamente de que el territorio donde este ocurra se encuentre poblado, es decir del grado de exposición existente, por lo que se puede decir que los desastres constituyen fenómenos sociales mas no de tipo natural y que evidencian problemas no resueltos del desarrollo. En muchas poblaciones del país la falta de acceso a los recursos básicos generan condiciones de pobreza extrema, consecuencia de esto presentan una gran dificultad para recuperarse ante un desastre, el fortalecimiento social de estas poblaciones se puede tomar en cuenta como una medida eficaz de reducción del riesgo; pero ante todo lo expuesto la pregunta es ¿Por qué la inequidad en la repartición de la tierra condiciona la vulnerabilidad?, al entender a la vulnerabilidad como un proceso de construcción social nos damos cuenta que toda acción realizada por el ser humano puede aumentar o reducir el riesgo existente. Como se ha establecido en párrafos anteriores la concentración del recurso tierra en pocas manos ha ocasionado que las poblaciones menos favorecidas se encuentran expuestas a distintas amenazas, como pendientes escarpadas, riveras de ríos, cerca de zona de licuefacción y para lograr estos asentamientos se realiza en muchos casos deforestaciones e impactos negativos al medio ambiente aumentando así el factor de exposición ante las amenazas presentes; la Política de Estado en tema de servicios como educación y salud en los últimos 10 años fue la de mejorar la atención y el sistema en V. 4, n.1, 2020 http://periodicos2.uesb.br/index.php/geo Este é um artigo de acesso aberto sob a licença Creative Commons da CC BY 40 Ilegalidad de la tenencia y desigualdad en la distribución de la tierra en Ecuador como condiciones de vulnerabilidad PAZ,R.L. RIVERA, A. general, con la construcción de mega obras centralizadas, principalmente en la temática de educación, se eliminaron las escuelas unidocentes, y se crearon las llamadas “Escuelas del Milenio” en los poblados con mayor población obligando a una migración interna mal llamada voluntaria, puesto que los campesinos para que sus hijos puedan acceder a este recurso debieron movilizarse hacia las centralidades, lastimosamente la tierra ya estaba ocupada, entonces esta población flotante al no tener acceso al recurso se asientan literalmente donde hay espacio, con construcciones precarias y poco técnicas pero con el único objetivo de tener este servicio. Imagen 1- Modelo de Presión y Liberación del Desastre (PAR) CAUSAS DE PRESIONES FONDO DINAMICAS Acceso Limitado a: Recursos Tierra, Sistemas económicos de desarrollo Crecimiento Poblacional Urbanismo Acelerado Falta de Servicios CONDICIONES DESASTRE INSEGURAS AmbienteFrágil: Localización peligrosa. Infraestructura poco técnica. Economía Frágil: RIESGO Bajos ingresos lo que AMENAZA conlleva una precariedad en X la vida VULENRABIL Sociedad Vulnerable: IDAD Grupos vulnerables en riesgo. Acciones Públicas: Falta de personal capacitado y acciones de prevención AMENAZAS TERREMOTOS INUNDACIONE S ERUPCIONES VOLCANICAS DESLIZAMIEN TOS SEQUIAS Fuente: El Autor (2019) El mismo caso se presenta en la ganadería y agricultura, como las “buenas” tierras ya están ocupadas se realiza sembríos poco técnicos en pendientes lo que ocasiona que el suelo pierda su compactación y se erosione con las pisadas del ganado lo que conlleva a aumentar el riesgo de deslizamientos de tierra y crezca la vulnerabilidad de las poblaciones asentadas en las faldas de las montañas, otro problemática que se ha presentado en la última década es el crecimiento población y la expansión urbana desenfrenada, la falta de productividad por la mala calidad de las tierras hace que la gente migre y los terratenientes han cambiado el uso de suelo a un sistema de urbanización, dividiendo y vendiendo lotes pequeños en su mayoría que no exceden los 300mts, sin la entrega de servicios básicos como dicta la ley, pero la falta de organismos de control hace que no exista obligatoriedad en estos procesos. La mayoría de estos predios se encuentran en zonas inseguras, pero por la necesidad, desesperación y falta de información de las personas con menos recursos los V. 4, n.1, 2020 http://periodicos2.uesb.br/index.php/geo Este é um artigo de acesso aberto sob a licença Creative Commons da CC BY 41 Ilegalidad de la tenencia y desigualdad en la distribución de la tierra en Ecuador como condiciones de vulnerabilidad PAZ,R.L. RIVERA, A. adquieren, en muchos casos, si no es en todos, se convierten en asentamientos irregulares debido a su imposibilidad de ser legalizados por su ubicación de esta manera existe un crecimiento de la vulnerabilidad física por exposición territorial y la vulnerabilidad social por todos los fenómenos que conllevan este tipos de asentamientos. La ilegalidad en la Tenencia de la Tierra como condicionante de vulnerabilidad La ilegalidad en la tenencia de la tierra es uno de los principales problemas de nuestro país en temas catastrales, desde las herencias sin sustento hasta la venta de terrenos por los traficantes de tierra, son obstáculos con los que tienen que lidiar día a día los distintos GADM´s del Ecuador, las invasiones a causa de la falta de acceso al recurso y luego la exigencia de los usuarios para que dichas tierras sean legalizadas y se entreguen todos los servicios básicos causan un freno en el desarrollo territorial. Caso Pedernales La ilegalidad en la tenencia no era tomada en cuenta al momento de hablar de Gestión de Riesgos en nuestro país, a partir del 16 de abril de 2016 cuando ocurrió el terremoto en las provincias de Esmeraldas y Manabí, mismo que causo una seria afectación a toda la zona costera del Ecuador. Tabla 1- Población y Número de predios por parroquia CABECERA CANTONAL Y PARROQUIALES Pedernales NUMERO DE HABITANTES 24073 SUPERFICIE POR (has) 1510 NUMERO DE PREDIOS 9705 Cojimíes 6165 173 4134 10 de Agosto 210 26.36 144 Atahualpa 232 16.84 163 TOTAL 30680 1726.20 14464 Fuente: PROYECCIÓN INEC 2015. CENSO 2010. IEE, SENPLADES, IGM Las autoridades se dieron cuenta del serio obstáculo que presentaba la ilegalidad de la tenencia de la tierra, según datos entregados por el MIDUVIiv unos meses después del desastre se pudo determinar que el 53% de los predios afectados no estaban legalizados o catastrados, por lo que la entrega de ayuda para nuevas viviendas o para reparación de afectaciones leves en las construcciones se tuvieron que retrasar hasta solucionar el primer problema, aumentando la vulnerabilidad de la población que ya se encontraba en hogares temporales luego del desastre. V. 4, n.1, 2020 http://periodicos2.uesb.br/index.php/geo Este é um artigo de acesso aberto sob a licença Creative Commons da CC BY 42 Ilegalidad de la tenencia y desigualdad en la distribución de la tierra en Ecuador como condiciones de vulnerabilidad PAZ,R.L. RIVERA, A. El aumento poblacional del cantón es producto de la acción de los traficantes de tierras, cuyo modo de operación era el de promocionar a Pedernales cómo un lugar de grandes oportunidades laborales con la finalidad de ofrecer terrenos a precios asequibles. El recurso suelo invadido pertenecía a personas que no residían permanentemente en Pedernales, situación que facilitaba su ilegal posesión, a diferencia de varias familias terratenientes que poseen grandes extensiones de tierra. Los asentamientos humanos irregulares, la mayoría ubicados en zonas de riesgos, la construcción precaria obviando las normativas y procesos adecuados de construcción han llevado a que durante años se vaya gestando un contexto altamente vulnerable, que por falta de previsión, decisión política ocasionó que las invasiones se apropiaran del territorio. Mapa 2 - Viviendas semaforizadas en la zona urbana del Cantón Pedernales Fuente: Cartografía Básica del INEC, IGM, SNI, MIDUVI, SENPLADES 2016 Elaborado por: Unidad de Diseño #1 Pedernales Como se observa en el mapa número 2, la afectación con daños considerables, catalogados como inseguros o restringidos suman un numero de 4998 registros que representan al 50% del total de predios de la zona urbana, de estos tan solo el 47% V. 4, n.1, 2020 http://periodicos2.uesb.br/index.php/geo Este é um artigo de acesso aberto sob a licença Creative Commons da CC BY 43 Ilegalidad de la tenencia y desigualdad en la distribución de la tierra en Ecuador como condiciones de vulnerabilidad PAZ,R.L. RIVERA, A. tenían posesión legal y registrada en el Departamento de Avalúos y Catastros del Municipio de Pedernales, por lo que se determina que más 3000 predios afectados sin registro, como se mencionó en párrafos anteriores, el problema salió a la luz luego del sismo , instituciones nacionales e internacionales se encontraron con un serio problema al momento de entregar la asistencia a los damnificados Las primeras acciones que se tomaron fue el de legalizar todos estos predios olos que más se pudo como manifestó la persona encargada del avalúos del GAD, a otros tantos que no se pudo legalizar porque estaban ubicados en invasiones se les entrego predios y casa básicamente en el sector “La Chorrera” que después de tres años del desastre aún no se ha podido legalizar del todo dicha situación. Otra problemática como ya se hablo es la falta de accesibilidad al recurso, en la cabecera cantonal de Pedernales se construyeron dos escuelas del milenio, lo que obligo a los pobladores de comunidades como: La Playita, San Vicente, Pata de Pájaro, Tachina, entre otros tuvieron que desplazarse hacia la centralidad o cerca de la misma formando asentamientos irregulares para poder acceder al servicio, por la precariedad de las construcciones el momento del sismo todas fueron afectadas, dejando a estas personas a la intemperie, en busca de ayuda fueron ubicadas en los alojamientos temporales lo que al final ocasiona un perjuicio para el Estado y para la población en sí. Caso Rioverde El crecimiento poblacional de las ciudades costeras en el Ecuador se ha dado de manera desorganizada, siempre caracterizada por asentamientos irregulares y anclados a los distintos booms económicos que han aparecido a lo largo de la historia, se han identificado con los crecimientos económicos de los grandes terratenientes, generando asentamientos en las cercanías de haciendas o pequeños poblados de pescadores. Dicha ubicación geográfica de estos poblados han dado como resultado pequeñas ciudades con carencias de servicios básicos y una alta inseguridad ante las amenazas naturales propias de los procesos geomorfológicos que se dan en el litoral. Según el Plan de Desarrollo y Ordenamiento Territorial la zona urbana cuenta con una extensión de 123,63 has, y una proyección de expansión de la misma dando una totalidad de 317,53 has, hay que recalcar que las cabeceras parroquiales fueron declaradas zonas urbanas, pero no cuentan con la información cartográfica necesaria. V. 4, n.1, 2020 http://periodicos2.uesb.br/index.php/geo Este é um artigo de acesso aberto sob a licença Creative Commons da CC BY 44 Ilegalidad de la tenencia y desigualdad en la distribución de la tierra en Ecuador como condiciones de vulnerabilidad PAZ,R.L. RIVERA, A. Mapa 3- Multiamenazas del Cantón Rioverde Fuente: Cartografía Básica del INEC,BDE, IGM, SNI, MIDUVI, SENPLADES 2016 Elaboración propia. Al no poseer la cartografía para trabajar en el catastro rural, se ha optado por ingresar en un inicio solo los predios que son escriturados por sus propietarios sin V. 4, n.1, 2020 http://periodicos2.uesb.br/index.php/geo Este é um artigo de acesso aberto sob a licença Creative Commons da CC BY 45 Ilegalidad de la tenencia y desigualdad en la distribución de la tierra en Ecuador como condiciones de vulnerabilidad PAZ,R.L. RIVERA, A. existir aun la obligatoriedad de catastrarlos, en cantón se encuentran legalizados apenas el 23% del total de sus predios tanto en la zona urbana como en la zona rural lo que refleja un alto porcentaje de ilegalidad en la tenencia y la presencia de un sinnúmero de asentamientos considerados irregulares. En este cantón la tenencia de la tierra en pocas manos dedicadas en su gran mayoría a la ganadería y la industria camaronera ha dado como resultado el crecimiento de asentamientos irregulares cerca de las grandes haciendas principalmente a lado de las vías principales, en la parroquia de Montalvo, comunidades como Walte, La Muralla, Machin, ubicadas en las zonas altas del cantón, por falta de trabajo su población se ha terminado desplazando para buscar trabajos temporales, asentándose cerca de las riveras del Río Verde, aumentando la vulnerabilidad en caso de la amenaza latente de inundaciones. El caso de la parroquia Chontaduro es diferente ya que por estar dedicada en su gran mayoría a la agricultura de subsistencia la migración se ha dado por otras circunstancias, cuando el Gobierno Nacional decidió cerrar las escuelas unidocentes, comunidades como Las Cumbres, Chorreron, Papayal quedaron aisladas de recibir este servicio teniendo que caminar hasta 4 horas al día para poder llegar a clases, por lo que muchos pobladores realizaron invasiones cercanas a los caminos de acceso, que no pudieron ser controladas por el GAD, y que a lo largo del tiempo se ha transformado en un serio problema, tanto en el ámbito privado como en el público. Dichos asentamientos por ser irregulares se han asentado en las riberas de los ríos y en pendientes pronunciadas donde son altamente vulnerables por exposición física, especialmente en la temporada invernal que en este cantón suele ser muy severa, según datos entregados por la jefatura de riesgos del GAD Municipal solo este año se ha perdido cerca de 10 vidas y cuantiosas cifras económicas por los deslizamientos de tierra en asentamientos irregulares. Consideraciones finales Según la Constitución de la República del Ecuador del año 2008 la Gestión de Riesgo debe ser tomada como una transversalidad en la ejecución de proyectos y principalmente en el Ordenamiento Territorial de los GAD, por lo que es de suma importancia comenzar a trabajar de manera exhaustiva en una ley que regule la gestión de riesgos en el áís o legislación asociada, que contemple temas de tenencia de tierras en V. 4, n.1, 2020 http://periodicos2.uesb.br/index.php/geo Este é um artigo de acesso aberto sob a licença Creative Commons da CC BY 46 Ilegalidad de la tenencia y desigualdad en la distribución de la tierra en Ecuador como condiciones de vulnerabilidad PAZ,R.L. RIVERA, A. situaciones de desastres y facilite la aplicación de directrices dentro del GAD para que fortalezcan esta temática. La ilegalidad en la tenencia de la tierra es uno de los problemas más graves que tiene el Ecuador, más aun cuando este condiciona la vulnerabilidad creciente ante las amenazas existentes, post desastre los procesos de reconstrucción o entrega de ayuda para reconstrucción se vuelven casi imposibles por la irregularidad de los asentamientos. Los GAD´s deben hacer campañas masivas de legalización de tierras que sirvan para ayudar a los menos favorecidos y de esta manera mejorar el desarrollo del territorio en el que se encuentran. La Inequidad en la repartición de la riqueza y de los recursos naturales han condenado a países enteros a la pobreza generalizada, y más aún en el tema de la tierra a poblaciones menos favorecidas a exponerse ante los fenómenos naturales en búsqueda de un mejor estilo de vida, se debe establecer lineamientos a nivel nacional sobre la tenencia y ocupación de la tierra pública y privada. Se debe generar Planes de Gestión de Riesgos para poder minimizar el riesgo existente en localidades asentadas en zonas de riesgo y darles soluciones para que puedan llevar un modo de vida digno. Para todos los asentamientos que se deben regularizar o desplazar se tienen que tomar en cuenta los medios y los modos de vida de estas personas para así no transgredir sus derechos fundamentales. Los proyectos de desarrollo urbano deben ir de la mano con la temática de gestión de riesgos para que tenga un funcionamiento adecuado y cuando existan eventos peligrosos no ocurra una pérdida de infraestructura o vidas, y que los recursos invertidos en estos proyectos no sea dinero perdido. Referências Abhas K. Jha, Todd W. Miner, and Zuzana Stanton-Geddes (2013). Building Urban Resilience, The World Bank, Sidney Anne-Catherine Chardon & Juan Leonardo González (2002). Indicadores para la Gestión de Riesgos, Universidad Nacional de Colombia. Manizales, Ben Wisner, Piers Blaikie, (2005). At Risk. Routledge, Londres, V. 4, n.1, 2020 http://periodicos2.uesb.br/index.php/geo Este é um artigo de acesso aberto sob a licença Creative Commons da CC BY 47 Ilegalidad de la tenencia y desigualdad en la distribución de la tierra en Ecuador como condiciones de vulnerabilidad PAZ,R.L. RIVERA, A. Terry Cannon; Piers Blaikie; Ian Davis (2003). At Risk: Natural Hazards, People's Vulnerability and Disasters, Routledge, Londres, BRASSEL, HERRERA y LAFORGE (2008). ¿Reforma Agraria en el Ecuador?: viejos temas, nuevos argumentos. SIPAE. Quito, Pierre Gondard y Hubert Mazurek, 30 Años de Reforma Agraria y Colonización en el Ecuador (1964-1994): dinámicas espaciales, Estudios de Geografía, Vol. 10, CEN, CGE, IRD, PUCE 2001, p. 15-40. SIPAE (2011). Atlas, Tenencia de la Tierra en Ecuador, SIPAE. Quito, V. 4, n.1, 2020 http://periodicos2.uesb.br/index.php/geo Este é um artigo de acesso aberto sob a licença Creative Commons da CC BY 48
https://openalex.org/W2287080577	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0148160&type=printable	English	null	Absence of Tumor-Infiltrating Lymphocyte Is a Reproducible Predictive Factor for Sentinel Lymph Node Metastasis: A Multicenter Database Study by the Brazilian Melanoma Group	PloS one	2,016	cc-by	5,381	RESEARCH ARTICLE Aims Editor: Andrzej T Slominski, University of Alabama at Birmingham, UNITED STATES Received: June 30, 2015 Accepted: January 13, 2016 Published: February 9, 2016 The aim of this study is to confirm the function of tumor-infiltrating lymphocytes (TILs) in sen- tinel lymph node (SLN) metastasis. Results SLN metastasis was detected in 101 of 633 cases (16.1%) and in 93 of 428 patients (21.7%) when melanomas 1mm were excluded. By multiple logistic regression, the absence of TILs was as an independent risk factor of SLN metastasis (OR = 1.8; 95%CI: 1.1–3.0), in addition to Breslow index (greater than 2.00 mm), lymph vascular invasion, and presence of mitosis. Data Availability Statement: Data contain potentially identifying information and are unsuitable for public deposition. Data requests may be sent to the corresponding author. Funding: The authors have no support or funding to report. Materials and Methods This retrospective study included 633 patients with invasive melanoma who underwent sentinel lymph node biopsy in 7 referral centers certified by the Brazilian Melanoma Group. Independent risk factors of sentinel node metastasis (SNL) were identified by multiple logistic regression. Copyright: © 2016 Duprat 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. Absence of Tumor-Infiltrating Lymphocyte Is a Reproducible Predictive Factor for Sentinel Lymph Node Metastasis: A Multicenter Database Study by the Brazilian Melanoma Group João Pedreira Duprat1, Eduard René Brechtbülh1, Bianca Costa de Sá1, João Pedreira Duprat1, Eduard René Brechtbülh1, Bianca Costa de Sá1, Mauro Enokihara2, Jose Humberto Fregnani3, Gilles Landman2, Marcus Maia4, Felice Riccardi5, Francisco Alberto Belfort6, Alberto Wainstein7, Luciana F. Moredo1, Higino Steck8, Miguel Brandão9, Marcelo Moreno10, Eduardo Miranda11, Ivan Dunshee de Oliveira Santos2 , g , , , Felice Riccardi5, Francisco Alberto Belfort6, Alberto Wainstein7, Luciana F. Moredo1, 8 9 10 11 Higino Steck8, Miguel Brandão9, Marcelo Moreno10, Eduardo Miranda11, Ivan Dunshee de Oliveira Santos2 1 AC Camargo Cancer Center, São Paulo, Brazil, 2 Medical School, São Paulo Federal University, São Paulo, Brazil, 3 Barretos Cancer Hospital, Barretos, Brazil, 4 Santa Casa de Misericórdia, São Paulo, Brazil, 5 Santa Rita Hospital, Porto Alegre, Brazil, 6 Sirio Libanês Hospital, Oswaldo Cruz Hospital, São José Hospital, São Paulo, Brazil, 7 Minas Gerais School of Medicine, Belo Horizonte, Brazil, 8 Mario Gatti Hospital, Campinas, Brazil, 9 AMO Clinic, Salvador, Brazil, 10 Medical School, Community University of Chapecó Region, Chapecó, Brazil, 11 Oncology Department, Pernambuco University, Recife, Brazil OPEN ACCESS * joao.duprat@accarmargo.org.br Citation: Duprat JP, Brechtbülh ER, Costa de Sá B, Enokihara M, Fregnani JH, Landman G, et al. (2016) Absence of Tumor-Infiltrating Lymphocyte Is a Reproducible Predictive Factor for Sentinel Lymph Node Metastasis: A Multicenter Database Study by the Brazilian Melanoma Group. PLoS ONE 11(2): e0148160. doi:10.1371/journal.pone.0148160 * joao.duprat@accarmargo.org.br Introduction The Brazilian Melanoma Group (www.gbm.org.br) was founded in 1996 as a multidisciplinary team, comprising oncologists, dermatologists, plastic surgeons, pathologists, nurses, and any professional who was seeking the best strategies for the management of melanoma patients. In November 2000, a multicenter database was initiated, and after 14 years, the complete data on 1008 invasive melanomas have been analyzed, including sentinel lymph node biopsy (SLNB) findings. The criteria for SLNB remain unknown, especially in thin melanomas [1–6]. Developed and emerging countries must direct their economic resources to balance the costs and benefits of medical procedures—particularly with regard to the MSLT-1 results (Multicenter Selective Lymphadenectomy Trial), which showed no survival benefits but highlighted the importance of staging, quality of life by regional disease control, and indications for adjuvant therapy [7]. This database was analyzed to establish Brazilian guidelines for SLNB, because each country requires specific and tailored recommendations [6,8,9]. The racial diversity in Brazil, whose miscegenation is a significant characteristic, makes its population unique–including Cauca- sians, Africans, Asians, and indigenous Brazilians [10,11]. The criteria for SLNB remain unknown, especially in thin melanomas [1–6]. Developed and emerging countries must direct their economic resources to balance the costs and benefits of medical procedures—particularly with regard to the MSLT-1 results (Multicenter Selective Lymphadenectomy Trial), which showed no survival benefits but highlighted the importance of staging, quality of life by regional disease control, and indications for adjuvant therapy [7]. This database was analyzed to establish Brazilian guidelines for SLNB, because each country This database was analyzed to establish Brazilian guidelines for SLNB, because each country requires specific and tailored recommendations [6,8,9]. The racial diversity in Brazil, whose miscegenation is a significant characteristic, makes its population unique–including Cauca- sians, Africans, Asians, and indigenous Brazilians [10,11]. The importance of certain histologic variables, such as Breslow thickness, ulceration, and the presence of mitosis, has been demonstrated with regard to SLNB positivity [1,5,12–15]. In this study, we examined the presence of tumor-infiltrating lymphocytes (TILs) as a prognostic factor, which has not been studied extensively. Thomas and colleagues reported the value of TILs in primary lesions in a large cohort from Australia, Italy, Canada, and the US. They recently concluded that the grade of TILs influences melanoma-specific survival [16]. Azimi et al. [14] and Taylor et al. [17] recently published the value of this factor in SNLB in a large cohort from a single-institution database. Introduction Also, in metastatic disease, the value of TILs was demonstrated in adoptive T-cell therapy using autologous TILs; in this study, the reinfusion of TILs and infusion of IL2 constituted an effective therapy [18]. In this multicenter study, including dermatopathology analyses from several professionals, we sought to determine the risk factors of sentinel lymph node (SLN) metastasis, especially TILs, to confirm them to be a reproducible parameter. Conclusion SLNB can identify patients who might benefit from immunotherapy, and the determination of predictors of SLNB positivity can help select the proper population for this type of therapy. Competing Interests: The authors have declared that no competing interests exist. 1 / 9 PLOS ONE \| DOI:10.1371/journal.pone.0148160 February 9, 2016 Reproducibility of TIL in Melanoma for Sentinel Lymph Node Status The absence of TILs is a reproducible parameter that can predict SLNB positivity in mela- noma patients, since this study was made with several centers with different dermatopathologists. Reproducibility of TIL in Melanoma for Sentinel Lymph Node Status After a consensus meeting in Brazil, melanoma SLNB was indicated for patients with cuta- neous melanoma with a Breslow depth 0.75 mm or < 0.75 mm if presenting with Clark level IV or V involvement, regression, and ulceration. All cases < 0.75 presented those conditions [19]. All participating melanoma centers followed the Brazilian guidelines for the histological evaluation of sentinel lymph nodes (SLNs) [20]. Prior to surgery, patients were evaluated by lymphoscintigraphy to locate SLNs. In most cases, blue dye and a hand-held gamma probe were used to identify sentinel nodes intraoperatively (n = 583, 93.0%). The remaining cases used either one technique or the other. Sentinel lymph nodes were examined as follows: 2 mm cross-sections of the main axis from the entire lymph node were paraffin-embedded. Three levels of each paraffin block were obtained. Four-micron sections were stained with routine hematoxylin and eosin (H&E); three spare unstained sections were subjected to immunohistochemistry for S100 protein, HMB45, and, whenever possible, Melan-A if no micrometastases were noted in the H&E-stained sections [21]. The following variables were recorded: age, gender, race, location, histological type, Breslow thickness, Clark level, ulceration, regression, vertical or radial growth phase, mitosis, lymph vascular invasion, perineural invasion, tumor-infiltrating lymphocytes, peritumoral inflamma- tory infiltration, satellitosis, and sentinel node status. For the purpose of this investigation, intratumoral lymphocytic infiltration was compared present versus absent. Brisk and non- Brisk (mild lymphocytic infiltrate) were lumped together. The study population was characterized using descriptive statistics. The risk for sentinel node metastasis was estimated using the odds ratio (OR) and 95% confidence interval calculated by logistic regression using SPSS version 21.0. Stepwise forward selection was used for the multiple model. The confidence interval for the proportions was calculated by means of MedCalc 13.0. The medical records were analyzed per the Declaration of Helsinki and ethical standards. The patients’ identities were preserved, as were their data, which were assessed only by the investigators. Informed consent was not necessary as it was a retrospective study and only clini- cal data from medical records were analyzed. PLOS ONE \| DOI:10.1371/journal.pone.0148160 February 9, 2016 Materials and Methods This retrospective study included 633 patients with invasive melanoma who underwent SLNB in 7 referral centers certified by the Brazilian Melanoma Group from 2000 to 2015. It belongs to a larger database of 1,343 melanoma patients with and without sentinel lymph node biopsy. The data from each patient were stored in a provisional database reviewed by an attending phy- sician and sent back to the melanoma center for correction due to errors or incompleteness; corrected data were included in the definitive database. A statistician or physician was respon- sible for analyzing the data and responding to any questions raised by the authors. The data- base used for analyses identified patients only by numbers. The study EC29/15 has been approved by A.C. Camargo Cancer Center Ethics Committee on Research. 2 / 9 PLOS ONE \| DOI:10.1371/journal.pone.0148160 February 9, 2016 Results In the database for patients with and without SLNB, the proportion of patients that were sub- mitted to SLNB was shown in Table 1. SLN metastasis was identified in 102 cases, yielding a positivity rate of 16.1% (95%CI: 13.1%– 19.6%) for all cases and 21.7% (95% CI: 17.5%– 26.6%) when melanomas 1mm were excluded. Table 2 describes the risk of SLN metastasis by study variable. The risk of SLN metastasis was greater for nonwhite patients (OR = 2.6; 95% CI: 1.3– 5.03), melanoma in the leg (OR = 4.2; 95% CI: 1.3–14.4), nodular type (OR = 1.9; 95% CI: 1.1–3.3), acrolentiginous type (OR = 3.3; 95% CI: 1.8–6.0), presence of ulceration (OR = 3.1; Table 1. Proportion and positivity rate of sentinel lymph node biopsy according to Breslow categories (n = 1,073). Breslow N SLNB procedure Positive SLNB < 0.75 409 98 / 409 (24.0%) 4 / 98 (4.1%) 0.75–1.00 155 107 / 155 (69.0%) 5 / 107 (4.7%) 1.01–2.00 238 208 / 238 (87.4%) 27 / 208 (13.0%) 2.01–4.00 158 135 / 158 (85.4%) 32 / 135 (23.7%) > 4.00 113 85 / 113 (75.2%) 34 / 85 (40.0%) Total 1,073 633 / 1,073 (59.0%) 102 / 633 (16.1%) SLNB: Sentinel lymph node biopsy. doi:10.1371/journal.pone.0148160.t001 Table 1. Proportion and positivity rate of sentinel lymph node biopsy according to Breslow categories (n = 1,073). Proportion and positivity rate of sentinel lymph node biopsy according to Breslow categories ) PLOS ONE \| DOI:10.1371/journal.pone.0148160 February 9, 2016 3 / 9 Reproducibility of TIL in Melanoma for Sentinel Lymph Node Status Table 2. (Continued) Variable Description N (%) OR (95% CI) Present 417 (66.7%) 1.4 (0.9–2.2) Missing 8 Satellitosis Absent 608 (96.8%) 1.0 Present 20 (3.2%) 3.6 (1.5–9.2) Missing 5 doi:10.1371/journal.pone.0148160.t002 Table 2. (Continued) doi:10.1371/journal.pone.0148160.t002 95% CI: 2.0–4.8), absence of regression (OR = 2.2; 95% CI: 1.2–4.0), vertical growth phase (OR = 4.8; 95% CI: 1.5–15.6), presence of mitosis (OR = 11.5; 95% CI: 2.8–47.6), lymph vascu- lar invasion (OR = 6.9; 95% CI: 2.8–17.2), perineural invasion (OR = 3.6; 95% CI: 1.6–7.9), absence of TILs (OR = 1.5; 95% CI: 1.0–2.5), and satellitosis (OR = 3.6; 95% CI: 1.5–9.2). 95% CI: 2.0–4.8), absence of regression (OR = 2.2; 95% CI: 1.2–4.0), vertical growth phase (OR = 4.8; 95% CI: 1.5–15.6), presence of mitosis (OR = 11.5; 95% CI: 2.8–47.6), lymph vascu- lar invasion (OR = 6.9; 95% CI: 2.8–17.2), perineural invasion (OR = 3.6; 95% CI: 1.6–7.9), absence of TILs (OR = 1.5; 95% CI: 1.0–2.5), and satellitosis (OR = 3.6; 95% CI: 1.5–9.2). Melanoma thickness was associated with SLN metastasis as follows: 0.75–1.00mm (OR = 1.2; 95% CI: 0.3–4.4), 1.01–2.00mm (OR = 3.5; 95% CI: 1.2–10.3), 2.01–4.00mm (OR: 7.3; 95% CI: 2.5–21.4), and >4.00mm (OR: 15.7; 95% CI: 5.3–46.6). Clark levels IV (OR = 4.0; 95% CI: 1.6–9.6) and V (OR = 11.4, 95% CI: 3.9–33.4) also correlated with an increased risk of metastasis. No significant difference was observed in mean patient age according to SLN status between those with (52.2 years; 95% CI: 48.9–55.5) and without metastasis (51.4 years; 95% CI: 50.1–52.6). By multiple logistic regression, the absence of TILs (OR = 1.8; 95% CI: 1.1–3.0) was an inde- pendent risk factor for SLN metastasis, in addition to Breslow index (greater than 2.00 mm), lymph vascular invasion and presence of mitosis (Table 3). Reproducibility of TIL in Melanoma for Sentinel Lymph Node Status Table 2. Risk for sentinel lymph node metastasis by study variable (n = 633). Variable Description N (%) OR (95% CI) Race White 587 (92.9%) 1.0 Nonwhite 45 (7.1%) 2.6 (1.3–5.0) Missing 1 Gender Female 357 (56.4%) 1.0 Male 276 (43.6%) 1.0 (0.7–1.6) Location Head and Neck 44 (7.2%) 1.0 Trunk 257 (42.3%) 2.2 (0.7–7.6) Arm 116 (19.1%) 2.3 (0.7–8.4) Leg 190 (31.3%) 4.2 (1.3–14.4) Missing 26 Histological Type Superﬁcial spreading 426 (71.6%) 1.0 Nodular 99 (16.6%) 1.9 (1.1–3.3) Acrolentiginous 64 (10.8%) 3.3 (1.8–6.0) Lentigo maligna 6 (1.0%) NA Missing 38 Breslow depth <0.75 mm 98 (15.5%) 1.0 0.75–1.00 mm 107 (16.9%) 1.2 (0.3–4.4) 1.01–2.00 mm 208 (32.9%) 3.5 (1.2–10.3) 2.01–4.00 mm 135 (21.3%) 7.3 (2.5–21.4) >4.00 mm 85 (13.4%) 15.7 (5.3–46.6) Clark II 88 (14.1%) 1.0 III 285 (45.6%) 1.7 (0.7–4.1) IV 219 (35.0%) 4.0 (1.6–9.6) V 33 (5.3%) 11.4 (3.9–33.4) Missing 8 Ulceration Absent 465 (73.8%) 1.0 Present 165 (26.2%) 3.1 (2.0–4.8) Missing 3 Regression Absent 475 (75.9%) 2.2 (1.2–4.0) Present 151 (24.1%) 1.0 Missing 7 Growth phase Radial 70 (11.1%) 1.0 Vertical 559 (88.9%) 4.8 (1.5–15.6) Missing 4 Mitosis Absent 98 (16.0%) 1.0 Present 516 (84.0%) 11.5 (2.8–47.6) Missing 19 Lymph vascular invasion Absent 607 (96.8%) 1.0 Present 20 (3.2%) 6.9 (2.8–17.2) Missing 6 Perineural invasion Absent 598 (95.5%) 1.0 Present 28 (4.5%) 3.6 (1.6–7.9) Missing 7 Tumor inﬁltration lymphocytes Absent 401 (64.4%) 1.5 (1.0–2.5) Present 222 (35.6%) 1.0 Missing 10 Peritumoral inﬂammatory inﬁltration Absent 208 (33.3%) 1.0 (Continued) Table 2. Risk for sentinel lymph node metastasis by study variable (n = 633). (Continued) PLOS ONE \| DOI:10.1371/journal.pone.0148160 February 9, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0148160 February 9, 2016 4 / 9 of the four variables included in the model. sidered in the model: 101 (sentinel lymph node metastasis). el: 101 (sentinel lymph node metastasis). mplete information of the four variables included in the model. Number of events considered in the model: 101 (sentinel lymph node metastasis). Reproducibility of TIL in Melanoma for Sentinel Lymph Node Status indications for SLNB. Even if resources are not a concern, unnecessary surgeries can have undesirable side effects, such as lymphedema and paresthesia [24,25]. Based on MSLT I, which did not provide a clear survival benefit of the SLNB procedure, the primary goals are to achieve regional control and identify the patients who should receive adju- vant therapy [7]. The current indications for SLNB per the Brazilian Melanoma Group are Bre- slow depth 0.75 mm or <0.75 mm with Clark level IV or V, regression, and ulceration [19]. An analysis of SNLB parameters is essential for reestablishing its indications. In study popula- tion, in cooperation with several dermatopathologists, we found the absence of TILs to be a reproducible parameter for SLNB indication. But we have also reviewed other established indications. We found that the absence of TILs was a predictive factor of SLN metastasis, independent of Breslow index, lymph vascular, and mitosis status. The value of TILs in the prognosis of mel- anoma has been discussed. A survival benefit is observed for primary lesions with the presence of TILs [14,16]. In the lymph node, a brisk reaction appears to be a prognostic influence in lymph node metastasis, decreasing the occurrence of SLN metastasis [26,27]. Many of these studies are single institution series, but in the present paper there are a number of dermato- pathologists, so we have to stress the importance of the reproducibility. The value of TILs is evident in therapeutic procedures, such as adoptive T cell therapy, with which tumors can regress on re-injection of TILs, especially in association with IL2 [18]. How- ever, there are many types of lymphocytes (T cells, natural killers, dendritic cells, macrophages, and others) that are responsible for TILs—some of which promote tumor growth, whereas oth- ers impede it [28]. Thus, the prognostic significance of TILs must be demonstrated, in conjunc- tion with our findings (analyzed by many dermatopathologists), which will show the reproducibility of this factor. Notably, the presence of brisk TILs has been linked to a lower probability of positive SLNB in univariate and multivariate analyses; others authors have recently shown a similar associa- tion [14,17,29]. They specifically investigated marked intratumoral infiltration and found that the absence of TILs increased the risk of SLN positivity by 25-fold compared with their presence. PLOS ONE \| DOI:10.1371/journal.pone.0148160 February 9, 2016 Discussion The validity of SLNB has continued to be debated extensively [1–6,8,19–22]. For instance, there are no consensus criteria on the indications for thin and thick melanomas, even among physicians who advocate the use of SLNB [23]. In countries such as Brazil, that strive to provide the best medical care with limited resources, efforts must be directed towards optimizing the sentinel lymph node metastasis according to the multiple logistic regression analysis (n = 608). Table 3. Independent predictive factors of sentinel lymph node metastasis according to the multiple logistic regression analysis (n = 608). Variable Category N OR (95% CI) Breslow < 0.75 mm 95 1.0 Reference 0.75–1.0 mm 100 0.8 (0.2–3.5) 1.01–2.00 mm 202 2.7 (0.9–8.0) 2.01–4.00 mm 130 5.3 (1.8–15.8) >4.00 mm 81 10.2 (3.3–31.3) Lymph vascular invasion Absent 588 1.0 Reference Present 20 3.5 (1.3–9.1) Tumor-inﬁltrating lymphocytes Absent 388 1.8 (1.1–3.0) Present 220 1.0 Reference Mitosis No 98 1.0 Reference Yes 510 5.7 (1.3–24.3) OR: Odds ratio, CI: Conﬁdence interval. Number of events considered in the model: 101 (sentinel lymph node metastasis). () 608 cases had complete information of the four variables included in the model. doi:10.1371/journal.pone.0148160.t003 Table 3. Independent predictive factors of sentinel lymph node metastasis according to the multiple logistic regression analysis (n = 608). dependent predictive factors of sentinel lymph node metastasis according to the multiple logistic regressio Number of events considered in the model: 101 (sentinel lymph node metastasis). PLOS ONE \| DOI:10.1371/journal.pone.0148160 February 9, 2016 5 / 9 Reproducibility of TIL in Melanoma for Sentinel Lymph Node Status such thin melanomas. Moreover, Clark level and regression do not appear to be risk factors for SLNB (see below). In our series, melanomas with a thickness between 0.75 and 1 mm had a 4.7% rate of SLN positivity, indicating that there is little value in recommending SLNB in these cases, unless mitosis, ulceration, lymph vascular invasion, or satellitosis is present [12]. Nevertheless, SLN metastasis in thicknesses of 0.75–1 mm is debated. Han et al. (2012) recorded 8% SLN metasta- sis rate in this range of thicknesses versus 5% and 13% with mitosis or ulceration [5]. Over time, Clark level has lost significance and was downgraded in the last AJCC stage sys- tem update in 2009 [30]. In our study, 9 cases with a thickness of less than 1 mm had a positive SLNB, of whom only 1 was Clark level IV, and none was Clark level V. Thus, we believe that Clark level should not be considered in indicating SLNB in thin melanomas. This conclusion is supported by White et al. [31], who found no relationship between Clark level and sentinel sta- tus by multivariate analysis. There is no consensus on whether regression has an adverse or favorable impact on mela- noma survival [3,32–34]. The reproducibility of regression impact is usually poor, primarily because pathologists have failed to agree on the criteria for the diagnosis of regression [35,36]. Our regression criteria stated that there should be complete absence of full-thickness tumor cells (even the in situ component) in a tumor segment that has been replaced by fibrosis. In addition, fibrosis and inflammatory reactions in parts of the melanoma were not considered regression. In a review by Requena et al. [36], full-thickness regression was linked to a worse prognosis. In our series, by multivariate analysis, there was no impact of regression on SLN positivity, and there was only 1case of regression with positive nodes, compared with 7 cases without regression, in the thin melanoma group. Most centers no longer indicate SLNB for thin melanomas [3,5,13,37]. Ulceration is a strong prognostic factor, regardless of tumor thickness. Thin melanomas with ulceration are rare, and patients have a high probability of developing local, regional, and distant metastases [12,14,35]. In our series, ulceration was highly associated with SLN metasta- sis by univariate analysis but was not significant in the multivariate analysis. Based on larger series [1,5,13,14], we believe that patients with ulceration should undergo SLNB, regardless of tumor thickness. Mitotic activity was not discussed in previous recommendations as a predictive factor of SNL metastasis. Nevertheless, because mitotic activity is now considered an important prognostic fac- tor [14,30] that indicates a greater probability of node metastasis, we included it in our analysis. Although mitotic activity is a significant prognostic factor in tumors that are less than 0.75 mm thick, its presence alone is insufficient to justify its use as an indicator for SLNB for such depths [5]—the results regarding the correlation between mitotic index and SLNB metastasis in tumors that are greater than 1 mm are contradictory [13,15]. Moreover, SLNB should only be considered in tumors that are 0.75–1 mm thick if mitosis is present [1]. In this study, mitotic index was an important prognostic factor for SLN metastasis in the univariate and multivariate analysis, and no case with positive SLNs without mitosis was observed in thin melanomas. To sum up, the absence of TILs is a reproducible parameter that can be used as a predictive factor for positive SLNB in melanoma patients. The findings in the literature are controversial regarding tumor thickness as an indication for SLNB. Some studies advocate SLNB for patients with a thickness 1 mm [1–4] or 0.75 mm [5,19], whereas other studies suggest that SLNB should not be performed in cases with a depth of less than 0.75 mm but is indicated for cases with a thickness of 0.75–1 mm in which the mitotic index is greater than 0 and there are ulceration and lymph vascular invasion observed [1,6]. There is a consensus that for lesions with a Breslow thickness > 4mm, SLNB helps only in regional control [23]. In our cohort, SLNB was performed for 69% of tumors with a thickness between 0.75 and 1 mm, in most patients with intermediate thickness (1–4 mm) 87%, and in 75% of those with melanomas that were thicker than 4 mm. In the group with a thickness under 0.75 mm, surgery was performed due to adverse prognostic factors, such as regression, ulceration, and Clark level IV or V, regardless of whether they were substantiated. Even with these bad prognosis factors in this group under 0.75mm, the SLN metastasis rate was 4.1%. Kunte et al. [12] suggested SLNB for lesions that were <1mm thick only if they were associ- ated with a poor prognostic factor, such as ulceration, regression, and Clark level IV or V. These authors noted a 6% rate of SLN metastasis in cases with a depth of less than 0.75 mm and an 8% metastasis rate in cases with a thickness between 0.75 and 1 mm. In the authors view, data do not justify the recommendation of SLNB for melanomas with a thickness of less than 0.75mm, even if mitosis is observed. Ulceration and lymph vascular invasion are rare in 6 / 9 PLOS ONE \| DOI:10.1371/journal.pone.0148160 February 9, 2016 References 1. Wong SL, Balch CM, Hurley P, Agarwala SS, Akhurst TJ, Cochran A et al. Sentinel lymph node biopsy for melanoma: American Society of Clinical Oncology and Society of Surgical Oncology joint clinical practice guideline. 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https://openalex.org/W2007555800	http://diposit.ub.edu/dspace/bitstream/2445/53108/1/584451.pdf	English	null	Moderately Oxidized Oils and Dietary Zinc and α-Tocopheryl Acetate Supplementation: Effects on the Oxidative Stability of Rabbit Plasma, Liver, and Meat	Journal of agricultural and food chemistry	2,010	public-domain	10,719	INTRODUCTION a result of the presence of these compounds in the gastrointestinal tract (5, 8) and in tissues, regardless of whether they are formed in vivo or absorbed from diet (7, 8). Whereas the absorption of some secondary oxidation compounds is possible (4, 5, 7, 9), the absorption of intact LHP is more limited because they seem to be decomposed in the gastrointestinal tract to com- pounds containing hydroxy, keto, or epoxy groups, which can be absorbed (9). There is currently interest in supplementing animal feed with unsaturated fats. This procedure seeks to improve the nutritional value of meat and other animal products not only by increasing the content of polyunsaturated fatty acids (PUFA) but also by reducing the n-6/n-3 ratio (1). However, feed and animal producers should consider not only the fatty acid (FA) composition of fat ingredients added to feeds but also their degree of oxidation because unsaturated fats are prone to oxidization. At low temperatures, for instance, during the storage of these ingredients, the content of lipid hydroper- oxides (LHP) increases until reaching a plateau (2). Thereafter, LHP decompose into secondary oxidation compounds. At high temperatures, the degradation of LHP is accelerated, so mostly secondary oxidation compounds are found in fat, as well as some polymeric compounds (2). High temperatures are achieved during common processes such as refining or frying. In fact, several fat co- and byproducts from the food chain obtained from processes involving high temperatures are currently used as animal feed ingredients (3). ( ) Therefore, evaluating the oxidation of fat is essential before its addition to feeds otherwise it might contain a high amount of primary and/or secondary oxidation compounds that may be absorbed by the animal. This absorption would alter tissue composition and favor its susceptibility to oxidation, which, apart from the negative biological effects related to oxidation compounds, would lead to a decrease in shelf life and meat nutritional quality (1, 10). However, the oxidative status of fat products added to feeds is not always globally assessed (3). Scientific literature is lacking in studies dealing with the incor- poration of moderately oxidized oils in rabbit feeds. Several studies in which heated oils were added to chicken feed reported reduced oxidative stability of tissues (11). Corresponding author (phone þ34-93-4024508; fax þ34-93- 4035931; e-mail atres@ub.edu). Moderately Oxidized Oils and Dietary Zinc and r-Tocopheryl Acetate Supplementation: Effects on the Oxidative Stability of Rabbit Plasma, Liver, and Meat ALBA TRES, RICARD BOU, RAFAEL CODONY, AND FRANCESC GUARDIOLA Nutrition and Food Science Department, XaRTA-INSA, Faculty of Pharmacy, University of Barcelona, Avinguda Joan XXIII s/n, 08028 Barcelona, Spain The aim of this study was to assess the alterations in plasma, liver, and meat oxidative stability and R-tocopherol content when moderately oxidized sunflower oils were added to feeds and when feeds were supplemented with R-tocopheryl acetate (100 mg/kg) and Zn (200 mg/kg). The effects of cooking the meat and its subsequent refrigeration were also studied. When the content of primary oxidation compounds of the oil was high, rabbit plasma, liver, and meat R-tocopherol content was reduced and meat susceptibility to oxidation increased. The addition of oil with a high content of secondary oxidation compounds (oil heated at 140 C, 31 h) to feed also led to an increase in meat susceptibi- lity to oxidation, although it presented an R-tocopherol content similar to that of nonheated oil. Feed supplementation with R-tocopheryl acetate increased tissue R-tocopherol content and improved the oxidative stability of liver and meat. However, in the latter, it was less effective when oil heated at 55 C was added. KEYWORDS: Heated oils; zinc supplementation; R-tocopherol; meat oxidation; cooking; TBA value; FOX; lipid hydroperoxides KEYWORDS: Heated oils; zinc supplementation; R-tocopherol; meat oxidation; cooking; TBA value; FOX; lipid hydroperoxides J. Agric. Food Chem. XXXX, XXX, 000–000 A DOI:10.1021/jf101635b pubs.acs.org/JAFC MATERIALS AND METHODS At 63 days of age, Animals and Diets. The diets were prepared and the animals were housed in the Animal Science Department at the Polytechnic University of Valencia (Spain). This study received prior approval from the Animal Protocol Review Committee of the Polytechnic University of Valencia, and all animal housing, husbandry, and slaughtering conditions were in agreement with current European Union guidelines. Animals and Diets. The diets were prepared and the animals were housed in the Animal Science Department at the Polytechnic University of Valencia (Spain). This study received prior approval from the Animal Protocol Review Committee of the Polytechnic University of Valencia, and all animal housing, husbandry, and slaughtering conditions were in agreement with current European Union guidelines. For meat samples, a modification of the FOX method was used (24), because as explained in ref24 preliminary tests showed that samples could have reached similar final LHP values in different ways due to their different susceptibilities to oxidation. Thus, apart from the final LHP value (at 216 h of incubation) (mmol of CHP equiv/kg of meat), the induction time (IT, h), the maximum LHP value reached (MAXLHP, mmol of CHP equiv/kg of meat), the time to reach the MAXLHP (TMAX, h), and the area under the LHP curve [AUC, (mmol of CHP equiv/kg of meat) h] were also determined. These parameters might provide a deeper insight into the evolution of oxidation in each sample (24). Samples. Oils were taken immediately after the thermal treatment, and peroxide value and p-anisidine value were determined within the following 6 h. Oil samples were stored in glass vials closed with Teflon caps, filled with N2, and frozen at -25 C until the rest of the analyses were performed. Determination of TBA Value. The TBA value of rabbit meat was determined by an acid aqueous extraction method with third-derivative spectrophotometry (25). This method was also used to determine the TBA value of liver and plasma samples, using the modifications described in Tres et al. (22). Feed samples were taken at the end of the feeding trial. Feeds were ground and vacuum-packed in high-barrier multilayer bags (Cryovac BB325; permeability to O2 25 cm3 m-2 day-1 bar-1 at 23 C and 0% relative humidity (RH), ASTMD-3985; Cryovac Europe, Sealed Air S. L., Sant Boi de Llobregat, Spain; approximately 15 g of feed/bag) and stored at -25 C until analysis. Feed analyses were performed in triplicate. Statistics. Tres et al. Plasma samples from each cage were mixed and transferred into microtubes and stored at -80 C until analysis. Livers were immediately removed from carcasses and refrigerated for 5 h. Then, the six livers from each cage were mixed, ground, vacuum-packed in high- barrier multilayer bags (Cryovac BB325; approximately 15 g of liver/bag), and stored at -80 C until analysis. Carcasses were refrigerated for 24 h at 4 C. One leg was taken from each animal of each cage and was hand deboned. Meat from the six legs was mixed, ground, and divided into two parts: raw and cooked meat. Raw meat samples were vacuum-packed in high-barrier multilayer bags (Cryovac BB325; approximately 20 g of meat/ bag) and stored at -25 C until analysis. For cooked meat samples, raw meat was vacuum-packed in high-barrier multilayer bags (Cryovac CN330; permeability to O2 15 cm3 m-2 day-1 bar-1 at 23 C and 0% RH (ASTMD-3985); approximately 5 mm of ground meat paste thickness and 20 g of meat/bag) and then cooked in a water bath at 78 C for 5 min. Cooked meat samples were then stored at -25 C until analysis, whereas refrigerated cooked meat samples were stored at 5 C for 62 days and then frozen at -25 C until analysis. cooking (10). Others such as Se or Zn form part of antioxidant enzymes, such as superoxide dismutase and glutathione peroxi- dase in some animal species (1); thus, it is interesting to study whether the dietary supplementation with zinc alters meat oxida- tion. Dietary supplementation with R-tocopheryl acetate (TA) has been recommended as a measure to protect meat from oxidation during processing, cooking, and storage, especially when feeds include unsaturated fats (1). However, the effect of antioxidant compounds added to feeds might be altered by the concomitant presence of primary and/or secondary lipid oxida- tion compounds. Thus, the aim of this study was to assess the effect of supple- menting rabbit feed with moderately oxidized sunflower oils (SO) with different contents of primary and secondary oxidation compounds. The effects of dietary supplementation with TA (100 mg/kg) and Zn (200 mg/kg) on RT content and on the oxidation and susceptibility to oxidation of rabbit plasma, liver, and meat were also examined. In addition, it was also studied how these dietary factors affected meat RT content and oxidation after cooking and after refrigeration of the cooked meat. Reagents and Standards. Tres et al. Thiobarbituric acid (TBA), RT, and cumene hydroperoxide (CHP) were obtained from Sigma-Aldrich (St. Louis, MO). Xylenol orange was purchased from Scharlab (Barcelona, Spain). Solvents used in RT analysis, polymer content, p-anisidine value, and FOX method were of HPLC grade. Tres et al. Tres et al. cooking (10). Others such as Se or Zn form part of antioxidant enzymes, such as superoxide dismutase and glutathione peroxi- dase in some animal species (1); thus, it is interesting to study whether the dietary supplementation with zinc alters meat oxida- tion. Dietary supplementation with R-tocopheryl acetate (TA) has been recommended as a measure to protect meat from oxidation during processing, cooking, and storage, especially when feeds include unsaturated fats (1). However, the effect of antioxidant compounds added to feeds might be altered by the concomitant presence of primary and/or secondary lipid oxida- tion compounds. rabbits were electrically stunned and killed by cutting carotids and jugulars. From four rabbits in each cage, 20 mL of blood per animal was collected in heparinized tubes and immediately centrifuged at 1450g at 4 C for 10 min. Plasma samples from each cage were mixed and transferred into microtubes and stored at -80 C until analysis. Livers were immediately removed from carcasses and refrigerated for 5 h. Then, the six livers from each cage were mixed, ground, vacuum-packed in high- barrier multilayer bags (Cryovac BB325; approximately 15 g of liver/bag), and stored at -80 C until analysis. Carcasses were refrigerated for 24 h at 4 C. One leg was taken from each animal of each cage and was hand deboned. Meat from the six legs was mixed, ground, and divided into two parts: raw and cooked meat. Raw meat samples were vacuum-packed in high-barrier multilayer bags (Cryovac BB325; approximately 20 g of meat/ bag) and stored at -25 C until analysis. For cooked meat samples, raw meat was vacuum-packed in high-barrier multilayer bags (Cryovac CN330; permeability to O2 15 cm3 m-2 day-1 bar-1 at 23 C and 0% RH (ASTMD-3985); approximately 5 mm of ground meat paste thickness and 20 g of meat/bag) and then cooked in a water bath at 78 C for 5 min. Cooked meat samples were then stored at -25 C until analysis, whereas refrigerated cooked meat samples were stored at 5 C for 62 days and then frozen at -25 C until analysis. rabbits were electrically stunned and killed by cutting carotids and jugulars. From four rabbits in each cage, 20 mL of blood per animal was collected in heparinized tubes and immediately centrifuged at 1450g at 4 C for 10 min. MATERIALS AND METHODS Thermal Treatment of Oils. An unrefined SO was divided in three aliquots to perform the thermal treatment (Table 1). The first aliquot, named fresh sunflower oil (FSO), was not subjected to thermal treatment. A second aliquot, named peroxidized sunflower oil (PSO), was heated at 55 C for 245 h in a heat exchanger under agitation. The third aliquot, named oxidized sunflower oil (OSO), was heated ina direct heating fryer at 140 C for 31 h. Immediately after the thermal treatments, butyl hydro- xytoluene was added to the oils at 100 mg/kg to prevent development of oxidation during oil storage. Taking into account the antioxidants included in the vitamin premix of basal feeds (Table 1), the global antioxidant amount included in the basal feeds was 7 mg (butyl hydro- xytoluene þ butyl hydroxyanisole þ ethoxyquin)/kg of feed, which is much lower than the upper limit allowed by European Regulation (13). Determination of rT in Feed, Plasma, Liver, and Meat. The content of RT in feed, liver, and meat was determined after saponification by HPLC with fluorescence detection (21). Plasma RT content was measured by HPLC-UV detection, without a previous saponification step, as described in Tres et al. (22). Plasma RT content was measured by HPLC-UV detection, without a previous saponification step, as described in Tres et al. (22). Susceptibility to Oxidation of Plasma, Liver, and Meat. To assess the susceptibility of plasma, liver, and meat to lipid oxidation, LHP were measured by the induced FOX method (23). In this procedure, a methanolic extract of samples is mixed with the FOX reagents in glass cuvettes capped with Teflon caps. The mixture (the final volume of the reaction mixture was always 2 mL) is then incubated in the dark to induce LHP formation until absorbance at 560 nm is steady. Absorbance at 560 nm is measured with a spectrophotometer (Shimadzu UV-160A, Shimadzu, Kyoto, Japan), and the LHP value is determined by means of a calibration curve using CHP as standard. The oxidation statusofoilswas evaluated bydetermining their peroxide value [DGF method C-VI 6a (14)], their LHP content using the non- induced ferrous oxidation-xylenol orange (FOX) method (15), and their p-anisidine value [AOCS official method Cd 18-90 (16)]. The polymer content of oils [DGF method C-III 3d (<3%) (17), IUPAC 2508 (>3%) (18)] and their RT content (19) were also determined. Results are shown in Table 1. INTRODUCTION These findings were attributed to the decrease in tissue R-tocopherol (RT) content caused by RT reaction with oxidation compounds in diets, in the gastrointestinal tract or in tissues (11, 12). The addition of heated oils to animal feed does not lead to toxic effects when the fat content of the diets is not above the recommended dose for each animal species and when oil contains <25% polar compounds (4). Some effects on digestibility occur when the polymer content of fats is high (5), but it should be taken into account that several detrimental biological effects have been reported for some oxidation compounds (6-8). These effects are Apart from the fat added to feeds, other factors such as meat mineral content, meat processing, and its storage conditions influence meat oxidation (10). Meat is a source of minerals with high bioavailability, and furthermore, some of them are involved in oxidation processes both in vivo and post-mortem (1, 10). Minerals such as Fe might act as a pro-oxidant, especially after pubs.acs.org/JAFC pubs.acs.org/JAFC © XXXX American Chemical Society J. Agric. Food Chem., Vol. XXX, No. XX, XXXX B MATERIALS AND METHODS The level of oxidation reached was below the limits established in most regulations for discarding frying oils (20). The procedure used for plasma and liver samples was as described by Tres et al. (22). In these samples, only the LHP value reached at the end of the incubation period (final LHP) was measured (116 h for plasma; 90 h for liver), which was enough to assess the different susceptibilities to oxidation among samples. (>3%) (18)] and their RT content (19) were also determined. Results are shown in Table 1. The level of oxidation reached was below the limits established in most regulations for discarding frying oils (20). Animals and Diets. The diets were prepared and the animals were housed in the Animal Science Department at the Polytechnic University of Valencia (Spain). This study received prior approval from the Animal Protocol Review Committee of the Polytechnic University of Valencia, and all animal housing, husbandry, and slaughtering conditions were in agreement with current European Union guidelines. Twelve dietary treatments were prepared from a basal diet by the combination of the dietary factors of interest, following a factorial design (3 2 2), replicated four times: 3 types of oxidized sunflower oil (FSO, PSO, and OSO) added to feeds at 3% (w/w); 2 doses of TA (0 or 100 mg TA/kg of feed) and 2 doses of Zn (0 or 200 mg/kg, added as zinc oxide). Samples. Oils were taken immediately after the thermal treatment, and peroxide value and p-anisidine value were determined within the following 6 h. Oil samples were stored in glass vials closed with Teflon caps, filled with N2, and frozen at -25 C until the rest of the analyses were performed. Feed samples were taken at the end of the feeding trial. Feeds were ground and vacuum-packed in high-barrier multilayer bags (Cryovac BB325; permeability to O2 25 cm3 m-2 day-1 bar-1 at 23 C and 0% relative humidity (RH), ASTMD-3985; Cryovac Europe, Sealed Air S. L., Sant Boi de Llobregat, Spain; approximately 15 g of feed/bag) and stored at -25 C until analysis. Feed analyses were performed in triplicate. Two hundred and eighty-eight rabbits (cross of New Zealand and Californian rabbit) were weaned at 28 days; they were randomly divided into 48 cages (12 dietary treatments, 4 replicates, 6 rabbits per cage) and fed ad libitum with the corresponding experimental diet. MATERIALS AND METHODS One-way ANOVA was used to determine whether there were any differences in the RT content and the oxidation parameters assessed in the oils added to feeds, as a result of heating conditions (n = 9, oils were analyzed in triplicate). Multifactor ANOVA was used to determine significant differences due to the dietary factors (degree of oil oxidation, TA and Zn supplementation) on the RT content of feeds (n= 36), and the RT content, susceptibility to oxidation, and TBA values of plasma (n = 48), liver (n = 48), raw meat (n = 48), cooked meat (n = 48), and refrigerated cooked meat (n = 48). Multifactor ANOVA was used to Two hundred and eighty-eight rabbits (cross of New Zealand and Californian rabbit) were weaned at 28 days; they were randomly divided into 48 cages (12 dietary treatments, 4 replicates, 6 rabbits per cage) and fed ad libitum with the corresponding experimental diet. At 63 days of age, Two hundred and eighty-eight rabbits (cross of New Zealand and Californian rabbit) were weaned at 28 days; they were randomly divided into 48 cages (12 dietary treatments, 4 replicates, 6 rabbits per cage) and fed ad libitum with the corresponding experimental diet. At 63 days of age, J. Agric. Food Chem., Vol. XXX, No. XX, XXXX C Article C Table 1. MATERIALS AND METHODS Thermal Treatment Applied to Oils Added to Feeds, Assessment of Oil Oxidation, Content of R-Tocopherol in Oils, Feed, Plasma, Liver, and Meat, and Oxidation (TBA Value) and Susceptibility to Oxidation (FOX Values) of Plasma, Liver, and Meat for the Diets Containing Sunflower Oils with Different Degrees of Oxidation, plus Supplementation with R-Tocopheryl Acetate (0 or 100 mg/kg) and Zn (0 or 200 mg/kg)a-c added oil TA (mg/kg) Zn (mg/kg) FSO PSO OSO 0 100 0 200 thermal treatment of oil no treatment 55 C, 245 h 140 C, 31 h assessment of oil oxidation peroxide value (mequiv of O2/kg of oil) 10.4 y 83.0 z 9.8 x LHP content (mmol of CHP equiv/kg of oil) 10.5 y 89.0 z 5.84 x polymer content (%, w/w) 2.8 x 2.7 x 124.5 y p-anisidine value 0.09 x 0.25 y 9.9 z R-tocopherol content oils (mg of RT/kg of oil) 621.5 y 510.6 x 605.1 xy feeds (mg of RT/kg of feed) 74.5 xy 67.6 x 77.4 y 33.7 x 112.6 y 75.0 71.3 plasma (mg of RT/L of plasma)d 4.21 y 3.59 x 4.02 xy 2.40 x 5.48 y 3.98 3.90 liver (mg of RT/kg of liver) 10.1 y 8.0 x 8.9 xy 4.6 x 13.5 y 8.8 9.2 raw meat (mg of RT/kg of meat) 2.82 y 2.18 x 2.72 y 1.27 x 3.88 y 2.61 2.53 cooked meat (mg of RT/kg of meat) 2.89 y 2.23 x 2.77 y 1.33 x 3.93 y 2.68 2.58 refrigerated cooked meat (mg of RT/kg of meat) 2.80 y 2.07 y 2.65 y 1.27 x 3.75 y 2.53 2.48 oxidation (TBA value) plasma (μg of MDA/L of plasma) nd nd nd nd nd nd nd livere (μg of MDA/kg of liver) 64 59 57 64 y 55 x 60 60 raw meat (μg of MDA/kg of meat) 21 21 23 25 21 22 24 cooked meatf (μg of MDA/kg of meat) 29 33 27 36 y 23 x 27 x 32 y refrigerated cooked meat (μg of MDA/kg of meat) 778 787 783 808 758 814 751 susceptibility to oxidation (FOX values) plasma (final LHP, mmol of CHP equiv/L of plasma) 0.044 0.048 0.045 0.047 0.044 0.046 0.045 liver (final LHP, mmol of CHP equiv/kg of liver) 5.72 6.59 5.60 9.23 y 2.71 x 6.24 5.70 raw meat ITd,f (h) 118i z 38 x 89i y 10 x 153i y 88i 75i MAXLHPd,f (mmol of CHP equiv/kg of meat) 0.41 x 0.71 z 0.55 y 0.84 y 0.28 x 0.55 0.56 TMAXd (h) 144i y 96 x 129i y 58 x 188i y 119i x 127i y final LHPd,f (mmol of CHP equiv/kg of meat) 0.29 x 0.52 z 0.41 y 0.56 y 0.25 x 0.39 0.42 AUCf [(mmol of CHP equiv/kg of meat) h] 62.2 x 101.4 y 81.4 y 130 y 33.2 x 82.7 80.6 cooked meat ITd,h (h) 121.5i y 64.8i x 114i y 16 x 184i y 100i 100i MAXLHP (mmol of CHP equiv/kg of meat) 0.44 x 0.65 y 0.48 x 0.84 y 0.20 x 0.52 0.52 TMAXg,h (h) 135i 129i 135i 51 x 215i y 132i 134i final LHPd,h (mmol of CHP equiv/kg of meat) 0.35 x 0.50 y 0.37 y 0.61 y 0.20 x 0.40 0.41 AUC [(mmol of CHP equiv/kg of meat) h] 66.5 x 88 y 69 x 130 y 19 x 74 75 refrigerated cooked meat ITd,e (h) 120i 61.7 109.6i 14.2 x 180i y 86i x 108i y MAXLHP (mmol of CHP equiv/kg of meat) 0.38 x 0.63 y 0.43 x 0.79 y 0.17 x 0.51 0.45 TMAXh (h) 138i y 109.5 x 135i x 56 x 199i y 123i 132i final LHP (mmol of CHP equiv/kg of meat) 0.25 x 0.47 y 0.29 x 0.54 y 0.14 x 0.37 0.31 AUC [(mmol of CHP equiv/kg of meat) h] 54.4 x 95 y 61.7 x 119 y 21.4 x 73.6 67.3 a Abbreviations: FSO, fresh sunflower oil; PSO, peroxidized sunflower oil; OSO, oxidized sunflower oil; TA, R-tocopheryl acetate; RT, R-tocopherol; CHP, cumene determine whether the RT content, FOX, and TBA values differed between plasma, liver, and raw meat samples as a result of the studied dietary factors (n = 144). a Abbreviations: FSO, fresh sunflower oil; PSO, peroxidized sunflower oil; OSO, oxidized sunflower oil; TA, R-tocopheryl acetate; RT, R-tocopherol; CHP, cumene hydroperoxide; TBA, thiobarbituric acid; MDA, malondialdehyde; LHP, lipid hydroperoxide; IT, induction time; MAXLHP, maximum LHP value; TMAX, time to reach the MAXLHP; AUC, area under the curve; nd, not detected. Values in the same row for a certain factor bearing no common letters (x-z) are statistically different (P e 0.05). P values were obtained from one-way ANOVA for oils (n = 9) and from multifactor ANOVA for feeds, plasma, liver, and meats (n = 36 for feeds, n = 48 for plasma, liver, and meats). Letters were obtained by means of the Scheffe´ test (R = 0.05). b Ingredients of diets: beet pulp, 28%; alfalfa, 25%; sunflower meal, 20%; wheat bran, 15%; soybean meal, 6%; fat (FSO, PSO, or OSO according to each dietary treatment), 3%; dicalcium phosphate, 1.2%; sodium chloride, 0.5%; L-lysine, 0.3%; calcium carbonate, 0.2%; DL-methionine, 0.1%; L-threonine, 0.1%; robenidine, 0.1% (not included in the feeds given to rabbits during their last week of life); sodium selenite, 0.1 mg/kg of feed; trace mineral-vitamin mix L-510 (Trouw Nutrition, Spain), 0.5% [supplied the following per kg of feed: 290 mg of magnesium oxide; 330 mg of sodium; 275 mg of sulfur; 700 μg of cobalt carbonate monohydrate; 10 mg of copper sulfate pentahydrate; 76 mg of ferrous sulfate monohydrate; 20 mg of manganese oxide; 59.2 mg of zinc oxide; 1.25 mg of potassium iodide; 8375 IU of vitamin A; 750 IU of vitamin D3; 20 mg of R-tocopherol; 1 mg of vitamin B1; 2 mg of vitamin B2; 1 mg of vitamin B6; 1 mg of vitamin K; 20 mg of niacin; 250 mg of choline chloride; 4 mg of butylated hydroxyanisoleþ ethoxyquin; 2.5 mg of flavophospholipol (80 mg/kg)]. c Values correspond to means (n = 9) for oils, and to least-squares means obtained from multifactor ANOVA for the main factors for feed (n = 36), plasma (n = 48), liver (n = 48), and meat (n = 48). d Interaction between oil added and TA supplementation, significant at P e 0.05. See Table 2. e Interaction between TA and Zn supplementation, significant at P e 0.05. f Interaction between oil added and Zn supplementation, significant at P e 0.05. See Table 4. g Interaction between cooking and oil added, significant at P e 0.05. (n = 96, including raw and cooked meat). h Interaction between cooking or refrigeration and TA supplementation, significant at P e 0.05 [n = 96, for the effect of cooking (raw þ cooked meat); n = 96 for the effect of refrigerating cooked meat (cooked þ refrigerated cooked meats)]. i If oxidation had not increasedin the samples by the end of reaction time, the final time (216 h) was considered to be the TMAX for these samples. Therefore, some values could be underestimated. RESULTS AND DISCUSSION These observations could be explained by the lower RT content of the TA-PSO feed than the TA-OSO and TA-FSO feeds, together with a greater loss of RT in the gastrointestinal tract as a result of the presence of a high amount of primary oxidation compounds in PSO feed. In addition, several oxidation compounds could be absorbed from diets (4,9,12), which would also explain why meat from animals on the TA-OSO diet was slightly more suscepti- ble to oxidation than meat from animals on theTA-FSO diet, although the diet had a similar RT content. Thus, our results show that when oxidized oils are added to feed, TA supplementation is required to reduce tissue susceptibility to oxidation. However, thissupplementation mustbeadjusteddepending onthe degree of oxidation of the oil added. The addition of PSO to feeds at 3% (w/w) favored meat susceptibility to oxidation and reduced the RT content of plasma, liver, and meat (Table 1). Heat treatment of PSO decreased its RT content, thereby affecting the RT content of the feed (Table 1). This reduced dietary RT content would explain both the de- creased RT content found in tissues and their increased suscept- ibility to oxidation. In addition, meat from rabbits on the OSO diet also tended to be more susceptible to oxidation than meat from rabbits on the FSO diet, as shown by the IT, MAXLHP, TMAX, and AUC in the FOX assay (Table 1). However, the RT content of OSO meats was similar to that of FSO meats; thus, the greater susceptibility to oxidation of the former could be attributed to the high content of oxidation compounds in feeds containing OSO oil. Increases in meat RT content with concomitant decreases in meat oxidation (TBA values) or in its susceptibility to oxidation (measured by means of the FOX method as in our study or by the TBA value after the induction of oxidation under standard conditions) have been reported in rabbit and other animal species as a result of the dietary supplementation with TA (19, 22, 26, 28-31). In our study, meat TBA values were low and not significantly reduced by TA supplementation (Table 1). RESULTS AND DISCUSSION The RT contents in our oils and feeds differed, and we were therefore unable to attribute the effects observed on rabbit liver and meat RT content and susceptibility to oxidation to all or to only some of these factors. Plasma, Liver, and Raw Meat. Plasma, liver, and meat oxida- tion was assessed by means of determining the TBA value, which measures their malondialdehyde (MDA), a secondary oxidation compound, content (25). In addition, the susceptibility of these samples to oxidation was assessed using the FOX method, which consists of an induced method that measures the LHP formed in the FOX reaction media over time (23, 24). Supplementation with TA can improve meat oxidative stabi- lity, shelf life, and overall quality, especially when unsaturated fat sources are added to feed (1). In our study, dietary supplementa- tionwith 100 mgofTA/kg reduced liver TBA values and also liver and meat susceptibility to oxidation (FOX method) as a result of the increase in RT in plasma, liver, and meat (Table 1). Oxidation (TBA values) and susceptibility to oxidation (final LHP values obtained from the FOX assay) decreased in the following order: liver, meat, and plasma (Table 1). This order is consistent with the same decreasing order of fat content in these samples (approximately 4.5% for liver, 3% for meat, and <0.8% for plasma). The TBA and final LHP values were relatively low compared to those of other animals fed more unsaturated diets (19,26).Infact, plasmaTBA was below thedetection limit in all samples (13 μg of MDA/L plasma) (Table 1). This finding might be related to plasma high RT content compared to its fat content. Despite the efficiency of TA supplementation in increasing the RT content in tissues and decreasing susceptibility to oxidation, significant interactions were found between the type of sunflower oil added to the feed and TA supplementation (Table 2). The higher susceptibility to oxidation of meat from animals on PSO diet than of meat from animals on OSO and FSO diets was evident after TA supplementation. This was reflected by shorter IT and TMAX and higher MAXLHP, AUC, and final LHP of meats from animals on diets with PSO and TA (Table 2). RESULTS AND DISCUSSION The lower oil content in rabbit feed, the low fat con- tent of rabbit meat compared to meat from other species, and its low content of FA having more than two double bonds [from which MDA is produced (2)] explained why meat oxidation (TBA values) was not high and why TA supplementation did not reduce it even more. However, the RT contents of plasma, liver, and meat correlated with each other and with the FOX parameters (IT, TMAX, MAXLHP, final LHP, and AUC) of meat (Table 3). This observation supports the notion that the FOX parameters reflected the protective effect of TA supplemen- tation. Therefore, the FOX method is useful to assess the suscep- tibility to oxidation of meat, especially meat with a low oxida- tion (TBA values). Correlations also showed that the RT content of plasma, liver, and rabbit meat was positively correlated between these tissues. This finding indicates that plasma RT content is a good indicator of RT content in tissues (Table 3) and is consistent with observations reported in rabbit and other species (11, 12, 22, 30, 31). Literature from studies assessing the oxidative stability of tissues from rabbits fed heated oils is lacking. Some studies dealing with chickens fed fresh and heated oils (containing a high amount of primary oxidation compounds) that had a similar RT content showed a reduced meat RT content and an increased susceptibility to oxidation (11,12). In previous studies (19,26), we found no significant differences in the RT content, oxidation (TBA value), and susceptibility to oxidation (LHP value) of raw chicken meat when chickens received feeds containing FSO or SO heated for different times and at distinct temperatures (leading to a high content of either primary or secondary oxidation com- pounds). However, results obtained by Bou et al. (19) showed a trend similar to those of the present study. All of these observations indicate that, apart from the lower RT supply, other mechanisms might contribute to the lower RT content of plasma, liver, and meat and to the higher susceptibility of liver and meat to oxidation. Indeed, RT could be lost in the gastrointestinal tract because of its reaction with radical species present in higher amounts in PSO oil than in OSO and FSO oils, thereby reducing the amount of RT available for absorption (12). D J. Agric. Food Chem., Vol. XXX, No. XX, XXXX D J. Agric. Food Chem., Vol. XXX, No. XX, XXXX Tres et al. D MATERIALS AND METHODS Multifactor ANOVA was used to deter- mine whether there were significant differences in the RT content, and FOX and TBA values of meat as a result of cooking (n = 96, raw vs cookedmeatsamples) and of refrigerating cookedmeat(n = 96, cooked vs refrigerated cooked meat samples). Interactions between more than two factors were ignored. Least-squares means for the main factors that had a significant effect were separated by Scheffe´ test. Pearson correlation coefficients were calculated between the RT content and FOX and TBA values in plasma, liver, and meat and between raw, cooked, and refriger- ated cooked meat. In all cases, P e 0.05 was considered to be significant. Statistical analysis was performed using SPSS 15.0 (SPSS Inc., Chicago, IL) software. factors were ignored. Least-squares means for the main factors that had a significant effect were separated by Scheffe´ test. Pearson correlation coefficients were calculated between the RT content and FOX and TBA values in plasma, liver, and meat and between raw, cooked, and refriger- ated cooked meat. In all cases, P e 0.05 was considered to be significant. Statistical analysis was performed using SPSS 15.0 (SPSS Inc., Chicago, IL) software. RESULTS AND DISCUSSION b Values given are least-squares means obtained from multifactor ANOVA for the interactions (n = 48). c If oxidation had not increased in the samples by the end of reaction time, the final time (216 h) was considered to be the TMAX for these samples. Therefore, some values could be underestimated. a Abbreviations: TA, R-tocopheryl acetate; FSO, fresh sunflower oil; PSO, peroxidized sunflower oil; OSO, oxidized sunflower oil; RT, R-tocopherol; LHP, lipid hydroperoxide; IT, induction time; MAXLHP, maximum LHP value; TMAX, time to reach the MAXLHP value; AUC, area under the curve. b Values given are least-squares means obtained from multifactor ANOVA for the interactions (n = 48). c If oxidation had not increased in the samples by the end of reaction time, the final time (216 h) was considered to be the TMAX for these samples. Therefore, some values could be underestimated. a Abbreviations: TA, R-tocopheryl acetate; FSO, fresh sunflower oil; PSO, peroxidized sunflower oil; OSO, oxidized sunflower oil; RT, R-tocopherol; LHP, lipid hydroperoxide; IT, induction time; MAXLHP, maximum LHP value; TMAX, time to reach the MAXLHP value; AUC, area under the curve. b Values given are least-squares means obtained from multifactor ANOVA for the interactions (n = 48). c If oxidation had not increased in the samples by the end of reaction time, the final time (216 h) was considered to be the TMAX for these samples. Therefore, some values could be underestimated. Table 3. Pearson Correlation Coefficients between the R-Tocopherol Content, Oxidation (TBA Value), and Susceptibility to Oxidation (FOX Values) of Rabbit Plasma (n = 48), Liver (n = 48), and Meat (n = 48)a Table 3. RESULTS AND DISCUSSION Furthermore, in tissues, RT could be reduced as a result of the promotion of oxidation by some of the oxidation products absorbed (12). Secondary oxidation compounds are absorbed in the gastrointestinal tract (5,7). However, LHP are not likely to be absorbed in their intact form. After previous breakdown of LHP in the stomach and reduction by gluthatione peroxidase in the intestinal epithelium, several compounds containing hydroxyl, epoxy, or keto groups derived from LHP might be absorbed (9). Therefore, the in vivo pro-oxidative effect of the oxidation com- pounds from the diet is difficult to explain (27). Supplementation with 200 mg of Zn/kg of feed did not alter the RT content of rabbit tissues or meat TBA (Table 1), although it slightlydecreased meatCucontent (32),which in somestudies has led to decreases in the activities of some antioxidant enzymes, such as copper-zinc superoxide dismutase (33). However, meat TBA values were not affected by Zn supplementation in previous studies in chickens (19, 21, 34). J. Agric. Food Chem., Vol. XXX, No. XX, XXXX Article E Table 2. Effect of the Degree of Oxidation of the Oil Added to Feed and Supplementation with R-Tocopheryl Acetate (0 or 100 mg/kg of Feed) on R-Tocopherol Content and FOX Values of Rabbit Plasma, Liver, and Meata,b Table 2. RESULTS AND DISCUSSION Effect of the Degree of Oxidation of the Oil Added to Feed and Supplementation with R Tocopheryl Acetate (0 or 100 mg/kg of Feed) on R Tocopherol Content and FOX Values of Rabbit Plasma, Liver, and Meata,b 0 mg of TA/kg 100 mg of TA/kg P FSO PSO OSO FSO PSO OSO feed RT content (mg of RT/kg of feed) 0.792 35.2 26.9 38.9 113.7 108.3 115.9 plasma RT content (mg of RT/L of plasma) 0.056 2.82 1.77 2.61 5.60 5.40 5.43 final LHP (mmol of CHP equiv/L of plasma) 0.233 0.044 0.052 0.045 0.043 0.044 0.045 liver RT content (mg of RT/kg of liver) 0.683 5.65 3.23 4.82 14.6 12.7 13.0 final LHP (mmol of CHP equiv/kg of liver) 0.672 8.66 10.32 8.72 2.77 2.87 2.48 raw meat RT content (mg of RT/kg of meat) 0.847 1.49 0.90 1.49 4.26 3.55 4.07 IT (h) 0.000 21 0.5 9 216c 75 168c MAXLHP (mmol of CHP equiv/kg of meat) 0.000 0.74 0.88 0.86 0.08 0.53 0.22 TMAX (h) 0.013 72 45 57 216c 147 201c final LHP (mmol CHP eq/kg meat) 0.000 0.50 0.58 0.61 0.08 0.46 0.21 AUC [(mmol of CHP equiv/kg of meat) h] 0.057 111 141 138 13 62 24 cooked meat RT content (mg of RT/kg of meat) 0.520 1.59 0.86 1.56 4.19 3.61 3.98 IT (h) 0.000 27 0.5 21 216c 129c 207c MAXLHP (mmol of CHP equiv/kg of meat) 0.222 0.79 0.92 0.81 0.10 0.38 0.14 TMAX (h) 0.602 54 45 54 216c 213c 216c final LHP (mmol of CHP equiv/kg of meat) 0.013 0.59 0.63 0.60 0.10 0.38 0.14 AUC [(mmol of CHP equiv/kg of meat) h] 0.718 119 145 126 14 31 13 refrigerated cooked meat RT content (mg of RT/kg of meat) 0.669 1.51 0.81 1.48 4.09 3.33 3.81 IT (h) 0.010 24 0.5 18 216c 123 201c MAXLHP (mmol of CHP equiv/kg of meat) 0.974 0.69 0.94 0.74 0.07 0.32 0.11 TMAX (h) 0.222 60 48 60 216c 171 209c final LHP (mmol of CHP equiv/kg of meat) 0.922 0.45 0.68 0.48 0.06 0.27 0.10 AUC [(mmol of CHP equiv/kg of meat) h] 0.099 98 152 108 11 38 15 a Abbreviations: TA, R-tocopheryl acetate; FSO, fresh sunflower oil; PSO, peroxidized sunflower oil; OSO, oxidized sunflower oil; RT, R-tocopherol; LHP, lipid hydroperoxide; IT, induction time; MAXLHP, maximum LHP value; TMAX, time to reach the MAXLHP value; AUC, area under the curve. RESULTS AND DISCUSSION Pearson Correlation Coefficients between the R-Tocopherol Content, Oxidation (TBA Value), and Susceptibility to Oxidation (FOX Values) of Rabbit Plasma (n = 48), Liver (n = 48), and Meat (n = 48)a FOX values meat RT plasma RT liver RT meat TBA liver TBA meat final LHP plasma final LHP liver IT MAXLHP TMAX final LHP AUC RT plasma 1 0.899 0.953 -0.448** -0.149 -0.307* 0.022 0.802 -0.816 0.865 -0.722 -0.857 RT liver 1 0.916 -0.226 -0.280* -0.292* -0.104 0.810 -0.830 0.849 -0.750 -0.853** RT meat 1 -0.322* -0.211 -0.311* -0.003 0.878 -0.875 0.896 -0.803 -0.887** TBA liver 1 -0.075 -0.153 -0.233 -0.216 0.184 -0.260 0.125 0.228 TBA meat 1 0.165 -0.072 -0.059 0.120 -0.032 0.098 0.125 final LHP plasma 1 -0.047 -0.221 0.312* -0.229 0.265 0.319* final LHP liver 1 -0.031 -0.041 -0.059 -0.047 -0.071 FOX values meat IT 1 -0.952 0.934 -0.938 -0.911 MAXLHP 1 -0.923 0.977 0.984 TMAX 1 -0.850 -0.934 final LHP 1 0.933 AUC 1 a Abbreviations: TBA, thiobarbituric acid; FOX, ferrous oxidation-xylenol orange; RT, R-tocopherol content; IT, induction time; MAXLHP, maximum LHP value; TMAX, time to reach the MAXLHP; AUC, area under the curve. , P value e 0.05; , P value e 0.001. a Abbreviations: TBA, thiobarbituric acid; FOX, ferrous oxidation-xylenol orange; RT, R-tocopherol content; IT, induction tim to reach the MAXLHP; AUC, area under the curve. , P value e 0.05; *, P value e 0.001. a Abbreviations: TBA, thiobarbituric acid; FOX, ferrous oxidation-xylenol orange; RT, R-tocopherol content; IT, induction time; MAXLHP, maximum LHP value; TMAX, time to reach the MAXLHP; AUC, area under the curve. , P value e 0.05; , P value e 0.001. (Table 4) (32) and, thus, in its susceptibility to oxidation, as indicated by the shorter IT and TMAX and higher MAXLHP, final LHP, and AUC (Table 4). Conversely, Zn supplementation in feed containing PSO led to a slight decrease in meat FA content (32) and thus in meat susceptibility to oxidation (Table 4). Zn supplementation did not alter the susceptibilityto oxidation of meat from animals on the FSO diet. Plasma and liver FA composition was not altered by this interaction, and thus neither Zn supplementation, however, altered meat susceptibility to oxidation depending on the degree of oxidation of oils added to feeds (Table 4). This was consistent with an interaction observed between these two factors for the FA content of meats (Table 4) (32). RESULTS AND DISCUSSION d Its Subsequent Refrigeration on Meat R-Tocopherol Content, Oxidation (TBA Value), and Oxidative Stability (FOX Values)a raw meat cooked meat refrigerated cooked meat a Abbreviations: TBA, thiobarbituric acid; FOX, ferrous oxidation-xylenol orange; RT, R-tocopherol; MDA, malondialdehyde; LHP, lipid hydroperoxide; IT, induction time; MAXLHP, maximum LHP value; TMAX, time to reach the maximum LHP value; AUC, area under the curve. Values in the same row bearing no common letters (x-z) are statistically different (P e 0.05). Letters were obtained by means of Scheffe´ test (R = 0.05). Values correspond to least-squares means obtained from multifactor ANOVA (raw meat vs cooked meat, n = 96; cooked meat vs refrigerated cooked meat, n = 96). was their susceptibility to oxidation. More research is needed to ascertain why Zn supplementation altered rabbit meat FA con- tent depending on the oxidation status of the oil added to feeds (32). together with the time, temperature, and cooking conditions used (35), might promote meat oxidation. In our study, cooking conditions were mild (vacuum-packed meat, at 80 C, for 5 min), thus leading to a slight increase in meat TBA value after cooking. However, during refrigerated storage (5 C, 62 days) of cooked meat, the TBA value increased (Table 5), although these values were not as high as those reported in other studies (26,37). Furthermore, we did not find differences in meat RT content after cooking or after the refrigerated storage of cooked meat (Table 5). Furthermore, an effect of the interaction between dietary supplementation with TA and Zn was observed for liver TBA values. The highest TBA values were found in liver from animals that did not receive TA or Zn supplementation [67 μg of MDA/kg for liver from 0 TA-0 Zn diets vs 61, 59, and 53 μg of MDA/kg for 0 TA-200 Zn, 100 TA-200 Zn, and 100 TA-0 Zn diets, respectively (SEM, 2.9 μg of MDA/kg)]. The lowest TBA values were detected when 100 mg of TA/kg of feed was added without Zn supplementation; thus, it seems that Zn supplementation diminished somehow the protective effect of TA supplementation against oxidation in liver. Although it was expected that the changes in meat induced by cooking might increase its susceptibility to oxidation, results from the FOX method showed that it slightly decreased (Table 5). However, the comparison between raw and cooked samples should bemade with care. RESULTS AND DISCUSSION c If oxidation had not increased in samples by the end of the FOX reaction time, the final time (216 h) was considered the TMAX for these samples. Therefore, some values could be underestimated. a Abbreviations: FSO, fresh sunflower oil; PSO, peroxidized sunflower oil; OSO, oxidized sunflower oil; FA, fatty acids; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; RT, R-tocopherol; FOX, ferrous oxidation-xylenol orange; IT, induction time; MAXLHP, maximum lipid hydroperoxide value; CHP, cumene hydroperoxide; TMAX, time to reach the MAXLHP value; AUC, area under the curve. b Values given are least-squares means for the interactions obtained from multifactor ANOVA (n = 48). Interaction between oil added to feeds and Zn supplementation significant at P e 0.05. c If oxidation had not increased in samples by the end of the FOX reaction time, the final time (216 h) was considered the TMAX for these samples. Therefore, some values could be underestimated. Table 5. Effect of Cooking Meat and Its Subsequent Refrigeration on Meat R-Tocopherol Content, Oxidation (TBA Value), and Oxidative Stability (FOX Values)a raw meat cooked meat refrigerated cooked meat R-tocopherol content (mg of RT/kg of meat) 2.63 2.63 2.51 oxidation TBA value (μg of MDA/kg of meat) 23 x 29 y 783 z susceptibility to oxidation IT (h) 81.7 x 100.1 y 97 y MAXLHP (mmol of CHP equiv/kg of meat) 0.56 0.52 0.48 TMAX (h) 123 x 133 y 128 y final LHP (mmol of CHP equiv/kg of meat) 0.41 y 0.41 y 0.34 x AUC [(mmol of CHP equiv/kg of meat) h] 82 y 74 x 70 x a Abbreviations: TBA, thiobarbituric acid; FOX, ferrous oxidation-xylenol orange; RT, R-tocopherol; MDA, malondialdehyde; LHP, lipid hydroperoxide; IT, induction time; MAXLHP, maximum LHP value; TMAX, time to reach the maximum LHP value; AUC, area under the curve. Values in the same row bearing no common letters (x-z) are statistically different (P e 0.05). Letters were obtained by means of Scheffe´ test (R = 0.05). Values correspond to least-squares means obtained from multifactor ANOVA (raw meat vs cooked meat, n = 96; cooked meat vs refrigerated cooked meat, n = 96). RESULTS AND DISCUSSION Meat susceptibility to oxidation depends on the balance between antioxidants, pro-oxidants, and substrates (10). Zn supplementation did not modify the RT content of meat from animals on OSO feed, but led to a slight increase in its FA content F J. Agric. Food Chem., Vol. XXX, No. XX, XXXX Tres et al. Table 4. Effect of the Degree of Oxidation of the Oil Added to Feeds and the Supplementation with Zinc (0 or 200 mg/kg of Feed) on the Fatty Acid Composition, R-Tocopherol Content, and Susceptibility to Oxidation of Raw Rabbit Meata,b R-Tocopherol Content, and Susceptibility to Oxidation of Raw Rabbit Meata,b FSO PSO OSO P 0 Zn 200 Zn 0 Zn 200 Zn 0 Zn 200 Zn fatty acid composition (mg of FA/100 g of meat) SFA 0.008 830 750 880 780 720 860 MUFA 0.032 580 530 600 530 510 590 n-6 PUFA 0.016 1120 1050 1180 1090 990 1130 n-3 PUFA 0.009 58 54 65 58 54 62 total PUFA 0.016 1180 1100 1240 1150 1050 1190 total trans FA 0.017 14.3 12.2 14.0 12.7 12.3 13.6 RT content (mg of RT/kg of meat) 0.328 3.05 2.70 2.22 2.23 2.72 2.84 susceptibility to oxidation (FOX values) IT (h) 0.003 120c 117c 30 45 114c 63c MAXLHP (mmol of CHP equiv/kg of meat) 0.001 0.41 0.42 0.78 0.63 0.46 0.65 TMAX (h) 0.000 147c 141c 72 120 138c 120c final LHP (mmol of CHP equiv/kg of meat) 0.000 0.29 0.29 0.56 0.47 0.33 0.49 AUC [(mmol of CHP equiv/kg of meat) h] 0.001 62 63 117 86 69 93 a Abbreviations: FSO, fresh sunflower oil; PSO, peroxidized sunflower oil; OSO, oxidized sunflower oil; FA, fatty acids; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA polyunsaturated fatty acids; RT R-tocopherol; FOX ferrous oxidation-xylenol orange; IT induction time; MAXLHP maximum lipid hydroperoxide value; a Abbreviations: FSO, fresh sunflower oil; PSO, peroxidized sunflower oil; OSO, oxidized sunflower oil; FA, fatty acids; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; RT, R-tocopherol; FOX, ferrous oxidation-xylenol orange; IT, induction time; MAXLHP, maximum lipid hydroperoxide value; CHP, cumene hydroperoxide; TMAX, time to reach the MAXLHP value; AUC, area under the curve. b Values given are least-squares means for the interactions obtained from multifactor ANOVA (n = 48). Interaction between oil added to feeds and Zn supplementation significant at P e 0.05. RESULTS AND DISCUSSION Pearson Correlation Coefficients between Raw Meat R-Tocopherol Content and TBA and FOX Values of Raw (n = 48), Cooked (n = 48), and Refrigerated Cooked Meat (n = 48)a cients between Raw Meat R-Tocopherol Content and TBA and FOX Values of Raw (n = 48), Cooked (n = 48), and Refrigerated Cooked Meat (n = 48)a FOX values RM FOX values CM FOX values RCM RT RM TBA RM TBA CM TBA RCM IT TMAX final LHP AUC IT TMAX final LHP AUC IT TMAX final LHP AUC RT RM 1 -0.211 -0.614 -0.179 0.878 0.896 -0.803 -0.887 0.918 0.935 -0.840 -0.913 0.887 0.904 -0.849 -0.901 TBA RM 1 0.109 0.295* -0.059 -0.032 0.098 0.125 -0.190 -0.257 0.259 0.267 -0.148 -0.171 0.043 0.113 TBA CM 1 -0.085 -0.522 -0.553 0.440 0.539 -0.603 -0.580 0.542 0.598 -0.531 -0.586 0.542 0.589 TBA RCM 1 -0.052 -0.039 0.083 0.050 -0.142 -0.115 0.053 0.016 -0.212 -0.152 0.186 0.153 FOX values RM IT 1 0.934 -0.938 -0.911 0.811 0.818 -0.819 -0.824 0.868 0.846 -0.828 -0.832 TMAX 1 -0.850 -0.934 0.884 0.882 -0.797 -0.869 0.922 0.907 -0.879 -0.895 final LHP 1 0.933 -0.814 -0.742 0.760 0.734 -0.807 -0.786 0.763 0.776 AUC 1 -0.872 -0.877 0.792 0.854 -0.885 -0.889 0.845 -0.911 FOX values CM IT 1 0.929 -0.895 -0.929 0.912 0.928 -0.874 -0.911 TMAX 1 -0.811 -0.947 0.872 0.923 -0.818 -0.903 final LHP 1 0.788 -0.856 -0.864 0.747 0.784 AUC 1 -0.876 -0.912 0.805 0.883 FOX values RCM IT 1 0.963 -0.912 -0.915 TMAX 1 -0.890 -0.932 final LHP 1 0.970 AUC 1 a Abbreviations: RM, raw meat; CM, cooked meat; RCM, refrigerated cooked meat, RT, R-tocopherol content; TBA, thiobarbituric acid; FOX, ferrous oxidation-xylenol orange; LHP, lipid hydroperoxide; IT, induction time; TMAX, time to reach the maximum LHP value; AUC, area under the curve. , P value e 0.05; , P value e 0.001. a Abbreviations: RM, raw meat; CM, cooked meat; RCM, refrigerated cooked meat, RT, R-tocopherol content; TBA, thiobarbituric acid; FOX, ferrous oxidation-xylenol orange; LHP, lipid hydroperoxide; IT, induction time; TMAX, time to reach the maximum LHP value; AUC, area under the curve. , P value e 0.05; **, P value e 0.001. method, is a useful predictor of meat oxidation (TBA value) after cooking. although not significant, was observed in raw meat, whereas the oppositetrend was detectedinrefrigeratedcookedmeat(Table 1). RESULTS AND DISCUSSION The FOX method is an induced method that measures the formation of LHP in a methanolic extract of the sample. In general, it is advisable to compare samples (methanolic extracts) with similar characteristics when this type of method is used (23). Given that the composition of the methanolic extract might differ slightly between raw and cooked samples, the devel- opment of the FOX reaction might be affected and, thus, the FOX values and parameters obtained. This would explain the changes in meat susceptibility to oxidation after cooking. However, as shown by the Pearson’s correlation coefficients (Table 6), the susceptibility of raw meat to oxidation, as measured by the FOX Effect of Cooking and Refrigeration of Cooked Meat on the Oxidation and Oxidative Stability of Meat. The cooking and storage of raw and cooked meat have been reported to affect meat oxidation (10, 26, 28, 29), but the values reached depend on the cooking and storage conditions (35, 36). The effects of cooking on meat oxidation have been related to protein dena- turation (loss of antioxidant enzyme activity and release of pro- oxidant metal ions such as nonheme iron), myoglobin oxidation, or the disruption of cell membranes, which might bring PUFA into contact with pro-oxidant compounds (10,26). These factors, Effect of Cooking and Refrigeration of Cooked Meat on the Oxidation and Oxidative Stability of Meat. The cooking and storage of raw and cooked meat have been reported to affect meat oxidation (10, 26, 28, 29), but the values reached depend on the cooking and storage conditions (35, 36). The effects of cooking on meat oxidation have been related to protein dena- turation (loss of antioxidant enzyme activity and release of pro- oxidant metal ions such as nonheme iron), myoglobin oxidation, or the disruption of cell membranes, which might bring PUFA into contact with pro-oxidant compounds (10,26). These factors, J. Agric. Food Chem., Vol. XXX, No. XX, XXXX Article G Table 6. ACKNOWLEDGMENT We thank Frit Ravich and Laboratorios Salvat for use of their facilities for heating the experimental oils. We also thank the Department of Animal Science at the Polytechnic University of Valencia for housing the animals and for slaughtering facilities and E. Carmona, M. Redecker, and M. Gonzalo for their help in the analyses. RESULTS AND DISCUSSION In previous studies, no differences in the TBA value were observed after cooking and after storage of meat from chickens fed diets supplemented with Zn (19, 21). During the refrigeration of cooked meat, the low oxygen availability and refrigeration temperature may have slowed the formation of new LHP, which may have been decomposed more quickly into secondary oxidation products, such as MDA, thus leading to an increase in TBA value and to similar FOX para- meters (Table 6). In summary, the presence of a high content of primary and secondary oxidation compounds in fats added to feeds increased meat susceptibility to oxidation. The highest increase was ob- served when PSO oil was used in feeds, which reduced the RT content of rabbit plasma, liver, and meat, facts that could be related to the lower RT supply from feeds when this oil was used. However, other mechanisms might also be involved. Dietary supplementation with TA reduced meat and liver susceptibility to oxidation; however, this supplementation must be adjusted depending on the oxidation of the fat added to feeds. The dietary supplementation with Zn slightly decreased the susceptibility to oxidation of meat from rabbit on PSO diets and increased that of meat from rabbits on OSO diets. These effects of Zn supplemen- tation were according to alterations of meat FA content; how- ever, more research is needed to discern the mechanisms that led to such alterations. The FOX method was useful to assess raw meat susceptibility to oxidation, which in turn was a good predictor of meat oxidation after cooking. ( ) Dietary supplementation with 100 mg of TA/kg of feed in- creased the RT content of cooked and refrigerated cooked meat, as it did in raw meat (Table 1). This led to reductions of meat susceptibility to oxidation (reflected by all of the parameters from the FOX method) both after cooking and after the refrigeration of cooked meat (Table 1). TA supplementation also led to a 35% reduction in meat TBA after cooking. However, given the high variability among samples, no significant differences were found after refrigeration (Table 1). Thus, the RT contents of all raw, cooked, and refrigerated cooked meat were strongly correlated with the parameters that measure the susceptibility of meat to oxidation after cooking and subsequent refrigeration (Table 6). RESULTS AND DISCUSSION The lower RT content in raw meat as a result of the addition of PSO oils to feed was still significant in cooked and in refrigerated cooked meat (Table 1). Similarly to raw meat, TBA content did not differ, but meat susceptibility to oxidation increased with the addition of this oil, as shown by the FOX parameters (Table 1). In some studies with chickens (29,38), differences in meat oxidation after cooking or after storage were found as a result of admin- istering diets containing oxidized oils (high in primary oxidation compounds). However, the RT content of those oils had been reduced during heating, thereby leading to a lower meat RT content (29,38). In other studies, in which RT losses in oil caused by heating were corrected, heated oils did not modify the oxidation (TBA values) of cooked chicken meat or the suscept- ibility of this meat to oxidation (Final LHP values) (19). Similar results were found by Grau et al. (26), although they did not correct RT losses in heated SO oil. They attributed this lack of a significant effect to the low LHP content of their heated oil tested. Di l i i h Z (200 /k ) did ff (3) Nuchi, C. D.; Guardiola, F.; Bou, R.; Bondioli, P.; Della Bella, L.; Codony, R. Assessment of the levels of degradation in fat co- and (2) Frankel, E. N. Lipid Oxidation; Oily Press: Dundee, U.K., 1998. LITERATURE CITED (1) Bou, R.; Codony, R.; Tres, A.; Guardiola, F.; Decker, E. A. Dietary strategies to improve nutritional value, oxidative stability and sensory properties of poultry products. Crit. Rev. Food Sci. Technol. 2009, 49, 800–822. Dietary supplementation with Zn (200 mg/kg) did not affect the TBA content of refrigerated cooked meat; however, it slightly increased this parameter in cooked meat. The same tendency, Dietary supplementation with Zn (200 mg/kg) did not affect the TBA content of refrigerated cooked meat; however, it slightly increased this parameter in cooked meat. 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(13) European Commission List of the authorized additives in feeding- stuffs published in application of Article 9t (b) of Council Directive 70/524/EEC concerning additives in feedingstuffs. Off. J. Eur. Union 2004, C50/1. (32) Tres, A.; Bou, R.; Codony, R.; Guardiola, F. Received for review April 28, 2010. Revised manuscript received July 14, 2010. Accepted July 20, 2010. This work was funded by the Ministerio de Educacio´ n y Ciencia (Spain) and by a research grant from the Instituto Danone awarded to A.T. LITERATURE CITED Effect of dietary fish oil, R-tocopheryl acetate, and zinc supplementation on the composition and consumer acceptability of chicken meat. Poult. Sci. 2004, 83, 282–292. (38) Jensen, C.; Engberg, R.; Jakobsen, K.; Skibsted, L. H.; Bertelsen, G. Influence of the oxidative quality of dietary oil on broiler meat storage stability. Meat Sci. 1997, 47, 211–222. (22) Tres, A.; Bou, R.; Codony, R.; Guardiola, F. 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https://openalex.org/W2889888667	https://iris.uniroma1.it/bitstream/11573/1213212/1/La%20Torre_Passive_2018.pdf	English	null	Passive Smoking Indicators in Italy: Does the Gross Domestic Product Matter?	International journal of environmental research and public health/International journal of environmental research and public health	2,018	cc-by	5,060	Received: 7 August 2018; Accepted: 14 September 2018; Published: 18 September 2018 Abstract: Background: The aim of this study is to analyse the correlation between regional values of Gross Domestic Product (GDP) and passive smoking in Italy. Methods: The outcome measures were smoking ban respect in public places, workplaces and at home, derived from the PASSI surveillance for the period 2011–2017. The explanatory variable was GDP per capita. The statistical analysis was carried out using bivariate and linear regression analyses, taking into consideration two different periods, Years 2011–2014 and 2014–2017. Results: GDP is showed to be positively correlated with smoking ban respect in public places (r = 0.779 p < 0.001; r = 0.723 p < 0.001 in the two periods, respectively), as well as smoking ban respect in the workplace (r = 0.662 p = 0.001; r = 0.603 p = 0.004) and no smoking at home adherence (r = 0.424 p = 0.056; r = 0.362 p = 0.107). In multiple linear regression GDP is signiﬁcantly associated to smoking ban respect in public places (adjusted β = 0.730 p < 0.001; β = 0.698 p < 0.001 in the two periods, respectively), smoking ban in workplaces (adjusted β = 0.525 p = 0.020; β = 0.570 p = 0.009) and no smoking at home (adjusted β = 0.332 p = 0.070; β = 0.362 p = 0.052). Conclusions: Smoking ban is more respected in Regions with higher GDP. For a better health promotion, systematic vigilance and sanctions should be maintained and strengthened, particularly in regions with low compliance with smoking bans. Keywords: passive smoking; Italy; Gross Domestic Product ceived: 7 August 2018; Accepted: 14 September 2018; Published: 18 September 2018 Int. J. Environ. Res. Public Health 2018, 15, 2045; doi:10.3390/ijerph15092045 International Journal of Environmental Research and Public Health Giuseppe La Torre , Cristina Sestili, Rosario Andrea Cocchiara , Sara Cianfanelli, Lorenza Lia and Alice Mannocci Giuseppe La Torre , Cristina Sestili, Rosario Andrea Cocchiara , Sara Cianfanelli, Lorenza L and Alice Mannocci Department of Public Health and Infectious Diseases, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy; cristina.sestili@uniroma1.it (C.S.); rosario.cocchiara@uniroma1.it (R.A.C.); sara.cianfanelli@uniroma1.it (S.C.); lorenza.lia@uniroma1.it (L.L.); alice.mannocci@uniroma1.it (A.M.) * Correspondence: giuseppe.latorre@uniroma1.it Keywords: passive smoking; Italy; Gross Domestic Product www.mdpi.com/journal/ijerph 1. Introduction In 1998, the CDC and WHO developed the Global Youth Tobacco Survey (GYTS) included in the Global Tobacco Surveillance System (GTSS); it reports the widest collection of data of secondhand smoke exposure in children, based on national school surveys [7]. In Europe the major source of data on SHS exposure is provided by Eurobarometer surveys that collect information from 2006 to 2017 in all UE countries. According to the Eurobarometer survey published in March 2017, with regard to SHS exposure in public places, a ﬁfth of respondents declare that they have come across people smoking inside bars, with a decrease of 5% since the survey of 2015. However, this report shows a huge difference among European countries, with high rates of SHS exposure in drinking establishments in countries of Southern Europe such as Greece, where the response of SHS exposure in places such as bars has increased to 87%, Croatia with 77% of the respondents, and Czech Republic with 73%. Concerning the eating establishments such as restaurants, the proportion of respondents that observed indoor smoke is lower, namely 9%. In these settings the rates differ less among countries; nevertheless, Greece shows a high rate (78% of respondents) and Cyprus 51% [8]. Due to the difﬁculties to collect direct measures of SHS exposure in each country, the prevalence of smoking in male and female population can be considered as surrogate measure of passive smoking exposure [9]. The knowledge of adverse health effects and the considerable costs of associated treatment for passive smoke led to many countries banning smoking in workplaces and in indoor and outdoor public places. Nevertheless, only 20% of the world’s population is protected by national anti-smoking bans. In many countries there is no smoking ban intervention yet or they are not reported, especially in Africa (i.e., Burundi, South Sudan, Sierra Leone). There are also countries where laws are applied only partially or in a limited way (South-East Asia Region) and exposure to passive smoke remains a serious problem [10]. In line with the trend of the prevalence distribution, it can be speculated that future diseases caused by tobacco smoke will concern mainly people in worse social and economic conditions, increasing health inequalities [2]. In Italy the tobacco prevalence discloses relevant differences among regions, and the decreasing trend is less prominent in Southern regions. According to Gallus et al. 1. Introduction Tobacco smoke is the major cause of premature deaths due to several diseases that could be prevented worldwide [1,2]. Second-hand smoke (SHS), known also as ‘environmental tobacco smoke’, ‘passive smoking’ or ‘involuntary smoking’ [3], is a phenomenon inherently derived from tobacco smoke, potentially present in all places such as at home, in the workplace and in public places. It consists of smoke released from the burning of tobacco products between the sidestream smoke (SM), which is the amount of smoke that spreads throughout the air from the lighted end without being inhaled by the smokers, and the mainstream smoke (MS), the smoke breathed out by the smoker. Sidestream smoke and mainstream smoke have similar constituents but in different quantities, the ratios of sidestream to mainstream smoke differ widely depending on the considered component [4]. At the physical-chemical level there are no particular differences between active and passive smoking; the only differences are the combustion temperature and the percentage of oxygen available (higher for active smoke). In both cases there are about 4000 different chemicals, carcinogens (polycyclic hydrocarbons, benzene, nitrosamines), irritants and allergenic substances such as formaldehyde, harmful gases such as carbon monoxide or irritants such as sulfur and nitrogen oxides, in addition Int. J. Environ. Res. Public Health 2018, 15, 2045; doi:10.3390/ijerph15092045 www.mdpi.com/journal/ijerph 2 of 8 Int. J. Environ. Res. Public Health 2018, 15, 2045 to nicotine. In enclosed spaces cigarette smoke can create very high concentrations of ﬁne dust, up to 100 times higher than the legal limits allowed for the external environment and prolonged indoor exposure to the 4000 substances present in the smoke can represent a source of pollution higher than the atmospheric of the most polluted metropolises [5]. Secondhand exposure to tobacco smoke causes cardiovascular diseases and lung cancer in nonsmoking adults and sudden infant death syndrome, acute respiratory infections, middle ear disease, exacerbation of asthma and decreased lung function in children [6]. According to the data of the Centers for Disease Control and Prevention (CDC) there is no safe threshold of secondhand smoke exposure, so short exposure can also threaten population’s health. Since 1964, in USA approximately 2,500,000 nonsmokers have died from diseases caused by exposure to secondhand smoke [6]. Indicators measuring exposure to second-hand tobacco smoke can be direct, indirect and surrogate. It seems difﬁcult to have extensively direct data of SHS exposure of a population. 1. Introduction in 2010, 21.7% of Italians were current smokers, including 23.9% of men and 19.7% of women. The prevalence in men was always higher than in women, but for the ﬁrst time, the prevalence in women in the middle-aged population (45–64 years old) is higher than in men, with 25.9% in women and 25.6% in men. Actually, the male and female smoking prevalence does not differ among individuals with high degrees of education and in the northern and central Italian regions [11]. Gorini et al. studied social determinants in smoking habits during the 30 years up to 2009 in Italy. According to their ﬁndings, the prevalence of male smoking dropped constantly from 56.1% in 1980 to 30.2% in 2009, despite concurrent increasing differences among different educational groups. In fact in 2009, the prevalence in poorly educated men was 53% higher than in graduates. On the other hand, female smoking rates shows a different trend, remaining ﬁrm around 18% but, as in male population, the results show a decreasing prevalence in highly educated women compared to low educated ones, in a pattern starting Int. J. Environ. Res. Public Health 2018, 15, 2045 3 of 8 from North to South and from younger women to older ones. Moreover, the educational inequalities in female population from the north and central regions seem to have decreased in the last decade, in contrast with the trend in the male populatio [12]. Many studies have documented social differences in passive sqke exposure. In the countries where tobacco smoke prevalence is high, SHS exposure occurs more often among the poorest families. References [13–18] found that male sex, lower education levels, lower individual income, and living in a small rural area are associated with increased exposure to secondhand smoke in workplaces [15]. In the Chinese study by Yang et al. [19], passive smoke exposure was signiﬁcantly related to Gross Domestic Product (GDP) per capita. In particular exposure was highest in cities with the lowest GDP per capita. In the study carried out by Martinez-Sanchez and coll. [20], the population of southern Italy showed more exposure to SHS, and even Minardi et al., in their paper, conﬁrmed a higher compliance with the smoking bans in northern and central Italy compared to the south. 1. Introduction According to them, there was an increase of respect for bans in work places (5%) and in hospitality premises (3%), and of the percentage of smoke-free homes (9%) 8 years after the smoking ban implementation, but with a signiﬁcant variation from north-south [21]. In 2003 the Italian Government, as one of the ﬁrst countries in Europe, approved the so-called “Sirchia Law”, which banned smoking in all indoor public places. The prohibition included all indoor public places, such as ofﬁces and restaurants [22]. The implementation of the smoking ban after the law, in January 2005, led to a considerable reduction of tobacco consumption, and consequently of passive smoke [23–27]. However, this evidence is based on sample populations. In Italy in 2006, the Ministry of Health established an overall health status surveillance system named PASSI (Progressi delle Aziende Sanitarie per la Salute in Italia) concerning lifestyles and behavioral risk factors associated with chronic diseases, such as tobacco smoke [28]. This surveillance made it possible to quantify the levels of compliance with smoking bans in Italy, understood as the effective application of indoor smoking bans, through interviews that investigated the respondents’ perception of compliance with bans in public places and workplaces frequented in the previous 30 days and at home. Data collected by PASSI are aggregated for the twenty-one Italian regions, providing a picture at the national level. So, the aim of this paper is to analyse the correlation between Gross Domestic Product (GDP) per capita by region and perceptions of compliance with the smoking ban in public avenues, at workplace and at home, according to data from the PASSI surveillance system. 3. Results A descriptive analysis of the GDP distribution among 21 Italian Regions was performed. Data concerning year 2015 (Eurostat), showed a GDP mean value of 26,714.29 €, with a SD of 7256.6. The rate of ban respect through each region was assessed, considering ban in public places, in workplaces and no smoking at home between year 2011–2014 and year 2014–2017. Table 1 shows the rates of adherence to each category [21]. Table 1. Rate of adherence to smoking ban in public places, workplaces and no smoking at home. Respect for Smoking Ban in Public Places (%) Respect for Smoking Ban in the Workplace (%) Adherence to No Smoking at Home (%) 2011–2014 89.7 91.4 78.7 2014–2017 91.0 93.1 81.7 Table 1. Rate of adherence to smoking ban in public places, workplaces and no smoking at home. The bivariate analysis concerning the association between GDP and smoking ban respect in public places showed a correlation coefﬁcient of 0.779 (p ≤0.001) concerning the period 2011–2014 and 0.723 (p ≤0.001) for the years 2014–2017. Furthermore, the analysis of the association between GDP and smoking ban respect in the workplace showed also a signiﬁcant correlation coefﬁcient of r = 0.662 (p = 0.001) for the years 2011–2014, and r = 0.603 (p = 0.004) concerning years 2014–2017. Adherence to no smoking at home showed that the correlation coefﬁcient was not statistically signiﬁcant considering the years 2011–2014 (0.424; p = 0.056) and 2014–2017 (0.362; p = 0.107). In Table 2 the results of the bivariate analysis are presented. Table 2. Results of the bivariate analysis of correlates of GDP per capita. Variables Correlation Coefﬁcient p-Value Smoking ban respect in public places 2011–2014 0.779 <0.001 Smoking ban respect in the workplace 2011–2014 0.662 0.001 No smoking at home 2011–2014 0.424 0.056 Smoking ban respect in public places 2014–2017 0.723 <0.001 Smoking ban respect in the workplace 2014–2017 0.603 0.004 No smoking at home 2014–2017 0.362 0.107 Table 2. Results of the bivariate analysis of correlates of GDP per capita. The linear regression analysis (Table 3) conﬁrmed the results from the bivariate analysis. The simple linear regression model considering smoking ban compliance in public places showed a beta coefﬁcient for GDP per capita of 0.735 (p < 0.001) considering the years 2011–2014 and of 0.710 (p < 0.001) considering the years 2014–2017. 2. Materials and Methods The outcome measures indicating passive smoking in different settings were, at the regional level, the prevalence of: - Perception of compliance with the smoking ban at public places; - Perception of compliance with the smoking ban in the workplaces; - No smoking at home. The source of these measures were data for the periods 2011–2014 and 2014–2017 that were retrieved from the PASSI surveillance system. This project, by administering surveys, continuously collects information from the Italian adult population (18–69 years) concerning smoking habits, physical inactivity, excess weight, alcohol consumption, diet, cardiovascular risk, access to cancer screening and the quality of life connected to health [28]. The main explanatory variable was GDP per capita (Eurostat 2015), also recorded at the regional level [29]. The bivariate analysis between these variables was performed using the Spearman correlation coefﬁcient (r). The linear regression analysis (simple and multiple) was performed using three different models considering the outcome measures as dependent variables and GDP per capita as the main independent variable. In the multivariate analysis, the models were adjusted for the total population Int. J. Environ. Res. Public Health 2018, 15, 2045 4 of 8 of the Region and its male percentage [30]. The results of the linear regression analysis are presented as standardized beta coefﬁcient and p-value. The goodness of ﬁt of the models was assessed using R2. The statistical signiﬁcance was set at p < 0.05. The analysis was carried out using IBM-SPSS, release 25.0 for Windows. 4. Discussion Passive smoking still represents one of the most important and most widespread health threatening forms of exposure. According to the PASSI Surveillance data, in Italy around 9 adults out of 10 stated that the ban of smoking in public places and in workplaces, in the thirty days prior to interview, is always or almost always respected (Table 1). However, there are clear regional differences and a clear North-South contrast, with compliance with the smoking ban s less likely to occur the southern regions. GDP per capita is considered an indicator of the standard of living. In Italy GDP is lower in southern regions where socio-economic indicators (high unemployment rate and low educational level) are less favourable. This study reveals that regions with higher GDP per capita have higher percentages of compliance with smoking bans. GDP per capita is signiﬁcantly related to respect for the smoking ban in public places and in workplaces, but this correlation is not signiﬁcant for smoking restrictions at home. GDP in fact becomes a parameter that describes the cultural awareness of the individual in the commitment to protect the health of the community in which he lives. It should also be noted that the concomitance of various economic and social factors contributes in determining the correlation between wealth and compliance with regulatory obligations. These ﬁndings are consistent with the results of the study by Yang [19]. Other researches focused on second-hand smoke and socioeconomic determinants. In the study carried out by Liao et al. [31], parents with lower education and lower annual incomes smoke more frequently in the presence of their children. Similar results about children’s passive smoke exposure were found by Kuntz et al. [32] and by Hajizadeh and Nandi [33]. Moreover, King et al. [34] and Tsai and colleagues [35] found that of SHS exposure was signiﬁcantly lower among graduates and with higher annual household income. More efforts should be realised to sensitize the population to the harmful effects of passive smoke on health. According to Martinez-Sanchez et al. [20] the lower rate of smoking restriction at home corresponds to an erroneous perception of SHS harmful effect by population. The lower rate of no smoking at home highlighted in the latest PASSI report can be linked to the absence of the type of legislation concerning private houses that can pose a deterrent in public places and in workplaces. 3. Results The multiple regression model showed a beta coefﬁcient of 0.730 (p < 0.001) considering the years 2011–2014 and of 0.698 (p < 0.001) considering the years 2014–2017. The simple linear regression model considering smoking ban respect in workplace showed a beta coefﬁcient of 0.549 (p = 0.01) concerning the years 2011–2014 and of 0.597 (p < 0.001) concerning the years 2014–2017. The multiple regression model showed a beta coefﬁcient of 0.525 (p = 0.020) and 0.570 (p = 0.009) respectively concerning the years 2011–2014 and 2014–2017. The simple linear regression model considering no smoking at home showed a beta coefﬁcient of 0.427 (p = 0.054) considering years 2011–2014 and of 0.446 (p = 0.043) considering years 2014–2017. Int. J. Environ. Res. Public Health 2018, 15, 2045 5 of 8 The multiple regression model showed a beta coefﬁcient of 0.332 (p = 0.070) and 0.362 (p = 0.052) respectively for years 2011–2014 and 2014–2017. Table 3. Results of the linear regression analysis. Dependent Variable GDP Per Capita Standardized Beta (p) * R2 of Simple Linear Regression Model GDP Per Capita Standardized Beta (p) ˆ R2 of Multiple Linear Regression Model Smoking ban respect in public places 2011–2014 0.735 (<0.001) 0.540 0.730 (<0.001) 0.579 Smoking ban respect in public places 2014–2017 0.710 (<0.001) 0.504 0.698 (<0.001) 0.544 Smoking ban respect in the workplace 2011–2014 0.549 (0.01) 0.301 0.525 (0.020) 0.318 Smoking ban respect in the workplace 2014–2017 0.597 (<0.001) 0.356 0.570 (0.009) 0.379 No smoking at home 2011–2014 0.427 (0.054) 0.182 0.332 (0.070) 0.517 No smoking at home 2014–2017 0.446 (0.043) 0.199 0.362 (0.052) 0.508 * simple linear regression model; ˆ multiple linear regression model, adjusted for male percentage and regional population. Table 3. Results of the linear regression analysis. 4. Discussion This concern could reﬂect a low awareness of the dangerous risk represented by SHS for health in public opinion. We need to acknowledge that this paper has some limitations. First of all, this is an ecological study, and the association between GDP and passive smoking indicators has to be considered with Int. J. Environ. Res. Public Health 2018, 15, 2045 6 of 8 caution. Data by PASSI are collected through questionnaires, and information bias could be an issue. However, as reported by Samet and Yang [36], SHS can be assessed through indirect parameters that consist in survey conducted through questionnaires. Finally, this study analyses data from all the Italian regions and it is therefore possible that selection bias could have an impact on the considered variables within the same region. However, the PASSI surveillance has been demonstrated to be a reliable system for lifestyle data. 5. Conclusions Thanks to the enforcement of the law on smoking bans, important results have been achieved but areas of action to be further strengthened remain, not only in the domestic context, but also in open spaces in the presence of minors, such as public parks, playgrounds, beaches, sports centres, and train stations, which could be regulated. This study shows that within the national context, adherence rates to smoking bans are variable and follow the distribution of the economic wealth of the regions. It will be necessary to carry out studies to highlight the factors inﬂuencing the different adherence to legislation, and on the basis of this, implement targeted programs to increase adherence rates. Furthermore, for a better health promotion, systematic vigilance and sanctions should be maintained and strengthened, mostly whenever low compliance towards smoking bans is registered. Author Contributions: Conceptualization, G.L.T.; Methodology, G.L.T., A.M.; Software, G.L.T.; Formal Analysis, G.L.T., C.S.; Investigation, G.L.T., C.S., R.A.C., S.C., L.L.; Resources, C.S., R.A.C., S.C., L.L.; Data Curation, G.L.T., A.M.; Writing—Original Draft Preparation, G.L.T., C.S., R.A.C., S.C., L.L.; Writing—Review & Editing, G.L.T., C.S., R.A.C., S.C., L.L., A.M.; Supervision, G.L.T. Author Contributions: Conceptualization, G.L.T.; Methodology, G.L.T., A.M.; Software, G.L.T.; Formal Analysis, G.L.T., C.S.; Investigation, G.L.T., C.S., R.A.C., S.C., L.L.; Resources, C.S., R.A.C., S.C., L.L.; Data Curation, G.L.T., A.M.; Writing—Original Draft Preparation, G.L.T., C.S., R.A.C., S.C., L.L.; Writing—Review & Editing, G.L.T., C.S., R.A.C., S.C., L.L., A.M.; Supervision, G.L.T. Author Contributions: Conceptualization, G.L.T.; Methodology, G.L.T., A.M.; Software, G.L.T.; Formal Analysis, G.L.T., C.S.; Investigation, G.L.T., C.S., R.A.C., S.C., L.L.; Resources, C.S., R.A.C., S.C., L.L.; Data Curation, G.L.T., A.M.; Writing—Original Draft Preparation, G.L.T., C.S., R.A.C., S.C., L.L.; Writing—Review & Editing, G.L.T., C.S., R.A.C., S.C., L.L., A.M.; Supervision, G.L.T. Funding: This research received no external funding. Acknowledgments: We are grateful to Emmy McDermott for the linguistic revision of the manuscript. Furthermore, we want to thank the PASSI Working group and in particular Maria Masocco and Valentina Minardi (National Center for Diseases Prevention and Health Promotion—National Health Institute). Conﬂicts of Interest: The authors declare no conﬂict of interest. 7. CDC and World Health Organization. Global Youth Tobacco Survey (GYT S). Atlanta, GA. Available online: https://www.cdc.gov/tobacco/data_statistics/surveys/yts/ (accessed on 2 August 2018). 6. U.S. Department of Health and Human Services. The Health Consequences of Smoking—50 Years of Progress: A Report of the Surgeon General; U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion, Ofﬁce on Smoking and Health: Atlanta, GA, USA, 2014. References 1. World Health Organization. Second-Hand Smoke: Assessing the Burden of Disease at National and Local Levels; World Health Organization: Geneva, Switzerland, 2008. 1. World Health Organization. Second-Hand Smoke: Assessing the Burden of Disease at National and Local Levels; World Health Organization: Geneva, Switzerland, 2008. 2. Giovino, G.A. The Tobacco Epidemic in the United States. Am. J. Prev. Med. 2007, 33, S318–S326. [CrossRef] [PubMed] 2. Giovino, G.A. The Tobacco Epidemic in the United States. Am. J. Prev. Med. 2007, 33, S318–S326. [CrossRef] [PubMed] 3. World Health Organization, and International Agency for Research on Cancer. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, Tobacco Smoke and Involuntary Smoking, Vol. 83, Lyon, France 2004. Available online: https://monographs.iarc.fr/wp-content/uploads/2018/06/mono83.pdf (accessed on 1 August 2018). 3. World Health Organization, and International Agency for Research on Cancer. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, Tobacco Smoke and Involuntary Smoking, Vol. 83, Lyon, France 2004. Available online: https://monographs.iarc.fr/wp-content/uploads/2018/06/mono83.pdf (accessed on 1 August 2018). 4. Jenkins, R.A.; Guerin, M.R.; Tomkins, B.A. The Chemistry of Environmental Tobacco Smoke: Composition and Measurement, 2nd ed.; Jenkins, R.A., Guerin, M.R., Tomkins, B.A., Eds.; Lewis Boca Raton: London, UK, 2000. 4. Jenkins, R.A.; Guerin, M.R.; Tomkins, B.A. The Chemistry of Environmental Tobacco Smoke: Composition and Measurement, 2nd ed.; Jenkins, R.A., Guerin, M.R., Tomkins, B.A., Eds.; Lewis Boca Raton: London, UK, 2000. 5. Bofﬁ, R.; Ruprecht, A.; Invernizzi, G. Istituto Nazionale dei Tumori. Il laboratorio per lo studio degli 5. Bofﬁ, R.; Ruprecht, A.; Invernizzi, G. Istituto Nazionale dei Tumori. Il laboratorio per lo studio degli inquinanti ambientali e del fumo di tabacco. Tabaccologia 2007, 2, 16–18. 6. U.S. Department of Health and Human Services. The Health Consequences of Smoking—50 Years of Progress: A Report of the Surgeon General; U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion, Ofﬁce on Smoking and Health: Atlanta, GA, USA, 2014. 7 of 8 Int. J. Environ. Res. Public Health 2018, 15, 2045 8. European Commission. Special Eurobarometer 458 “Attitudes of Europeans towards Tobacco and Electronic Cigarettes”. Available online: https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1& cad=rja&uact=8&ved=2ahUKEwi0rpD3vcPdAhUMiLwKHTtcDCEQFjAAegQIABAC&url=https%3A% 2F%2Fec.europa.eu%2Fcommfrontofﬁce%2Fpublicopinion%2Findex.cfm%2FResultDoc%2Fdownload% 2FDocumentKy%2F79002&usg=AOvVaw1wpjzABTIkKRn_gPUbXnIB (accessed on 1 August 2018). 9. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Tobacco Smoke and Involuntary Smoking; IARC Monogr Eval Carcinog Risks Hum: Lyon, France, 2004; Volume 83, pp. 1–1438. 10. World Health Organization. References WHO Report on The Global Tobacco Epidemic, 2017: Monitoring Tobacco Use and Prevention Policies. Available online: http://www.who.int/tobacco/global_report/2017/en/ (accessed on 1 August 2018). 11. Gallus, S.; Muttarak, R.; Martínez-Sánchez, J.M.; Zuccaro, P.; Colombo, P.; La Vecchia, C. Smoking prevalence and smoking attributable mortality in Italy. Prev. Med. 2010, 52, 434–438. [CrossRef] [PubMed] 12. Gorini, G.; Carreras, G.; Allara, E.; Faggiano, F. Decennial trends of social differences in smoking habits in Italy: A 30-year update. Cancer Causes Control 2013, 24, 1385–1391. [CrossRef] [PubMed] 13. Fischer, F.; Minnwegen, M.; Kaneider, U.; Kraemer, A.; Khan, M.H. Prevalence and determinants of secondhand smoke exposure among women in Bangladesh, 2011. Nicot. Tob. Res. 2015, 17, 58–65. [CrossRef] [PubMed] 14. Butler, K.M.; Rayens, M.K.; Ashford, K.; Adkins, S.; Gombeski, B.; Britt, J.; Hahn, E.J. Smoke-free homes, strength of smoke-free law and children in the home. Nicot. Tob. Res. 2014, 16, 485–490. [CrossRef] [PubMed] 15. Harris, J.K.; Geremakis, C.; Moreland-Russell, S.; Carothers, B.J.; Kariuki, B.; Shelton, S.C.; Kuhlenbeck, M. Demographic and geographic differences in exposure to secondhand smoke in Missouri workplaces, 2007–2008. Prev. Chronic Dis. 2014, 8, A135. 16. Huang, J.; King, B.A.; Babb, S.D.; Xu, X.; Hallett, C.; Hopkins, M. Sociodemographic characteristics in local smoke-free law coverage in 10 states. Am. J. Public Health 2015, 105, 1806–1813. [CrossRef] [PubMed] 17. Abdullah, A.S.; Hitchman, S.C.; Driezen, P.; Nargis, N.; Quah, A.C.; Fong, G.T. Socioeconomic differences in exposure to tobacco smoke pollution (TSP) in Bangladeshi households with children: Findings from the International Tobacco Control (ITC) Bangladesh Survey. Int. J. Environ. Res. Public Health 2011, 8, 842–860. [CrossRef] [PubMed] 18. Kurtz, M.E.; Kurtz, J.C.; Contreras, D.; Booth, C. Knowledge and attitudes of economically disadvantaged women regarding exposure to environmental tobacco smoke: A Michigan, USA study. Eur. J. Public Health 2003, 13, 171–176. [CrossRef] [PubMed] 19. Yang, T.; Jiang, S.; Barnett, R.; Peng, S.; Yu, L. Individual and city-level determinants of secondhand smoke exposure in China. Int. J. Health Geogr. 2015, 14, 36. [CrossRef] [PubMed] 20. Martínez-Sánchez, J.M.; Gallus, S.; Zuccaro, P.; Colombo, P.; Fernández, E.; Manzari, M.; La Vecchia, C. Exposure to secondhand smoke in Italian non-smokers 5 years after the Italian smoking ban. Eur. J. Public Health 2011, 22, 707–712. [CrossRef] [PubMed] 21. Minardi, V.; Gorini, G.; Carreras, G.; Masocco, M.; Ferrante, G.; Possenti, V.; Quarchioni, E.; Spizzichino, L.; Galeone, D.; Vasselli, S.; et al. Compliance with the smoking ban in Italy 8 years after its application. Int. J. 26. Gallus, S.; Zuccaro, P.; Colombo, P.; Apolone, G.; Paciﬁci, R.; Garattini, S.; Bosetti, C.; La Vecchia, C. Smoking in Italy 2005–2006: Effects of a comprehensive National Tobacco Regulation. Prev. Med. 2007, 45, 198–201. [CrossRef] [PubMed] References Public Health 2014, 59, 549–554. [CrossRef] [PubMed] 22. Disposition of the Legal System in the Field of Public Administration (It Official JN. 15–20 January 2003). Available online: http://www.salute.gov.it/imgs/C_17_normativa_366_allegato.pdf (accessed on 1 August 2018). 23. Valente, P.; Forastiere, F.; Bacosi, A.; Cattani, G.; Di Carlo, S.; Ferri, M.; Figà-Talamanca, I.; Marconi, A.; Paoletti, L.; Perucci, C.; et al. Exposure to ﬁne and ultraﬁne particles from secondhand smoke in public places before and after the smoking ban, Italy 2005. Tob. Control 2007, 16, 312–317. [CrossRef] [PubMed] 24. Tramacere, I.; Gallus, S.; Fernandez, E.; Zuccaro, P.; Colombo, P.; La Vecchia, C. Medium-term effects of the Italian smoke-free legislation: Findings from 4 annual population based surveys. J. Epidemiol. Commun. Health 2009, 63, 559–562. [CrossRef] [PubMed] 25. Gallus, S.; Zuccaro, P.; Colombo, P.; Apolone, G.; Paciﬁci, R.; Garattini, S.; La Vecchia, C. Effects of new smoking regulations in Italy. Ann. Oncol. 2006, 17, 346–347. [CrossRef] [PubMed] 26. Gallus, S.; Zuccaro, P.; Colombo, P.; Apolone, G.; Paciﬁci, R.; Garattini, S.; Bosetti, C.; La Vecchia, C. Smoking in Italy 2005–2006: Effects of a comprehensive National Tobacco Regulation. Prev. Med. 2007, 45, 198–201. [CrossRef] [PubMed] 8 of 8 Int. J. Environ. Res. Public Health 2018, 15, 2045 27. Gorini, G.; Moshammer, H.; Sbrogi, L.; Gasparrini, A.; Nebot, M.; Neuberger, M.; Tamang, E.; Lopez, M.J.; Galeone, D.; Serrahima, E. Italy and Austria before and after study: Second-hand smoke exposure in hospitality premises before and after 2years from the introduction of the Italian smoking ban. Indoor Air 2008, 18, 328–334. [CrossRef] [PubMed] 28. PASSI. Progressi Delle Aziende Sanitarie per la Salute in Italia. Available online: http://www.epicentro.iss. it/passi/ (accessed on 26 July 2018). 29. Eurostat. 2015 GDP per Capita in 276 EU Regions. 52/2017–30 March 2017. Available online: http://ec.europa.eu/eurostat/documents/2995521/7962764/1-30032017-AP-EN.pdf/4e9c09e5- c743-41a5-afc8-eb4aa89913f6 (accessed on 26 July 2018). 30. ISTAT. Italian Population Census 2011. Available online: http://dati-censimentopopolazione.istat.it/Index. aspx?lang=it (accessed on 26 July 2018). 31. Liao, Y.M.; Chen, Y.T.; Kuo, L.C.; Chen, P.L. Factors associated with parental smoking in the presence of school-aged children: A cross-sectional study. BMC Public Health 2013, 13, 819. [CrossRef] [PubMed] 32. Kuntz, B.; Lampert, T. Social disparities in parental smoking and young children’s exposure to secondhand smoke at home: A time-trend analysis of repeated cross-sectional data from the German KiGGS study between 2003–2006 and 2009–2012. BMC Public Health 2016, 16, 485. [CrossRef] [PubMed] 33. Hajizadeh, M.; Nandi, A. References The socioeconomic gradient of secondhand smoke exposure in children: E from 26 low-income and middle-income countries. Tob. Control 2016, 25, e146–e155. [CrossRef] [Pu 34. King, B.A.; Homa, D.M.; Dube, S.R.; Babb, S.D. Exposure to secondhand smoke and attitudes toward smoke-free workplaces among employed US adults: Findings from the National Adult Tobacco Survey. Nicot. Tob. Res. 2014, 16, 1307–1318. [CrossRef] [PubMed] 35. Tsai, Y.W.; Chang, L.C.; Sung, H.Y.; Hu, T.W.; Chiou, S.T. The impact of smoke-free legislation on reducing exposure to secondhand smoke: Differences across gender and socioeconomic groups. Tob. Control 2015, 24, 62–69. [CrossRef] [PubMed] 36. Samet, J.; Yoon, S.Y. Passive smoking, women and children. In Women and the Tobacco Epidemic, Challenges for the 21st Century; WHO/NMH/TF1; World Health Organization: Geneva, Switzerland, 2001. © 2018 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/W2048763120	https://publications.goettingen-research-online.de/bitstream/2/24548/1/c1cp20883g_Otto.pdf	English	null	Temperature-dependent intensity anomalies in amino acid esters: weak hydrogen bonds in protected glycine, alanine and valine	Physical chemistry chemical physics/PCCP. Physical chemistry chemical physics	2,011	cc-by	12,234	a Institut fu¨r Physikalische Chemie, Universita¨t Go¨ttingen, Tammannstr. 6, D-37077 Go¨ttingen, Germany. E-mail: msuhm@gwdg.de b Institut fu¨r Organische und Biomolekulare Chemie, Universita¨t Go¨ttingen, Tammannstr. 2, D-37077 Go¨ttingen, Germany w Electronic supplementary information (ESI) available. See DOI: 10.1039/c1cp20883g z Present address: Laboratoire de Chimie Physique Mole´ culaire, E´ cole Polytechnique Fe´ de´ rale de Lausanne, CH-1015 Lausanne, Switzerland. y Present address: Department of Chemistry, University of Basel, CH-4056 Basel, Switzerland. z Present address: Interfacultary Institute of Biochemistry, University of Tuebingen, D-72076 Tuebingen, Germany. Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G Received 23rd March 2011, Accepted 8th June 2011 DOI: 10.1039/c1cp20883g Esters of glycine, alanine and valine are investigated by FTIR and Raman spectroscopy in supersonic jets as gas phase model systems for the neutral peptide N-terminus. The NH-stretching vibrations exhibit very large temperature- and substitution-dependent intensity anomalies which are related to weak, bifurcated intramolecular hydrogen bonds to the carbonyl group. Comparison to theory is only satisfactory at low temperature. Spectral NH aggregation shifts are small or even negligible and the associated IR intensity is remarkably low. In the case of valine, chirality recognition eﬀects are nevertheless detected and rationalized. Comparison to quantum-chemical calculations for dimers shows that dispersion interactions are essential. It also rules out cooperative hydrogen bond topologies and points at deﬁciencies in standard harmonic treatments with the linear dipole approximation. Fig. 1 Scheme for the nomenclature of the diﬀerent conformers of the examined amino acid esters. The 1st (NC), 2nd (CC) and 3rd (OC) dihedral angles are used, yielding ttt in this case. PCCP PCCP Cite this: Phys. Chem. Chem. Phys., 2011, 13, 14119–14130 Cite this: Phys. Chem. Chem. Phys., 2011, 13, 14119–14130 Katharina E. Otto,a Susanne Hesse,a Tobias N. Wassermann,za Corey A. Rice,ya Martin A. Suhm,a Thorsten Staﬀorstzb and Ulf Diederichsenb Katharina E. Otto,a Susanne Hesse,a Tobias N. Wassermann,za Corey A. Rice,ya Martin A. Suhm,a Thorsten Staﬀorstzb and Ulf Diederichsenb PCCP Dynamic Article Links View Article Online / Journal Homepage / Table of Contents for this issue PCCP Dynamic Article Links View Article Online / Journal Homepage / Table of Contents for this issue Dynamic Article Links View Article Online / Journal Homepage / Table of Contents for this issue PAPER PAPER www.rsc.org/pccp This journal is c the Owner Societies 2011 1 Introduction The concentration was adjusted by letting the rare gas ﬂow through a thermostated saturator ﬁlled with the liquid esters. Variations in concentration could be obtained by setting the saturator to diﬀerent temperatures. For details, see ref. 27 and the ESI.w The reported approximate gas phase concentrations are based on comparison with quantum chemical intensity calculations in the IR carbonyl stretching region on the MP2/6–311+G* level of approximation. coordination leading to a folded ring involving 8 heavy atoms was predicted. Hydrogen bond-induced aliphatic N–H stretching fundamental shifts in dimers were found to be small, in rather pronounced contrast to aromatic N–H stretching modes.17 The purpose of the present contribution is to explore this weak vibrational signature of NH2 hydrogen bonding for ethyl esters of the elementary amino acids glycine, alanine and valine. Ethyl esters are preferred over methyl esters because of their increased stability with respect to polymerization. For the study of neutral aliphatic amino acids and their esters, detection techniques based on UV-absorption like (two-photon) ionization or laser-induced ﬂuorescence,18 IR-UV double resonance techniques19,20 or other mass spectrometric action- spectroscopy16,21,22 are less feasible. Instead, for the elementary model systems studied here, direct FTIR absorption spectroscopy and spontaneous Raman scattering appear to be the most suitable vibrational techniques available.17,23 As will be demon- strated, there are remarkable and fairly systematic temperature- dependent intensity eﬀects, combined with the phenomenon of non-shifting hydrogen bonds and with subtle chirality recognition. Raman jet spectroscopy turns out to be crucial in uncovering these phenomena, as is the consideration of dispersion eﬀects in the interaction. The results are of relevance for the potential role of N-terminal amino acids in peptide recognition and catalysis under non-protonated conditions. Such conditions may exist in solution, with typical pKa values of the (protonated) amino group between 7 and 8 and sometimes as low as 6.24 However, solution studies of the NH stretching dynamics are strongly aﬀected by the intense OH stretching band of the water solvent. Therefore, the present gas phase spectra oﬀer new insights into the properties of this functional group. Our spectra also provide new test cases for the various theories on red-, blue- and non-shifting hydrogen bonds.25,26 Spontaneous Raman scattering measurements28 were carried out using similar gas mixtures of the amino acid esters in He. They were expanded via a 4 0.15 mm2 slit nozzle, pumped by a 500 m3 h1 Roots pump. 1 Introduction Amino acids oﬀer a wide range of intra- and intermolecular interactions, which change with the aggregation stage, charge, side chain and peptide incorporation.1 They have been extensively studied in the gas phase,2,3 in supersonic jets,4–6 in matrix isolation7–11 and in polar environments.12,13 Here, we block the acidic OH group by esteriﬁcation.14 What remains is a weak intramolecular interaction between the NH2 group and the carbonyl group, which may be replaced by intermolecular variants as well as NH NH contacts upon aggregation. In contrast to unprotected amino acids,15 zwitterion formation16 is suppressed even in a polar environment. Fig. 1 Scheme for the nomenclature of the diﬀerent conformers of the examined amino acid esters. The 1st (NC), 2nd (CC) and 3rd (OC) dihedral angles are used, yielding ttt in this case. The conformational freedom is illustrated in Fig. 1. Rotation around the ﬁrst torsional angle (nitrogen lone pair–N–C–C, in the following abbreviated as NC) directly modulates the interaction between the N–H bonds and the electron density at the carbonyl group. Rotation around the second torsional angle (N–C–C–O, abbreviated as CC) is most feasible and allows for a switch from carbonyl to ester oxygen coordination. Torsion around the ester bond (C–C–O–C) is rather stiﬀ, partially mimicking the amide bond, whereas torsion around the third dihedral angle shown (C–O–C–C, abbreviated as OC) should be considered if one goes beyond the methyl ester. By using t for torsional angles of 1801 301 (g for 601 301 and c for 01 301), the conformation shown in Fig. 1 is denoted ttt for the ﬁrst, second and third torsional angles in this sequence. The interplay between intra- and intermolecular contacts is normally best probed in the N–H stretching fundamental range,14 but the very low IR intensity of uncoordinated pyramidal NH2 group vibrations and the oscillator coupling between the two geminal N–H bonds present additional challenges. The former can be met by the inclusion of Raman spectroscopy, whereas the latter was avoided chemically in an earlier study of proline esters.17 The key result of that study was a weak aggregation signature in the IR. In proline ester dimers, a slight preference for mutual N–H OQC Phys. Chem. Chem. Phys., 2011, 13, 14119–14130 14119 This journal is c the Owner Societies 2011 View Article Online The compounds were highly diluted in a carrier gas (99.996% He, Linde). 3 Results and discussion The results are organized according to increasing complexity of the amino acid, always comparing the experimental spectra with quantum chemical spectral predictions. FTIR spectra at 2 cm1 resolution were recorded in a pulsed slit jet expansion.17 The jet chamber was crossed by a mildly focussed and recollimated Bruker IFS 66v/S probe beam over a total length of 0.78 m. Either the equilibrated gas phase or a supersonic jet expansion emerging from a 0.6 m slit nozzle with a slit width of 0.2 mm was probed close to the nozzle over a varying cross section, which averages around 15 mm 15 mm. Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G g p Quantum chemical calculations (structure optimizations and harmonic frequency calculations) were explored for these systems using the Gaussian0931 package. The computational methods include B3LYP/6-311++G** (abbreviated B3LYP), B97D/TZVP/TZVPFit (abbreviated B97D), MP2/6-311+G* (MP2) and MP2/aug-cc-pVTZ (MP2/aug). For some MP2 levels, Raman intensities were not available but rather adopted from the smaller basis set MP2 prediction or, for the dimers, from B97D predictions. At levels where zero-point energy corrections were not available because of the involved computational eﬀort beyond that of the structure optimization, these were adopted from a lower level, as indicated in the corresponding tables and ﬁgures. 2 Methods Due to their instability regarding polymerization, the amino acid esters were always freshly prepared from their hydro- chlorides (glycine ethyl ester hydrochloride, 99%, ABCR; L-alanine ethyl ester hydrochloride, 99%, Aldrich; L-valine ethyl ester hydrochloride, 99%, Acros Organics; DL-valine ethyl ester hydrochloride, 99%, ABCR). The hydrochlorides were dissolved in water, cooled to 0 1C and Z 1 equivalent of aqueous K2CO3 (Gru¨ ssing GmbH, 99.5%) was added. The mixture was stirred at 0 1C for ﬁve minutes and brought to room temperature. After another ﬁve minutes of stirring the solution was extracted with dichloromethane (Roth, Z 99%), dried over Na2SO4 (ABCR, 99%), and the solvent was evaporated under reduced pressure. The esters were obtained as colourless oil with overall yields of Z 90% and their purity was checked by NMR spectroscopy (for detailed information see ESIw). 1 1 Introduction The beam of a frequency doubled cw NdYVO4-Laser (Coherent Verdi V18, 18 W, l = 532 nm) was focused onto the expansion at distances from the nozzle exit of 1 and 3 mm. Variation of the nozzle distance can lead to relaxation eﬀects by collisions of the molecules with the carrier gas atoms.29,30 Substance concentrations were adopted from the IR experiment. At 901 angle, the scattered light was collected and collimated using a fast camera lens (Nikon, += 50 mm, f/1.2). It was then focused onto the entrance slit of the monochromator (McPherson Model 2051 f/8.6, f = 1000 mm) using an achromatic planoconvex lens (Edmund Optics, += 50 mm, f/7). A Raman edge ﬁlter (L.O.T.,+ = 25 mm, OD 6.0, T 4 90%, 535.4–1200 nm) was used to eliminate Rayleigh scattered photons before the dispersion by the monochromator. A liquid N2 cooled back-illuminated CCD camera (PI Acton, Spec-10: 400 B/LN, 1340 400 pixel) served as the detection device. The primary wavelength calibration of the spectra was carried out using the lines of a Ne I emission light source. Cosmic ray signals were removed by the comparison of block- averaged spectra. Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G 14120 Phys. Chem. Chem. Phys., 2011, 13, 14119–14130 Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G Fig. 2 The most stable structures obtained at the MP2/aug-cc-pVTZ level of approximation for monomeric glycine ethyl ester along with their relative energies including zero-point energy (DE0). The amino group clearly prefers a symmetric double coordination of the carbonyl group by both N–H bonds.32,33 We note that this is the same arrangement as in neutral gas phase glycine itself, according to high level quantum chemical calculations,1,34,35 36 6 microwave structures36 and Raman spectroscopy.6 In contrast, microwave structures36 and Raman spectroscopy.6 In contrast, N-terminal glycine in a peptide can also form a N–H NH2 hydrogen bond37 if the ﬁrst peptidic NH-group is available. Coordination of the carbonyl group by only one N–H (gtt) or coordination of the ester oxygen (tct) are both associated with an energy penalty of more than 4 kJ mol1 and should normally not be observed in a supersonic jet, except very close to the nozzle.6 One may speculate that the double coordination in ttt and ttg proﬁts from overlap with the p-orbital of the carbonyl group, in the presence of an unfavorable angle for interaction with the oxygen lone pair (see Fig. 1). Fig. 3 Experimental FTIR and Raman spectra of glycine ethyl ester in He in the NH-stretching region: (a) gas phase (IR), 170 mbar, 0.03% glycine ethyl ester; (b) CQO stretching fundamental on a 1.99 stretched wavenumber scale after 100-fold intensity compression; (c) jet (IR) scaled by a factor of 100 to match the CH-region of the gas phase spectrum, 0.03%, 0.7 bar stagnation pressure; (d) gas phase (Raman), 0.2 bar, 0.01%; (e) jet (Raman) scaled by a factor of 14 to match the CH-region of the gas phase, nozzle distance 3 mm, stagnation pressure 0.8 bar, 0.01%. yg p ( g ) In the N–H stretching region of isolated glycine ethyl ester, one thus expects two stretching bands due to symmetric and antisymmetric motion of the two geminal bonds. For an unsubstituted amine such as methylamine, they fall in the range of 3360–3424 cm1 and are separated by B64 cm1 38–41 with the lower band corresponding to symmetric stretching motion. This pattern is reproduced reasonably well by a range of harmonic quantum chemical calculations (see Table S1 in the ESIw). Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G The splitting is exaggerated (78 cm1 at the B3LYP level, 90–100 cm1 at B97D and MP2 levels). Nevertheless, such calculations allow for a relative prediction of the glycine ethyl ester spectra. Here, the symmetric/antisymmetric splitting is predicted consistently smaller by about 8 2 cm1, whereas the position of the symmetric stretch is always within 10 cm1 of the methylamine value. Harmonic quantum chemical calculations (see Table S2, ESIw) consistently indicate that the antisymmetric mode of the glycine ethyl ester has about 3–5 times more IR intensity than the symmetric one, whereas the Raman intensity is higher by a factor of 2–3 in the symmetric case. In the N–H stretching region of isolated glycine ethyl ester, one thus expects two stretching bands due to symmetric and antisymmetric motion of the two geminal bonds. For an unsubstituted amine such as methylamine, they fall in the range of 3360–3424 cm1 and are separated by B64 cm1 38–41 bands together with a very weak trace of the second-lowest (30–100 fold weaker relative intensity). The extra band at a higher wavenumber in the IR spectrum can be explained as in the case of proline esters.17 It is due to the weak overtone of the carbonyl stretching vibration, as shown in trace (b) after multiplication of the fundamental wavenumber by 1.99. This illustrates how weak the N-terminal NH stretching bands are in the IR, sometimes even preventing their identiﬁcation.3 The symmetric NH-stretching mode would be expected at 3360 10 cm1 and indeed it is observed at 3356 cm1. The antisymmetric NH-stretching mode would be expected B64–8 cm1 higher and indeed it is found at 3413 cm1. If there were no 10–30-fold attenuation of the antisymmetric Raman band and no B5-fold attenuation of the antisymmetric IR stretching transition compared to theoretical expectations, everything would be in perfect agreement for the gas phase spectrum at room temperature. bands together with a very weak trace of the second-lowest (30–100 fold weaker relative intensity). The extra band at a higher wavenumber in the IR spectrum can be explained as in the case of proline esters.17 It is due to the weak overtone of the carbonyl stretching vibration, as shown in trace (b) after multiplication of the fundamental wavenumber by 1.99. Phys. Chem. Chem. Phys., 2011, 13, 14119–14130 14121 This journal is c the Owner Societies 2011 3.1 Glycine The most stable structure of glycine ethyl ester (ttt) is shown in Fig. 2, together with three higher-lying conformations. Torsion of the ester group into a gauche position (ttg) comes with a small energy penalty but this is not expected to lead to separate spectroscopic features in the investigated range. 14120 Phys. Chem. Chem. Phys., 2011, 13, 14119–14130 This journal is c the Owner Societies 2011 View Article Online Fig. 3 Experimental FTIR and Raman spectra of glycine ethyl ester in He in the NH-stretching region: (a) gas phase (IR), 170 mbar, 0.03% glycine ethyl ester; (b) CQO stretching fundamental on a 1.99 stretched wavenumber scale after 100-fold intensity compression; (c) jet (IR) scaled by a factor of 100 to match the CH-region of the gas phase spectrum, 0.03%, 0.7 bar stagnation pressure; (d) gas phase (Raman), 0.2 bar, 0.01%; (e) jet (Raman) scaled by a factor of 14 to match the CH-region of the gas phase, nozzle distance 3 mm, stagnation pressure 0.8 bar, 0.01%. View Article Onli Fig. 2 The most stable structures obtained at the MP2/aug-cc-pVTZ level of approximation for monomeric glycine ethyl ester along with their relative energies including zero-point energy (DE0). Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G It leads to a small but distinct blue-shift of the symmetric NH-stretch and an enormous intensiﬁcation of the antisymmetric stretching mode both in Raman (25) and IR (7) spectra. In the Raman spectrum, the central band gains by a factor of 25 5, whereas the lowest frequency band increases by less than a factor of 2 and shifts somewhat to a higher wavenumber. Detailed values may be found in Table S3 (ESIw). As a consequence, jet cooling changes the intensity ratio of the central band to the low-wavenumber band from 1 to more than 5 in the IR and from 0.02 0.01 to 0.3 0.1 in the Raman case. To explain this temperature eﬀect, one might invoke cluster formation in the jet combined with a negligible clustering shift. While small frequency shifts are rare in hydrogen bonding, they are conceivable, given the existence of both red- and blue-shifted hydride stretching vibrations.25,26 Therefore, Fig. 4 shows sequences of jet expansions, with clustering conditions increasing from bottom to top within a given method. There are indeed weak cluster features to lower wavenumbers (marked with arrows), but the main peaks do not change substantially, thus ruling out a major intensity distortion by cluster formation. This is conﬁrmed by the scaling factors chosen to keep the monomer signal intensity more or less constant. They are approximately inversely proportional to concentration, as expected. Minor cluster contributions on top of the IR monomer bands should however not be ruled out and are highly probable. Independent of the nature of the intermolecular hydrogen bonds in these clusters To corroborate the origin of the intensity anomaly in glycine ethyl ester, we have carried out corresponding experiments with 2-methoxyethylamine,43 which lacks the carbonyl group. As elaborated in the ESIw, there is an analogous pattern of symmetric and antisymmetric NH-stretching vibrations (Fig. S2, ESIw). The splitting is closer to the methylamine case (Table S1, ESIw), fully in line with theoretical predictions, and there is a contribution from a secondary isomer. Most importantly, the s/as intensity ratio in the Raman spectrum is now independent of temperature within experimental error bars (Table S5, ESIw). Absolute Raman intensities change at most by a factor of 2 in the jet, as opposed to 25 5 in the case of the glycine ester. Furthermore, there is no blue-shift of the symmetric NH-stretching mode upon cooling. Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G This illustrates how weak the N-terminal NH stretching bands are in the IR, sometimes even preventing their identiﬁcation.3 The symmetric NH-stretching mode would be expected at 3360 10 cm1 and indeed it is observed at 3356 cm1. The antisymmetric NH-stretching mode would be expected B64–8 cm1 higher and indeed it is found at 3413 cm1. If there were no 10–30-fold attenuation of the antisymmetric Raman band and no B5-fold attenuation of the antisymmetric IR stretching transition compared to theoretical expectations, everything would be in perfect agreement for the gas phase spectrum at room temperature. In a supersonic jet, the spectral intensities change drastically. The jet spectra are displayed in Fig. 3 on a scale where the integral C–H band intensity is comparable to that in the gas phase. Assuming that the C–H transitions are not very temperature- and clustering-sensitive, this indicates that the central IR band gains in intensity by a factor of 7 2, whereas the other two do not and therefore disappear in the noise level. The experimental spectra recorded in the gas phase at room temperature fully conﬁrm the predicted NH band positions but provide a rather diﬀerent picture in terms of intensity and number of bands (Fig. 3). The IR spectrum (trace (a)) contains three weak bands of more or less similar intensity, whereas the Raman spectrum (trace (d)) only shows the lowest of these Phys. Chem. Chem. Phys., 2011, 13, 14119–14130 14121 This journal is c the Owner Societies 2011 View Article Online Fig. 4 Experimental FTIR and Raman spectra of glycine ethyl ester in He in the NH-stretching region with increasing concentration scaled to a similar integral band intensity of the antisymmetric NH-stretching vibration of the monomer. (a)–(c) Jet spectra (IR) measured at a stagnation pressure of 0.7 bar, (a) 0.01%, scaled by 3.6, (b) 0.03%, scaled by 1.9, (c) 0.06%; (d)–(f) jet spectra (Raman) measured at a stagnation pressure of 0.8 bar, (d) nozzle distance 1 mm, 0.01%, (e) nozzle distance 3 mm, 0.01%, scaled by 3.1, (f) nozzle distance 3 mm, 0.03%, scaled by 1.9. (N–HOQC or N–HO–C or N–HN–H), it is remarkable that the red-shifts do not exceed 15 cm1. The cluster-induced red-shift of the carbonyl stretching fundamental (10–15 cm1) is of comparable size and the blue-shift of the ester C–O stretching mode is as large as 14 cm1, both supporting a N–H OQC interpretation (see Fig. Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G S1, ESIw). We note that the NH bending mode gives rise to a single monomer band that is split into red- and blue-shifted bands in the clusters, also indicative of intermolecular N–H OQC hydrogen bonds. Further details may be found in the ESIw (Fig. S1 and Table S4). The monomer interpretation of the peaks observed in the jet spectra is further supported by quantum chemical calculations. Table S2 (ESIw) shows that the theoretical intensity predictions are matched quite well by the experimental jet spectra, whereas the room temperature gas phase spectra strongly disagree with theory. Evidently, the eﬀect must be related to the eﬀective temperature of the amino acid esters, with lower temperatures promoting the asymmetric stretching intensity. It is noteworthy that a He droplet experiment for glycine42 also revealed a dominance of the antisymmetric NH stretch, which in this case occurs at 3436 cm1. Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G We emphasize that microwave studies of the methyl ester of glycine33 at room temperature have identiﬁed the most stable bifurcated hydrogen bond structure (ttt), but also signiﬁcant population of a CC torsional state, the fundamental excitation of which was estimated at 40–70 cm1, along with at least 7 other populated states. This underscores the ﬂoppiness of the molecule at room temperature and our work suggests that some of these excited states have a much weaker antisymmetric NH stretching mode than the ground state. Indeed, a rough estimate of the vibrational ground state population of glycine ethyl ester at room temperature is 0.3%, which may rise to 430% in the jet expansions. Fig. 4 Experimental FTIR and Raman spectra of glycine ethyl ester in He in the NH-stretching region with increasing concentration scaled to a similar integral band intensity of the antisymmetric NH-stretching vibration of the monomer. (a)–(c) Jet spectra (IR) measured at a stagnation pressure of 0.7 bar, (a) 0.01%, scaled by 3.6, (b) 0.03%, scaled by 1.9, (c) 0.06%; (d)–(f) jet spectra (Raman) measured at a stagnation pressure of 0.8 bar, (d) nozzle distance 1 mm, 0.01%, (e) nozzle distance 3 mm, 0.01%, scaled by 3.1, (f) nozzle distance 3 mm, 0.03%, scaled by 1.9. If we attribute the jet cooling eﬀects to the strengthening of the bifurcated NH2 OQC hydrogen bond, this particular interaction has remarkable properties. 14122 Phys. Chem. Chem. Phys., 2011, 13, 14119–14130 This journal is c the Owner Societies 2011 Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G From this control experiment, it follows that the NH2 intensity and shift anomalies are correlated with the presence of a carbonyl group in the a position. One may now speculate that the intensity of the anti- symmetric stretching mode is promoted by the interaction 14122 Phys. Chem. Chem. Phys., 2011, 13, 14119–14130 This journal is c the Owner Societies 2011 View Article Online Fig. 6 Energy level scheme including zero-point energies (DE0) for diﬀerent dimer species of glycine ethyl ester optimized on diﬀerent calculation levels. with the p-orbital, where the stretching N–H bond may proﬁt from the polarization of the lobe pointing in its direction, while the contracting N–H bond recedes from the other lobe. In order to test this, we also compare IR and Raman intensities of methylamine at the same levels of approximation in Table S2 (ESIw). The non-systematic behaviour of the IR and Raman intensities, also across diﬀerent computational levels, suggests that this is not the case. Following the formal IR and Raman signal strengths along the NC and CC torsional angles (see Fig. S3–S6 in the ESIw) does not provide further insights into the observed intensity eﬀects, either. In contrast, these relaxed scans would indicate that the IR intensities of methoxyethylamine are more sensitive to thermal excitation than those of glycine ethyl ester. Either the employed electronic structure approach (MP2/6–311+G) is unable to describe the intensity trends or an anharmonic treatment of the torsional states is needed. Fig. 6 Energy level scheme including zero-point energies (DE0) for diﬀerent dimer species of glycine ethyl ester optimized on diﬀerent calculation levels. predict 8cc as the global minimum structure, closely followed by an unsymmetrical 8ec topology. Indications for the presence of diﬀerent dimer structures in the jet expansions can be expected from the IR spectra. In the carbonyl stretching region with its large oscillator strength, cluster bands can be used for a semiquantitative assessment of their concentration. It is likely (and conﬁrmed by theoretical calculations at several levels) that the average CQO IR-intensity does not change by more than a factor of two when this group engages in hydrogen bond formation. Based on this, the CQO stretching band shown in Fig. S1 (ESIw) and in the top trace of Fig. 7 indicates that around 60% of the CQO groups are present in the clustered form for a 0.06% expansion of glycine ethyl ester in He. Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G 3.2 Glycine ester aggregates Diﬀerent hydrogen-bonded dimer topologies were explored for the ethyl ester of glycine. Their nomenclature involves the size of the hydrogen-bonded ring (counting only heavy atoms). Two isolated N–H hydrogen bonds lead to 8-rings and can either engage carbonyl oxygen (c) or ester oxygen atoms (e). A cooperative N–H N–H O arrangement involves 5 heavy atoms including either a carbonyl (c) or an ester (e) oxygen. This gives rise to 5 diﬀerent topologies (8cc, 8ee, 8ec, 5c, 5e), of which the ﬁrst four are exempliﬁed in Fig. 5. Topology 5e was discarded because of its high energy. When dispersion inter- action is included, all four structures are predicted to be rather compact. This has major consequences for the predicted energy sequence, which is shown in Fig. 6 (including harmonic zero point energy). Cooperative 5-ring structures stop to be relevant when dispersion interaction is included. All methods Fig. 5 Diﬀerent dimer structures of glycine ethyl ester optimized on the MP2/6-311+G level of approximation including their zero- point-corrected dissociation energies (D0). The most striking result is obtained when transferring the 40% dimer simulation to the NH stretching range recorded under identical conditions. The predicted IR dimer features are far too strong. In the simulation shown in Fig. 8 they had to be attenuated by a factor of 10 relative to the monomer. Despite this attenuation the 5c prediction at the B3LYP level is still too strong and fails completely in matching experimental features. The 8cc intensity prediction is also too high. Similar remarks apply to the B97D intensities. In addition, qualitatively wrong blue-shifts are predicted. The MP2 simulation is qualitatively satisfactory but the reduction of the stick length by up to a factor of 10 still needs to be explained. One possibility is an increased width of the dimer bands. Indeed, there is some indication of a broad background at a lower wavenumber relative to the monomer absorptions. However, this may also be due to larger clusters and does not apply to the more structured features near 3400 cm1. The other possibility is a drastic overestimation of the IR band strength–– not unreasonable for such a weakly IR-active oscillator. However, the predicted increase of the low infrared NH Fig. 5 Diﬀerent dimer structures of glycine ethyl ester optimized on the MP2/6-311+G* level of approximation including their zero- point-corrected dissociation energies (D0). Phys. Chem. Chem. Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G Assuming that dimers dominate the cluster distribution, this corresponds to a dimer fraction of up to 40%. The value will be smaller if larger clusters are present as well. The fact that the cluster CQO band is red-shifted relative to the monomer band suggests the presence of inter- molecular N–H OQC hydrogen bonds which are stronger than the intramolecular ones. As a consequence, the C–O stretching transition near 1200 cm1 is blue-shifted because this bond is strengthened when the adjacent CQO bond is weakened. Simulated stick spectra (lower traces of Fig. 7) allow for a narrowing down of the conformational options. At the B3LYP level, where 8cc and 5c are predicted to be particularly stable, both ﬁt the experimental spectra at least qualitatively. At the B97D level this is only the case for 8cc, but not for the second most stable 8ec structure. At the MP2 level both 8cc and 8ec match the experimental shift qualitatively. Phys. Chem. Chem. Phys., 2011, 13, 14119–14130 14123 3.2 Glycine ester aggregates This is a strong indication of either band broadening or overestimated infrared dimer NH intensities. Colour code: black (thick): monomer (ttt), red: 8cc–, blue: 8ec–, green: 5c–dimers. The calculated spectra are shifted such that the harmonic monomer antisymmetric NH stretching fundamental matches the experiment (namely 167 cm1 (B3LYP), 84 cm1 (B97D), 238 cm1 (MP2) and 202 cm1 (MP2/aug)). Fig. 7 Comparison of the IR jet spectrum in the carbonyl stretching and ﬁngerprint region (0.06% glycine ethyl ester in He) to calculated spectra of the two most stable dimer structures of a given method assuming 40% dimer fraction visible in the IR spectrum. Colour code: black (thick): monomer (ttt), red: 8cc–, blue: 8ec–, green: 5c–dimers. The calculated spectra are shifted such that the harmonic monomer CQO stretching fundamental matches the experiment (namely 26 cm1 (B3LYP), +26 cm1 (B97D), 29 cm1 (MP2) and 16 cm1 (MP2/aug)). Fig. 7 Comparison of the IR jet spectrum in the carbonyl stretching and ﬁngerprint region (0.06% glycine ethyl ester in He) to calculated spectra of the two most stable dimer structures of a given method assuming 40% dimer fraction visible in the IR spectrum. Colour code: black (thick): monomer (ttt), red: 8cc–, blue: 8ec–, green: 5c–dimers. The calculated spectra are shifted such that the harmonic monomer CQO stretching fundamental matches the experiment (namely 26 cm1 (B3LYP), +26 cm1 (B97D), 29 cm1 (MP2) and 16 cm1 (MP2/aug)). oscillator strength upon coordination of a CQO group is very robust. We ﬁnd it in complexes of methylamine with methyl acetate (not shown) and we ﬁnd it at diﬀerent computational levels within the harmonic approximation. We conclude that glycine ethyl ester clusters can be generated in high abundance but narrow IR spectral features, attributable to dimers (most likely 8cc), in the NH stretching range are unexpectedly weak. B3LYP calculations fail completely, B97D calculations predict qualitatively wrong shifts, whereas MP2 predictions of dimers are consistent with the experiment apart from IR intensities which are too high by about an order of magnitude. In this situation, Raman spectroscopy aids in the analysis because it exhibits rather strong and therefore more robust N–H scattering strengths. Fig. 9 shows a dilute expansion for which one can roughly estimate a 20% dimer fraction. A more precise cluster quantiﬁcation is diﬃcult because the CQO stretching range is not very Raman active. Again, the B3LYP simulation fails completely in terms of shift and intensity. 3.2 Glycine ester aggregates Phys., 2011, 13, 14119–14130 14123 This journal is c the Owner Societies 2011 View Article Online oscillator strength upon coordination of a CQO group is very Fig. 7 Comparison of the IR jet spectrum in the carbonyl stretching and ﬁngerprint region (0.06% glycine ethyl ester in He) to calculated spectra of the two most stable dimer structures of a given method assuming 40% dimer fraction visible in the IR spectrum. Colour code: black (thick): monomer (ttt), red: 8cc–, blue: 8ec–, green: 5c–dimers. The calculated spectra are shifted such that the harmonic monomer CQO stretching fundamental matches the experiment (namely 26 cm1 (B3LYP), +26 cm1 (B97D), 29 cm1 (MP2) and 16 cm1 (MP2/aug)). Fig. 8 Comparison of the IR jet spectrum in the NH-stretching region (0.06% glycine ethyl ester in He) to calculated spectra of the two most stable dimer structures of a given method assuming a 4% dimer fraction instead of the 40% extracted from the CQO range (see the text). This is a strong indication of either band broadening or overestimated infrared dimer NH intensities. Colour code: black (thick): monomer (ttt), red: 8cc–, blue: 8ec–, green: 5c–dimers. The calculated spectra are shifted such that the harmonic monomer antisymmetric NH stretching fundamental matches the experiment (namely 167 cm1 (B3LYP), 84 cm1 (B97D), 238 cm1 (MP2) and 202 cm1 (MP2/aug)). View Article Online Fig. 8 Comparison of the IR jet spectrum in the NH-stretching region (0.06% glycine ethyl ester in He) to calculated spectra of the two most stable dimer structures of a given method assuming a 4% dimer fraction instead of the 40% extracted from the CQO range (see the text). This is a strong indication of either band broadening or overestimated infrared dimer NH intensities. Colour code: black (thick): monomer (ttt), red: 8cc–, blue: 8ec–, green: 5c–dimers. The calculated spectra are shifted such that the harmonic monomer antisymmetric NH stretching fundamental matches the experiment (namely 167 cm1 (B3LYP), 84 cm1 (B97D), 238 cm1 (MP2) and 202 cm1 (MP2/aug)). Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G Fig. 8 Comparison of the IR jet spectrum in the NH-stretching region (0.06% glycine ethyl ester in He) to calculated spectra of the two most stable dimer structures of a given method assuming a 4% dimer fraction instead of the 40% extracted from the CQO range (see the text). 3.2 Glycine ester aggregates However, the B97D simulation provides quite reasonable Raman intensities. Combining them with the more reliable MP2 band positions gives a plausible representation of the experimental Raman spectra and shows that dimers do not have excessive bandwidths. Therefore, the failure of all employed methods to reproduce the IR intensity of the clusters is most likely related to an overestimation of IR band strengths by an order of magnitude. Either more accurate dipole hypersurfaces or an anharmonic treatment will be needed to explain the low intensity of these cluster IR spectra. 14124 Phys. Chem. Chem. Phys., 2011, 13, 14119–14130 3.3 Alanine At this stage, it is advisable to switch to the homologous ester of alanine, discussing the analogies and diﬀerences in the glycine case. Based on the insights obtained, B3LYP calculations will not be considered further, and B97D predictions will only be used to borrow Raman intensities. The most stable structures of the monomer are shown in Fig. 10. The ttt structure is again particularly stable, consistent with an electron diﬀraction study of the methyl ester44 and both experimental and theoretical evidence for the parent compound alanine.4,45 14124 Phys. Chem. Chem. Phys., 2011, 13, 14119–14130 This journal is c the Owner Societies 2011 View Article Online Fig. 9 Comparison of the Raman jet spectrum (0.03% glycine ethyl ester in helium, 3 mm) to calculated spectra of the two most stable dimer structures of a given method assuming a 20% dimer fraction visible in the Raman spectrum. Colour code: black (thick): monomer (ttt), red: 8cc–, blue: 8ec–, green: 5c–dimers. The calculated spectra are shifted such that the harmonic monomer antisymmetric NH stretching fundamental matches the experiment. Fig. 10 The most stable structures obtained at the MP2/aug-cc- pVTZ level for the monomeric alanine ethyl ester along with their relative energies including zero-point energy (DE0) at the MP2/ 6–311+G* level and the most stable homochiral dimer (8cc) obtained at the MP2/6–311+G* level along with its zero-point corrected dissociation energy (D0) into two ttt monomers. Fig. 10 The most stable structures obtained at the MP2/aug-cc- pVTZ level for the monomeric alanine ethyl ester along with their relative energies including zero point energy (DE ) at the MP2/ Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G Fig. 10 The most stable structures obtained at the MP2/aug-cc- pVTZ level for the monomeric alanine ethyl ester along with their relative energies including zero-point energy (DE0) at the MP2/ 6–311+G* level and the most stable homochiral dimer (8cc) obtained at the MP2/6–311+G* level along with its zero-point corrected dissociation energy (D0) into two ttt monomers. Fig. 9 Comparison of the Raman jet spectrum (0.03% glycine ethyl ester in helium, 3 mm) to calculated spectra of the two most stable dimer structures of a given method assuming a 20% dimer fraction visible in the Raman spectrum. Colour code: black (thick): monomer (ttt), red: 8cc–, blue: 8ec–, green: 5c–dimers. 3.3 Alanine The calculated spectra are shifted such that the harmonic monomer antisymmetric NH stretching fundamental matches the experiment. phase IR spectrum, with the central one gaining intensity upon jet cooling and the others not (see Fig. S7, ESIw). This is consistent with the non-observation of the symmetric NH stretching mode in matrix isolation.46 In the Raman spectra, the antisymmetric band is again very weak in the gas phase but gains intensity in the jet. Compared to glycine gain factors of B7 (IR) and B25 (Raman), the gains are now both around 10 (Table S7, ESIw). However, comparison to CQO stretching spectra, in which the clustering extent can be quantiﬁed (Fig. S8, ESIw), shows that the antisymmetric NH band is now directly overlapped by cluster transitions. Therefore, the monomer IR gain is actually less than 10 and one may summarize the alanine ester monomer case as being qualitatively analogous and quantitatively somewhat less pronounced than the glycine case. This is also true for the slight blue-shift of the symmetric stretching band with jet cooling. Reference spectra with racemic alanine (not shown) do not exhibit signiﬁcant diﬀerences as one would expect from a monomer dominated spectrum. At this point, one can speculate about the reason for the reduced extent of the intensity anomalies upon introduction of the methyl group. Certainly, the bifurcation of the N–H OQC hydrogen bonds in the monomer is less perfect and the overlap with the p-orbitals is reduced in favor of the lone pair interaction. Yet, the extent of the symmetry breaking is quite modest. This is also true for the decrease of the largest rotational constant in the alanine case, which might otherwise explain some change in band contour in the room temperature gas phase spectrum. Interestingly, the ttg structure with two methyl groups on the same side now competes with ttt, presumably due to dispersive interactions. At the highest level considered here, it is even more stable than ttt, but only by a small margin. In contrast, rotation of the methyl group to the other side (ttg0) leads to a somewhat higher energy. As discussed for glycine, this subtle isomerism is not expected to appear in low resolution spectra. Similar to the glycine case, torsion of the NH2 group and coordination of the ester oxygen do not lead to competitive structures. This journal is c the Owner Societies 2011 3.3 Alanine 12 Comparison of the IR jet spectrum (0.06% alanine ethyl ester in He) and the Raman jet spectrum (0.06% alanine ethyl ester in He, 3 mm) to calculated spectra of the most stable monomer (black, thick) and dimer (red) obtained at the MP2 level. The dimer fraction is set to 20% as estimated from the carbonyl stretching range. (Raman dimer intensities borrowed from B97D calculations.) Fig. 12 Comparison of the IR jet spectrum (0.06% alanine ethyl ester in He) and the Raman jet spectrum (0.06% alanine ethyl ester in He, 3 mm) to calculated spectra of the most stable monomer (black, thick) and dimer (red) obtained at the MP2 level. The dimer fraction is set to 20% as estimated from the carbonyl stretching range. (Raman dimer intensities borrowed from B97D calculations.) Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G Fig. 11 Experimental FTIR and Raman spectra of alanine ethyl ester in He in the NH-stretching region with increasing concentration scaled to a similar integral band intensity of the asymmetric NH-stretching vibration of the monomer. (a)–(c) Jet spectra (IR) measured at a stagnation pressure of 0.7 bar, (a) 0.03%, scaled by 8.5, (b) 0.06%, scaled by 2.7, (c) 0.16%; (d)–(g) jet spectra (Raman) measured at a stagnation pressure of 0.8 bar, (d) nozzle distance 1 mm, 0.01%, scaled by 1.6, (e) nozzle distance 3 mm, 0.02%, scaled by 4.0, (f) nozzle distance 3 mm, 0.03%, scaled by 2.2, (g) nozzle distance 3 mm, 0.06%. Fig. 12 Comparison of the IR jet spectrum (0.06% alanine ethyl ester in He) and the Raman jet spectrum (0.06% alanine ethyl ester in He, 3 mm) to calculated spectra of the most stable monomer (black, thick) and dimer (red) obtained at the MP2 level. The dimer fraction is set to 20% as estimated from the carbonyl stretching range. (Raman dimer intensities borrowed from B97D calculations.) Fig. 11 shows the evolution of the alanine ethyl ester spectra with increasing cluster extent, normalized to comparable antisymmetric N–H stretching intensity. In the IR case, the cluster content can be followed in the carbonyl stretching region (Fig. S8, ESIw). It increases from about 10% in trace (a) to 20% in trace (b). Despite this strong increase, no substantial cluster absorptions are observed in the IR spectra. 3.3 Alanine However, due to the symmetry breaking of the alanine CH3 group, the ﬁrst torsional angle (Fig. 1) is now diﬀerent from 1801. Depending on the level of calculation, it ranges between 1751 and 1781 (see Table S6, ESIw). The symmetry breaking may also be expressed in terms of the diﬀerence between the two N–H OQC bond lengths. It amounts to 29–35 pm, which may be compared to the experi- mental value of 18(4) pm for the parent compound alanine.4 A substantial fraction of the eﬀect is due to torsion around the CC bond, which allows the ester oxygen to avoid contact with the alanine CH3 group. This should not aﬀect the monomer NH spectra signiﬁcantly and indeed they show just the same anomalies as the glycine case, namely three bands in the gas Phys. Chem. Chem. Phys., 2011, 13, 14119–14130 14125 This journal is c the Owner Societies 2011 This journal is c the Owner Societies 2011 View Article Online Fig. 11 Experimental FTIR and Raman spectra of alanine ethyl ester in He in the NH-stretching region with increasing concentration scaled to a similar integral band intensity of the asymmetric NH-stretching vibration of the monomer. (a)–(c) Jet spectra (IR) measured at a stagnation pressure of 0.7 bar, (a) 0.03%, scaled by 8.5, (b) 0.06%, scaled by 2.7, (c) 0.16%; (d)–(g) jet spectra (Raman) measured at a stagnation pressure of 0.8 bar, (d) nozzle distance 1 mm, 0.01%, scaled by 1.6, (e) nozzle distance 3 mm, 0.02%, scaled by 4.0, (f) nozzle distance 3 mm, 0.03%, scaled by 2.2, (g) nozzle distance 3 mm, 0.06%. Fig. 11 Experimental FTIR and Raman spectra of alanine ethyl ester in He in the NH-stretching region with increasing concentration scaled to a similar integral band intensity of the asymmetric NH-stretching vibration of the monomer. (a)–(c) Jet spectra (IR) measured at a stagnation pressure of 0.7 bar, (a) 0.03%, scaled by 8.5, (b) 0.06%, scaled by 2.7, (c) 0.16%; (d)–(g) jet spectra (Raman) measured at a stagnation pressure of 0.8 bar, (d) nozzle distance 1 mm, 0.01%, scaled by 1.6, (e) nozzle distance 3 mm, 0.02%, scaled by 4.0, (f) nozzle distance 3 mm, 0.03%, scaled by 2.2, (g) nozzle distance 3 mm, 0.06%. Fig. 3.3 Alanine In the Raman case one can see some cluster contributions on the low frequency wing of the symmetric stretching band, whereas contributions in the antisymmetric stretching region appear to coincide with the monomer peak. This is supported by the spectral simulations shown in Fig. 12 for the most stable dimer of two L-alanine ethyl esters, an 8cc cluster with C2 symmetry (see Fig. 10), which has two methyl groups pointing opposite to each other. As in the glycine case, the major discrepancy is the absence of a signiﬁcant symmetric NH stretching IR intensity in the experimental spectra. which we introduce as a fourth label, can be close to 1801 (tttt) or close to 601 (tttg). These valine ester structures are analogous to two out of the three lowest conformations of valine itself.5,47 The isomerism survives the jet cooling and leads to two partially resolved doublets at 3410 and 3403 cm1 for the antisymmetric (IR + Raman) and at 3353 and 3338 cm1 for the symmetric (Raman) NH stretching modes (Table S8 and Fig. S9, ESIw). This is in slight contrast to the microwave evidence for one valine isomer only.5 We note, however, some contributions from clusters, in particular, for the lower frequency peak. There is again g/t isomerism in the ester group (third label) and, depending on the level of calculation, the ttgt conformer is actually somewhat lower in energy than the tttt conformer. As this should not have spectral consequences in the NH or CQO range, it will subsequently be ignored, although the position of the ethyl group may have eﬀects on the molecular recognition between monomers. More interestingly, the bulky isopropyl group destroys the bifurcation of the NH2 group with respect to the carbonyl group to a signiﬁcant extent (91–191 deviation from 1801 for the NC torsional angle with the larger values corresponding to higher levels, 431–491 deviation from 1801 for the CC torsional angle; Table S6, ESIw). This should be viewed together with the observation that the intensity ratio between symmetric and antisymmetric stretch is 3.4 Valine Instead of a factor of B25 (glycine) or o10 (alanine), it only increases by less than a factor of 1.5 in the Raman spectrum. The IR intensity gain factor is slightly larger (3 2) but rather uncertain. The symmetric NH stretch does not shift with jet cooling, also in contrast to the smaller homologues. This strongly suggests that the anomalous temperature dependence of the intensity is related to the bifurcated intramolecular hydrogen bond situation in glycine esters which is gradually lost in favor of an unsymmetric hydrogen bond pattern in alanine and particularly in valine. Fig. 14 Experimental FTIR spectra of valine ethyl ester in He in the carbonyl stretching and ﬁngerprint region: (a) gas phase, 0.02%, 170 mbar, scaled by a factor of 0.1; (b) = 1.1 (d)–(c); (c) L-valine ethyl ester in He, 0.03%, stagnation pressure 0.7 bar; (d) DL-valine ethyl ester in He, 0.03%, stagnation pressure 0.7 bar. topologies for LL and DL pairings. In the homochiral case an 8cc dimer analogous to those for glycine and alanine is found, where the isopropyl groups point away from each other (Fig. 13). Such a structure cannot be realized easily for the DL dimer because of the associated steric hindrance. Instead, an even more stable 8ec structure is found, where the isopropyl groups can also avoid each other. Although this theoretical ﬁnding has to be conﬁrmed at higher levels of approximation and by more systematic sampling of the conformation space, it agrees nicely with the experimental observation in the CQO stretching range. Even the need to scale up the DL signals by 10–20% before subtraction ﬁts the assumption that the racemate is more strongly bound. Possibly, the dimer observations can be extrapolated to the liquid state. A simpliﬁed simulation of the diﬀerence spectrum based on a single heteroconﬁgured and a single homoconﬁgured dimer is shown in Fig. 16. It recovers the basic spectral features and therefore supports the interpretation that two enantiomeric valine ester molecules prefer a diﬀerent hydrogen bond topology than a pair of equal molecules. The most interesting diﬀerence refers to the cluster IR signals which appear at higher concentration both in the CQO/C–O and in the N–H stretching regions. They are shown in Fig. 14 and 15, respectively. The uppermost trace corresponds to DL-valine, while the second trace refers to L-valine. 3.4 Valine The case of valine ethyl ester is again analogous in many respects but it shows three noteworthy diﬀerences. First of all, its NH stretching spectrum is complicated by conformational isomerism of the isopropyl group (Fig. 13). Its dihedral angle, 14126 Phys. Chem. Chem. Phys., 2011, 13, 14119–14130 This journal is c the Owner Societies 2011 This journal is c the Owner Societies 2011 View Article Online Fig. 14 Experimental FTIR spectra of valine ethyl ester in He in the carbonyl stretching and ﬁngerprint region: (a) gas phase, 0.02%, 170 mbar, scaled by a factor of 0.1; (b) = 1.1 (d)–(c); (c) L-valine ethyl ester in He, 0.03%, stagnation pressure 0.7 bar; (d) DL-valine ethyl ester in He, 0.03%, stagnation pressure 0.7 bar. Fig. 13 The most stable structures obtained at the MP2/aug-cc- pVTZ level of approximation for monomeric valine ethyl ester. The nomenclature of the monomers includes the orientation of the isopropyl group and the relative energies including zero-point energy (DE0) at the MP2/6–311+G* level of approximation are enclosed. The hetero- (DL-8ec) and homochiral (LL-8cc) dimers were obtained at the MP2/6–311+G* level and are shown along with the energy needed for their dissociation into two tttt monomers including zero-point energy (D0) at the B97D/TZVP level. Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G Fig. 13 The most stable structures obtained at the MP2/aug-cc- pVTZ level of approximation for monomeric valine ethyl ester. The nomenclature of the monomers includes the orientation of the isopropyl group and the relative energies including zero-point energy (DE0) at the MP2/6–311+G* level of approximation are enclosed. The hetero- (DL-8ec) and homochiral (LL-8cc) dimers were obtained at the MP2/6–311+G* level and are shown along with the energy needed for their dissociation into two tttt monomers including zero-point energy (D0) at the B97D/TZVP level. Fig. 13 The most stable structures obtained at the MP2/aug-cc- pVTZ level of approximation for monomeric valine ethyl ester. The nomenclature of the monomers includes the orientation of the isopropyl group and the relative energies including zero-point energy (DE0) at the MP2/6–311+G* level of approximation are enclosed. The hetero- (DL-8ec) and homochiral (LL-8cc) dimers were obtained at the MP2/6–311+G* level and are shown along with the energy needed for their dissociation into two tttt monomers including zero-point energy (D0) at the B97D/TZVP level. now essentially temperature independent (Table S8, ESIw). 3.4 Valine The third trace shows the diﬀerence between the two, after adjusting for slight concen- tration diﬀerences. Where there is no chirality recognition,48 the peaks cancel. Where homoconﬁgurational dimers absorb, the diﬀerence is negative because they are statistically less abundant in the racemic mixture. In regions with speciﬁc heterochiral absorption, the diﬀerence is positive.49 One can easily see that the homochiral dimer has a stronger hydrogen bond to CQO, accompanied by a strengthening of the C–O bond. The hetero- chiral dimer has a lower C–O stretching wavenumber and a higher CQO stretching wavenumber, indicative of some coordination of the ester oxygen. The NH stretching range is less conclusive because of monomer/dimer overlap but also shows clear signs of chirality recognition (Fig. 15). This journal is c the Owner Societies 2011 3.5 Amino acid trends The intensity ratio between theory and experiment is ﬁxed between the two spectral ranges. along which the evolution of molecular properties can be followed. Several of these trends are documented in the ESIw, some of them shall be highlighted here: The bifurcation trend (Table S6, ESIw) may be most important for the present work. In glycine, the NH2 group forms two equivalent weak hydrogen bonds to the carbonyl group. In alanine the symmetry is slightly broken and in valine the torsion angles deviate by up to 191 and 491, respectively. This changes the way in which the two functional groups interact and may also have eﬀects on rovibrational coupling in this ﬂoppy system. double-harmonic approximation (linear restoring force and linear dipole change with normal mode displacement) nor on the basis of intensity studies along large amplitude coordinates (see Fig. S3–S6, ESIw). However, it is likely to be connected to the conformational dynamics of the NH2 group in the vicinity of a CQO group, as reference experiments for the CQO-deﬁcient analogue show. The carbonyl stretching wavenumber decreases with increasing residue size from 1764 to 1752 cm1. This is largely a through-bond electronic eﬀect of the substituents. The NH stretching vibrations decrease from glycine to alanine and split in the case of valine due to rotational isomerism of the isopropyl group. The most striking experimental observation is the intensity increase of the antisymmetric stretching mode in both the IR and the Raman spectra upon cooling. It can be inferred from a comparison to robust CH stretching modes and amounts to about 25, o10, and 1 for glycine, alanine and valine in the Raman spectra and to about 7, o10, and 3 in the IR spectra, respectively. For the symmetric stretching mode the trend is much less pronounced. This eﬀect is not easy to rationalize on the basis of ab initio calculations within the The electronic (De) and zero-point-corrected (D0) dissociation energies of the most stable dimers (all except for DL-valine of the 8cc type with isolated N–H OQC bonds and a sandwich- like character) are instructive (Table S9, ESIw). Most of the driving force for aggregation is seen to arise from dispersion- driven folding interactions. Increasing residues lead to steadily increasing interaction energies in particular at the MP2 level. 3.5 Amino acid trends On the computational side, the present study is only exploratory due to the large size of the accessible conformation space. There are indeed two diﬀerent particularly stable dimer Key to this work and the understanding of the observed phenomena is the series of glycine, L-alanine, L-valine, DL-valine, Phys. Chem. Chem. Phys., 2011, 13, 14119–14130 14127 This journal is c the Owner Societies 2011 Fig. 16 Comparison of the experimental valine spectra obtained by subtraction with calculated diﬀerence spectra for the homochiral LL-8cc dimer (downward peaks) and the heterochiral DL-8ec dimer (upward peaks) at the B97D level for the NH (above) and carbonyl (below) stretching regions. The calculated spectra were simulated with a Gaussian line shape and a FWHM of 5 cm1. The intensity ratio between theory and experiment is ﬁxed between the two spectral ranges. View Article Onl View Article Online Fig. 15 Experimental FTIR spectra of valine ethyl ester in He in the NH-stretching region: (a) gas phase, 0.02%, 170 mbar, scaled by a factor of 0.1; (b) = 1.2 (d)–(c); (c) L-valine ethyl ester in He, 0.03%, stagnation pressure 0.7 bar; (d) DL-valine ethyl ester in He, 0.03%, stagnation pressure 0.7 bar. Fig. 15 Experimental FTIR spectra of valine ethyl ester in He in the NH-stretching region: (a) gas phase, 0.02%, 170 mbar, scaled by a factor of 0.1; (b) = 1.2 (d)–(c); (c) L-valine ethyl ester in He, 0.03%, stagnation pressure 0.7 bar; (d) DL-valine ethyl ester in He, 0.03%, stagnation pressure 0.7 bar. Fig. 16 Comparison of the experimental valine spectra obtained by Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G Fig. 15 Experimental FTIR spectra of valine ethyl ester in He in the NH-stretching region: (a) gas phase, 0.02%, 170 mbar, scaled by a factor of 0.1; (b) = 1.2 (d)–(c); (c) L-valine ethyl ester in He, 0.03%, stagnation pressure 0.7 bar; (d) DL-valine ethyl ester in He, 0.03%, stagnation pressure 0.7 bar. Fig. 16 Comparison of the experimental valine spectra obtained by subtraction with calculated diﬀerence spectra for the homochiral LL-8cc dimer (downward peaks) and the heterochiral DL-8ec dimer (upward peaks) at the B97D level for the NH (above) and carbonyl (below) stretching regions. The calculated spectra were simulated with a Gaussian line shape and a FWHM of 5 cm1. 14128 Phys. Chem. Chem. Phys., 2011, 13, 14119–14130 Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G (i) Why is the antisymmetric NH-stretching intensity in the monomers so temperature dependent? Agreement with theory is only satisfactory at low temperatures or when the hydrogen bond bifurcation is removed by bulky substituents. 5 A. Lesarri, E. J. Cocinero, J. C. Lopez and J. L. Alonso, The Shape of Neutral Valine, Angew. Chem., Int. Ed., 2004, 43, 605–610. 6 R. M. Balabin, Conformational Equilibrium in Glycine: Experi- mental Jet-Cooled Raman Spectrum, J. Phys. Chem. Lett., 2010, 1, 20–23. (ii) Why are the cluster NH-bands nearly invisible in the IR spectra? Inspection of the CQO- stretching range and the Raman spectra leaves no doubt that clustering is extensive under appropriate conditions but seems to avoid cooperative arrangements. (ii) Why are the cluster NH-bands nearly invisible in the IR spectra? Inspection of the CQO- stretching range and the Raman spectra leaves no doubt that clustering is extensive under appropriate conditions but seems to avoid cooperative arrangements. 7 S. G. Stepanian, I. D. Reva, E. D. Radchenko and L. Adamowicz, Conformational Behavior of a-Alanine. Matrix-Isolation Infrared and Theoretical DFT and ab initio Study, J. Phys. Chem. A, 1998, 102, 4623–4629. 8 S. G. Stepanian, I. D. Reva, E. D. Radchenko and L. Adamowicz, Combined Matrix-Isolation Infrared and Theoretical DFT and ab initio Study of the Nonionized Valine Conformers, J. Phys. Chem. A, 1999, 103, 4404–4412. Furthermore, it was shown that B3LYP and B97D levels are unable to describe the spectral eﬀects upon clustering, by predicting the wrong structures or wrong frequency shifts. At the harmonic MP2 level only the weak IR intensity of NH stretching transitions in dimers is poorly described. In addition to identifying these theoretical challenges, which invite future high level studies,35,45 the present work allows for a few clearcut experimental conclusions: 9 R. Ramaekers, J. Pajak, M. Rospenk and G. Maes, Matrix-Isolation FTIR Spectroscopic Study and Theoretical DFT(B3LYP)/ 6-31++G** Calculations of the Vibrational and Conformational Properties of Tyrosine, Spectrochim. Acta, Part A, 2005, 61, 1347–1356. At the harmonic MP2 level only the weak IR intensity of NH stretching transitions in dimers is poorly described. In addition to identifying these theoretical challenges, which invite future high level studies,35,45 the present work allows for a few clearcut experimental conclusions: 10 A. Kaczor, I. D. Reva, L. M. Proniewicz and R. Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G Fausto, Importance of Entropy in the Conformational Equilibrium of Phenylalanine: Matrix-Isolation Infrared Spectroscopy and Density Functional Theory Study, J. Phys. Chem. A, 2006, 110, 2369–2379. (iii) Esters of amino acids can be prepared in a single conformation with respect to the weakly attractive NH2OQC arrangement. (iii) Esters of amino acids can be prepared in a single conformation with respect to the weakly attractive NH2OQC arrangement. 11 C. Espinoza, J. Szczepanski, M. Vala and N. C. Polfer, Glycine and Its Hydrated Complexes: A Matrix Isolation Infrared Study, J. Phys. Chem. A, 2010, 114, 5919–5927. (iv) Rearrangement to intermolecular NH2OQC hydrogen bonds upon aggregation leads to very small vibrational red-shifts of the involved NH2-groups. 12 R. Schweitzer-Stenner, T. Measey, L. Kakalis, F. Jordan, S. Pizzanelli, C. Forte and K. Griebenow, Conformations of Alanine-Based Peptides in Water Probed by FTIR, Raman, Circular Dichroism, Electronic Circular Dichroism and NMR Spectroscopy, Biochemistry, 2007, 46, 1587–1596. (v) If the amino acid residue is bulky enough such as in valine, chirality recognition in dimers can lead to diﬀerences in hydrogen bond topology. (v) If the amino acid residue is bulky enough such as in valine, chirality recognition in dimers can lead to diﬀerences in hydrogen bond topology. 13 G. D. Chumanov, R. G. Efremov and I. R. Nabiev, Surface- Enhanced Raman Spectroscopy of Biomolecules Part I-Water- Soluble Proteins, Dipeptides and Amino Acids, J. Raman Spectrosc., 1990, 21, 43–48. It is not clear whether hydrogen bonding in amino acid esters should be classiﬁed as weak. Intramolecularly, it certainly is. On the other hand, the cohesion energy between two molecules is quite substantial and almost comparable to that of carboxylic acid dimers.50 However, most of it seems to be of dispersive and electrostatic nature far from the hydrogen bond centers and the associated distortions of the inter- molecular hydrogen bonds may contribute to their weak IR signature in terms of small wavenumber shifts and almost negligible intensity. We hope that our experimental work will trigger a high level theoretical analysis of the intensity anomalies in the monomers and clusters of these model compounds. High resolution IR spectroscopy may also contribute to the elucidation of the monomer intensity anomaly. 14 M. Gerhards and C. Unterberg, Structures of the protected amino acid Ac–Phe–OMe and its dimer: A beta-sheet model system in the gas phase, Phys. Chem. Chem. Phys., 2002, 4, 1760–1765. 15 S. M. 3.5 Amino acid trends The trend is probably overestimated due to basis set superposition eﬀects but a binding energy per monomer of B20 kJ mol1 may be estimated. The DL combination for valine allows for an additional twist with extra binding energy 14128 Phys. Chem. Chem. Phys., 2011, 13, 14119–14130 This journal is c the Owner Societies 2011 View Article Online jet spectrometer and from support by the Fonds der Chemischen Industrie. jet spectrometer and from support by the Fonds der Chemischen Industrie. despite the realization of a less stable N–H O–C contact. This underscores the signiﬁcance of interactions beyond local hydrogen bonding in amino acid esters. Downloaded by University of Goettingen on 15 March 2013 Published on 28 June 2011 on http://pubs.rsc.org \| doi:10.1039/C1CP20883G Bachrach, Microsolvation of Glycine: A DFT Study, J. Phys. Chem. A, 2008, 112, 3722–3730. 16 J. T. O’Brien, J. S. Prell, J. D. Steill, J. Oomens and E. R. 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Borho and M. A. Suhm, Glycidol dimer: anatomy of a molecular handshake, Phys. Chem. Chem. Phys., 2002, 4, 2721–2732. S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski and D. J. Fox, Gaussian 09, Revision A.02, 2009. 32 L. Scha¨ fer, C. V. Alsenoy, J. N. Scarsdale, V. J. Klimkowski and J. D. Ewbank, Ab initio Studies of Structural Features not Easily Amenable to Experiment. 18. Conformational Analysis and 50 Z. Xue and M. A. Suhm, Adding more weight to a molecular recognition unit: the low-frequency modes of carboxylic acid dimers, Mol. Phys., 2010, 108, 2279–2288. 14130 Phys. Chem. Chem. Phys., 2011, 13, 14119–14130 This journal is c the Owner Societies 2011
https://openalex.org/W1018197725	https://digitalcommons.uri.edu/cgi/viewcontent.cgi?article=1623&context=theses	English	null	Maslow Interpreted for the Residential Environment	null	2,020	cc-by	16,422	Maslow Interpreted for the Residential Environment Maslow Interpreted for the Residential Environment Leslie Marie Poirier University of Rhode Island Follow this and additional works at: https://digitalcommons.uri.edu/theses Terms of Use All rights reserved under copyright. Recommended Citation Recommended Citation Recommended Citation Recommended Citation Poirier, Leslie Marie, "Maslow Interpreted for the Residential Environment" (1986). Open Access Master's Theses. Paper 621. https://digitalcommons.uri.edu/theses/621 Recommended Citation Recommended Citation Poirier, Leslie Marie, "Maslow Interpreted for the Residential Environment" (1986). Open Access Master's Theses. Paper 621. https://digitalcommons.uri.edu/theses/621 This Thesis is brought to you by the University of Rhode Island. It has been accepted for inclusion in Open Access Master's Theses by an authorized administrator of DigitalCommons@URI. For more information, please contact digitalcommons-group@uri.edu. For permission to reuse copyrighted content, contact the author directly. MASLOW INTERPRETED FOR THE RESIDENTIAL ENVIRONMENT BY LISA MARIE POIRIER MASTER OF COMMUNITY PLANNING RESEARCH PROJECT OF LISA MARIE POIRIER Approved: Major Professor Acknowledged: Director J I '(] MASTER OF COMMUNITY PLANNING RESEARCH PROJECT OF LISA MARIE POIRIER Approved: Major Professor Acknowledged: Director J I '(] UNIVERSITY OF RHODE ISLAND 1986 MASTER OF COMMUNITY PLANNING RESEARCH PROJECT OF LISA MARIE POIRIER MASTER OF COMMUNITY PLANNING RESEARCH PROJECT OF LISA MARIE POIRIER Acknowledged: Director TABLE OF CONTENTS List of Illustrations ............... . .. . .......... . ... i List of Tables. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii Preface .. . ............................................ iii CHAPTER I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Significance of the Issue: The Meaning of the House . . . . . . . . . . . . . . . . . . . . . . . . . . 3 II. Current Literature on the Relationship between Residential Quality and Residential Sa ti sf action . . . . • . . . . . . . . . . . . . . . . . 7 III. Issue Identification: Maslow's Hierarchy of Prepotent Needs .........•....... . ••.......•.•. 12 Physiological Needs .....•... . ... . ............ 15 Lighting . ................. . ................ 16 Sound . ................... . .. . .............. 19 The Thermal Environment .................... 21 Free Anatomical Movement . . .... . .........•.. 25 Need for Safety ................... . .......... 32 Need for Love and Belongingness ... . ... . •..... 40 Need for Self-Esteem ............. . ........... 47 Need for Self-Actualization . . ........... . .... 55 IV . Conclusion . ......... . ........... . ............... 58 Footnotes . ......................... . .................. 6 2 TABLE OF CONTENTS ILLUSTRATIONS 1. Maslow 1 s Hierarchy of Needs and Supportive Environmental Attributes ...... . . ... . . 14 2. Tolerance a nd Comfort with Temperature and Duration . . . ..... . .................... . . . ........ 24 3. Key to Dimensions of Body Sizes ..•......... . ....• . 28 i LIST OF TABLES 1. Distribution of Body Sizes ........................ 27 2. Conunon Hardware Problem Areas ..........•......... . 33 3. Peripheral Motives Derived from Safety Needs .................. . ........... . ..... 3 6 4. Peripheral Motives Derived from Love and Belongingness Needs ... . ... . ................... . . 42 5. Peripheral Needs Derived from Esteem Needs ..•..... 50 ii PREFACE The concept for this study emanated from an enlightening experience I once had while touring several "governmentally - planned" residential developments in northeastern Greece as part of the University of Illinoi s Summer Program in Greece, 1984. This was my first introduction to the devastating effects of poor planning. The Greek people in this part of the country have a close relationship with their land which is central to their way of life. The residential developments I witnessed were the antithesis of the residents' natural and rural nature which has been part of their culture for centuries. The houses were large, concrete structures of three, four, and five stories which seemed to isolate the residents from the land as well as from each other. The land lots which fronted the buildings were conspicuously small and rectangular, intersected by cement sidewalks and pathways . In my wildest imagination, I could not conceive of these natural people adapting to such foreign structures. It seemed to me that absolutely no consideration was made of precisely who would be living in l ll their buildings and what their needs and desires might be. Basic to my way of thinking, is the be lie f that designing for people and understanding their needs is the only sound approach to designing and planning residential environments. In adopting Maslow's Model of Prepotent Needs as a way of understanding basic human needs, and applying this model in the consideration of design criteria, I feel I have touched upon the fundamental rudiments of man-environment relationships. The field of Man-Environment Studies, or Environmental Psychology, is a relatively new yet rapidly growing discipline which has received a great amount of support. There are still many unanswered questions; however, pertaining to the ways in which man and his environment interact. The present study is made in an atte~pt to creatively respond to some of these questions. I would like to acknowledge my Research Committee, Dr. Howard H.Foster Jr., Dr.Irving Spaulding, and Dr. Farhad Atash for all their help and guidance in seeing me through this project. I am greatly indepted to Dr. Paul W. Dahlgren and the Office of Residential Life for giving me the time to complete this project and the support I needed. And finally, I would like to thank Mr. Christopher M. McGreavy and Ms. Louise A. Poirier for their unending encouragement which they generously gave from the beginning. PREFACE iv Introduction Planners and developers have been criticized for designing residential environments which do not meet the needs of the resident-user. There exists a gap between the expectations of the planner-developer and those of the resident-user. This condition results in residents' lack of satisfaction with the area in which they live. Residential satisfaction could be enhanced if consideration were given during the design of a residential area to the effects of the physical and social residential environment and the psychological needs of its inhabitants. Planners should understand the basic and inherent needs of residents who will live in the buildings they are designing; these needs are complex, individual and changing. However, Abraham Maslow's Hierarchy of Prepotent Needs · is a framework of use for generalizing about these needs. (1) The present study begins by introducing the meaning of the house in different cultures. A discussion follows l of current lite rature on the concepts of residential quality and satisfaction. The needs of the residents are described with Abraham Maslow's Hierarchy of Prepotent Needs which consists of the psychological needs; the needs for safety; the needs for love and belongingness; the needs for esteem of self and others; and the needs for self- actualization. 2 The Significance of the Issue: The Meaning of the House The house is an important and meaningful aspect of the built environment. It is argued that the house "embodies not only personal meaning but expresses and maintains the ideology of prevailing orders." (2) As a significant cultural object, the house is endowed with meaning according to the world view and ethos of the designers, builders, users, and observers of the house in its final form. ( 3 ) There are diverse meanings people ascribe to houses from one culture to another. (4) (5) In many traditional societies, for example, houses enclosed sacred space: deities dwelt there, religious rituals were performed while profane activities were proscribed, and the hearth , like the temple, helped to unify natural, social, and super-natural realms and to resolve symbolically the conflicts among them. (6) Attitudinal differences also exists within cultures. A 1980 study conducted by James s. Duncan of the social worlds of two elite groups within an Indian city discovered two strikingly opposing attitudes towards the house, 3 especially with regards to status achievement. elite achieve status through group-oriented demonstrated by spending large sums of money on The old activities funerals, Prestige and is large granted weddings, friends. parties for relatives and to those group members who bestow their wealth to their extended family and status peers. The old elite are sentimental towards their house. They hope that the house will be passed along from one generation of the family to another. However, despite its transfer from one family member to another, the house is considered a private container of the extended family rather than an arena of status display. Conversely, the new elite individualistic spending patterns. achieve status through They place an emphasis on purchasing consumer goods. The house serves as the most important status object for this group since it is a showcase for the other goods that this group collects for itself.(7) For most groups in our culture, the house is considered the central or "anchoring" point of the larger community setting. The typical person spends most of his time in the house; the house is one's most valuable possession; the house is endowed with a great emotional meaning; and the house is increasingly the center of recreation previously taking place elsewhere.(8) In a series of interviews with a panel of white, middle- income persons in the Seattle area, R.M. The Significance of the Issue: The Meaning of the House Rakoff 4 reveals attitudes towards the house reflective of mainstream American culture. For many panel members, the house is a commodity or an investment opportunity to be bought and sold for prof it in addition to being a place to live. The house is defined as the place where child rearing takes place, making the common distinction between house and a home. The house is an indicator of personal status and success. The house renders a sense of permanence and security. Panel members mention the desire of "sinking roots," "nesting," and generally settling down. Relative to permanence and security, the house is described as a powerful symbol of order, continuity, physical safety, and a sense of place or physical belonging. There exist sharp differences in these meanings; however, since some panel members consider the permanence of the home as "a positive antedate to the felt chaos of the outside world, while others experienced that permanence negatively as a trap, a symbol of lost hope for change, a mark of personal indecision or failure." (9) Closely tied in with this thought is the feeling that the house provides a refuge from the outside world. This search for refuge brings with it the following: A desire to escape from other people and from social involvement, the establishment of a place from which others could be excluded, and where, consequently, one could truly be onself in control, 'more of an individual,' capable of loving, and fully human. (10) 5 A fi nal attitude reported in the study i s that ownership is necessary for actualizing any or all of the above f eelings . Those panel members who rent their house s are also in agreement with this attitude. Ownership is considered a necessary ingredient of freedom. The freedom gained from ownership is expressed as the ability to change physical structures and personal behavior, the freedom from other's control, the freedom from the responsibility to others and their property, or the freedom from the perceived uncertainty of the outside world. Most importantly, the "freedom that people associate with owned houses express their belief, that these private spaces are a real and proper realm of self-fulfillment." (11) 6 6 6 CHAPTER II Current Literature 2.!! the Relationship Between Residential Quality and Residential Satisfaction As described in the previous section, one's attitude towards his life and the outer world are closely related to one's attitude towards his residential environment. Residential environment in this case refers to one's dw~lling unit plus the immediate area surrounding the dwelling unit. This relationship between attitudes is described in terms of satisfaction by Campbell, Converse and Rodgers in their 1976 study of the quality of American life. With respect to residential environments they write: Satisfaction with community is strongly related to satisfaction with the neighborhood, and satisfaction with neighborhood shows a strong relationship to housing satisfaction. Sa ti sf actiori' with these domains of the environment are also related to satisfaction with other domains of life experience. Finally, satisfactions with these residential environments, as well as satisfaction with other domains of life experiences, are related to expressed satisfaction with life as a whole. (12) Satisfaction with community is strongly related to satisfaction with the neighborhood, and satisfaction with neighborhood shows a strong relationship to housing satisfaction. Sa ti sf actiori' with these domains of the environment are also related to satisfaction with other domains of life experience. Finally, satisfactions with these residential environments, as well as satisfaction with other domains of life experiences, are related to expressed satisfaction with life as a whole. (12) The type of relationship between life satisfaction and The type of relationship between life satisfaction and 7 residential satisfaction, suggested by Weidemann, et al., is its relation to a larger concern for quality of life. However, the measurement of residential satisfaction doe s not depend on life satisfaction but rather on the satisfaction environment. of specific aspects of the residential Satisfaction with one's own residential environment is an important issue since "even though a person's assessment of his financial well-being, family life, and health are more germain to the overall quality of life... the residential environment is subject to greater alteration by design and planning than any of the other situations of the individual." (13) Evaluation of residential environments made in the past by planners and designers have been based on several criteria. Among these are economic criteria which consider the relationship between rent and income; physical criteria which consider the structural quality of the dwelling; and social criteria which consider the incidence of disease and the degree of resident crowding. Current Literature 2.!! the Relationship Between Residential Quality and Residential Satisfaction It is argued that these criteria are insufficient for evaluating residential environments since they ignore the quality criteria of the residents themselves. (14) The use of residential satisfaction as a criterion for evaluating the quality of the residential environment is a concept initially suggested by Fried and Gleicher in 1961; they state that "of the many varieties of resident 8 perceptions and behaviors, residents' satisfaction might be a more appropriate criteria for evaluating the quality of housing than characteristics such as plumbing or structure." (15) Since 1961, there have been many attempts to investigate and understand the physical as well as social aspects of the residential environment and how they relate to residential quality. (16) (17) (18) There is little disagreement that the residential environment is a highly complex concept. F.C. Ladd points out the "complexity and interdependency of attributes associated with residential environments as well as the groups that determine where residences are located, what they look like, and, to some extent, how they are used." F.C Ladd proposes three sets of factors to suggest the complex nature of residential environments: "perceiver groups" such as residents, developers, mortgage bankers, politicians, planners, architects, urban designers, and real estate board members; "environmental factors" such as the region, urban, suburban and rural neighborhood configuration, and house type; and "associated factors" such as density, crowding, privacy, security, location, and spatial layout. (19) To further socio-cultural complicate this system, context, in economic "changes in the events, media- influenced styles, and the moment in history may change the salience and evaluation of environmental qualities." (20) Yet another obstacle to consider in evaluating Yet another obstacle to consider in evaluating 9 r eside nts ' perceived e va luation of the ir e nvironme nts lies in problems with valid me as urement; among these is the problem of dissonance r educ tion. This concern i s related to the problem of dissonance reduction - the tendency for an individual to avoid conflict between past actions and the resultant current attitudes. It has been argued that people often refuse to recognize or admit their dissatisfaction with a past decision such as the selection of their present place of residence. This refusal to recognize or admit dissatisf actin may occur at the conscious or subconscious levels. Current Literature 2.!! the Relationship Between Residential Quality and Residential Satisfaction In a similar vein, it has been suggested that some people have a tendency to respond in ways which are socially desirable or positive. (21) A great deal of effort has been exerted to understand ways of improving and evaluating the quality of residential environments. The use of perceived residential satisfaction has received substantial support and is favored in comparison to objective measurements of physical characteristics. Attempts to assess residential satisfaction however, have faced several problems due to the complex, individual, and ever-changing concept of perceived residential satisfaction. Some authors have focused on user needs of residents to improve residential quality and this concept has also received some criticism . Campbell, et al., point out three problems in applying the concept of residential satisfaction to the quality of life experience based on user needs. First, individual needs differ among individuals from time to time. Second, Campbell, et al., point out three problems in applying the concept of residential satisfaction to the quality of life experience based on user needs. First, individual needs differ among individuals from time to time. Second, 10 a s ense of s atisfaction i s a pers ona l experience influenced by past experiences and current expectations of individuals . Third, the question must be raised about whether the quality of life of a society can be measured by the extent to which its overall needs are satisfied. Campbell sites John Stuart Mill's suggestion that "it is better to be a human being dissatisfied than a pig satisfied; better to be Socrates dissatisfied than a fool dissatisfied." (22) Campbell's criticisms against the use of user needs as criteria for residential qualitity are strong; however, there exists a psychological model for understanding human needs which responds favorably to these criticisms. Abraham Maslow's hierarchy of prepotent needs provides a model for understanding human needs which allows for individual differences yet maintains that humans possess five intrinsic needs common to all . These needs are hierarchially arranged in a sequence. A lower level need must be satisfied to an unspecified extent before the level above it can be satisfied. However, this sequence is not fixed and more than one need may dominate at the same time; therefore it follows the personal developmental pattern of the individual. Current Literature 2.!! the Relationship Between Residential Quality and Residential Satisfaction Maslow's model does not require that all needs be met at the same time but rather the individual strive at his own pace towards the satisfaction of the final and ultimate need, self-actualization. 11 Maslow's Hierarchy of Prepotent Needs Abraham Maslow is known as one of the founders of humanistic psychology; a "positive theory of human motivations". (23) By 1968, the humanistic movement had grown to such an extent that it is referred to as a strong third alternative to behavioristic psychology and orthodox Freudianism, two prevalent theories at that time. (24) Maslow developed five levels of human needs: physiological, safety, love and belongingness, esteem of others and self, and self-actualization. These needs are ~rranged in ascending order . As each level of attained and satisfied, people strive to reach levels, the ultimate being self-fulfillment. need is higher All people are born with these five basic needs . In his article entitled "Toward a Clarification of the Hierarchy heart of Theory," Maslow's Charles Brockett states that "at work are the assumptions that Need the 'the individual is an integrated, organized whole ' , that the primary motivational forces in human behavior are intrinsic 12 'instinctoid needs '." (25) Mas low's theory has received a substantia l amount of examination and support, and has been applied to other disciplines outside the psychological field. (26) Maslow's hierarchy of prepotent needs is applied as a framework for "encompassing the concept of human needs of the attributes of biological, social and psychological behavior" of people in the work environment. (27) Another application of Maslow's model is in D. Lester's development of a questionnaire to measure the level of people's satisfaction at each of the five levels in the hierarchy described by Maslow. The questionnaire was administered to 166 college undergraduate students. As expected, the level of basic needs satisfaction was found to be related to scores on measures of neuroticism and of belief in an internal locus of control.(28) The five basic needs within Maslow's hierarchy model are elaborated on individually in the following section. A framework for applying Maslow's model is borrowed from A. Kaplan and is used as a guideline in the following analysis . (See Figure 1) Each need is discussed relative to considerations that could be made in the planning and design of residential environments to create a supportive environment which encourages an individual in fulfilling his needs. The considerations proposed are from literary sources as well as personal creative input. Further suggestions for design ideas which support the efforts of 13 MA01-DW- l-\te.~c~I OF NEE:OS E:N" IRDNMC:NTA\.. '5UPP~l'/f= ATTRI eurecz, Levei. Maslow's Hierarchy of Prepotent Needs l - P'-'"t'S 101..o 1 CAL- !' l..l~~T 'SOt>~O Al~ f "f~~ITOR\Al-IT"f • O~l&r4TA1l°"4 VR.ITj" - r 9~T"{' PFtoY.! M \T,. Leve.i.. 4 - 1..e.vet- :? - PEOPL..E: -JI?! e>U-lT"( ~ ~WU::D~er DF \j\~ e,...N IR.ONME:s.)f ~ ~~ ---. loeNTIA!AUON C"\ 8 ;~ 'NC'M .,. 'z SN'l/J~ONM~ ~ ;: ~ ~ . A~ ~ ~ re - ~ ~" ........ ~·~ .t\ g~ ---' ~Retlt!O I\\ ~ ~~ ~Tl..le11C.. EYAW.t>.110"4 ~ ~~ CO~\'ROI.- -:::! 'OOC.IAL. p~ Tl CN I~ l PAnl.WAJ' <$e~C::.T10!'-I 11 ~Tl C:.IPA.L'10i..t • ($0'NO!lo-!Ct aJ- Ot>~<:> • sp..o.ce- . Mo"r::Me.0T SOURCE: Archie Kaplan, "Maslow Interpreted for the Work Environment," Man-Environment Systems 6(4) (July, 1976):p.248. \) Al z ~ ~ . ~ \)\ ~ !;) ~ I. ~ -· ......, 3: \ti .!\ ::;= c ~ \l ~ . .(, ~ ~ G I. E:N" IRDNMC:NTA\.. '5UPP~l'/f= ATTRI eurecz, MA01-DW- l-\te.~c~I OF NEE:OS SOURCE: Archie Kaplan, "Maslow Interpreted for the Work Environment," Man-Environment Systems 6(4) (July, 1976):p.248. 14 indivi dua l s in sati sfy i ng t heir basic needs are highly e ncouraged and a r e limited only by one's imagination. indivi dua l s in sati sfy i ng t heir basic needs are highly e ncouraged and a r e limited only by one's imagination. Physiologi cal Needs Physiologi cal Needs Maslow is quoted as saying that ''to survive, man needs food, clothing and shelter. These represent the most essential need." The need for shelter is related to the residential environment and is expressed as sufficient degrees of light, sound, thermal conditions, and free anatomical movement. The importance of considering these factors in the residential environment is reflected in A. Kaplan's interpretation of Maslow's physiological need as applied to the work environment: work environments: shelter for the work situation includes the ambient factors of sufficient intensities of light, sound, air, and heat. These factors must be considered in proper balance with human sensory stimulation requirements and tolerances for physiological well- being and productive work performance, for, no matter how well programmed and designed a space is, people cannot work well, or at all, without properly controlled ranges of ambient factors .... Anatomical movement should be included on this level, for, with lack of movement, there is stagnation of body fluids and its ensuing discomfort. (29) The physiological needs of adequate light, sound, thermal conditions, and anatomical movement are analyzed in their relation to design considerations of the residential 15 environment in the following section. Lighting The natural and artificial lighting of residential environments is an important design criterion as it potentially affects residents' health and safety, performance, comfort, aesthetic appreciation and color effects. (30) Sufficient lighting enhances residents' health and safety since "more illumination would increase the probability of seeing hazards and reduce the liklihood of accidents. In three European industrial site studies where in-force lighting was increased from about 15 to 100 footcandles, accidents dropped about 50 percent". (31) The information gained from this study can be incorporated into the design of residential environments. Extreme levels of light, bright and dark, affect the health of the eye. Extreme brightness may burn the retina of a human eye while extreme darkness may cause eye strain and produce damage over the long run. It is also suggested that insufficient lighting may have nonvisual health effects as well. (32) Human performance can be enhanced by planning various levels of lighting intensity according to the activities taking place in a specific area. If the activities generally taking place in an area are important and difficult requiring a lot of detail, illumination levels should be higher. Conversely, illumination should be lower 16 where place . mor e relaxed ( 33) The and l ess i mportant activities t ake us e of diffe rent light ing source s according to different activities is als o an e fficient, energy-saving concept. where place . mor e relaxed ( 33) The and l ess i mportant activities t ake us e of diffe rent light ing source s according to different activities is als o an e fficient, energy-saving concept. A design suggestion along the lines of practicality and lighting according to activity is that if 11 lighter surfaces (ceilings, walls, floors, furniture) are used they will reflect more light, and the room will be brighter. If dark woods, fabrics, and paints are used in places where important seeing is required, either more light will have to be provided, or there will be a risk of poor seeing." ( 3 4) Overly bright light alone, or together with contrasting light sources, creates a glare which results in discomfort and difficulty in seeing. If a source of light is bright enough, i ts background light is highly contrasting, and the light is directed at a person's eyes, that person experiences disability and possibly discomfort. Lighting A good example of this is the headlights on an approaching car in the evening hours. The amount of disability discomfort experienced by a person depends on the size and of the light source, the contrast of the light and its background, and the direction the light is coming from. To ·avoid the discomfort and difficulty in seeing caused by glare, the amount of luminance introduced into a room should reflect the direction a person is facing and the frequency with which the person looks in that 17 direction. For e xample, if a person looks up towards the ceiling from a seated postion and it is necessary to look in this direction frequently, the amount of luminance from a lighting fixture on the ceiling should be relatively low to prevent glare Additionally, windows may produce glare problems if the ground is snow-covered or if the sun is visable. To prevent such glare problems, standing and sitting positions should be located either where the user does not directly face the window or where he can pull the blinds.(35) Three factors are found to enhance aesthetic pleasantness: pleasantness, visability, and spaciousness. Pleasant lighting consists of a combination of different lighting systems. Best is the combination of overhead diffuse and downlighting and peripheral lighting. A visibility factor emerges - the more illumination the lighting system produces, the higher the judged visibility factor. The amount of illumination is unrelated, however, to lighting pleasantness. The third factor, spaciousness, is greater when peripheral lighting is used - when the walls were light. Spaciousness is also somewhat higher with illumination. (36) Pleasant lighting consists of a combination of different lighting systems. Best is the combination of overhead diffuse and downlighting and peripheral lighting. A visibility factor emerges - the more illumination the lighting system produces, the higher the judged visibility factor. The amount of illumination is unrelated, however, to lighting pleasantness. The third factor, spaciousness, is greater when peripheral lighting is used - when the walls were light. Spaciousness is also somewhat higher with illumination. (36) Color is considered an important aspect of the luminous environment; however, the majority of conclusions from research performed on the effects of color are loosely supported. It is suggested that this may be due to poor research or that many reactions to color are very small effects. Lighting (37) The most commonly studied reaction to color is color 18 pleasant ne ss or pref erence . Results from various studies gene rally s how that: warm col ors s uch as reds, yellows, and oranges a r e less preferred t han cool colors such as blues, greens, and purples; there is a growing preference for increasing lightness i n hues. Black and white is preferred to gray; and high-lightness-contrast combinations of object and background colors are highly liked, low-lightness- contrast combinations are liked less. That is, preferred combinations are light object colors with dark background colors, or vice-versa. (38) One study consisted of judges relating certain moods to color hues: blue connotes moods and feelings such as security, comfortableness, tenderness, soothing, calm, and serene; red connotes moods such as exciting, protective, defending, and defiant; orange connotes moods such as distressed and upset; black connotes moods such as despondent and powerful; purple connotes moods such as dignified; and yellow connotes moods such as cheerfullness. Although color is thought to convey emotions, there is little evidence in support of this notion.(39) Sound Noise is an incr eas ing problem in our civilization. Noise levels in the United States are reported to be doubling every ten years. However, sounds and sound levels can be controlled through proper planning and design with the purpose of enabling individuals to hear desired sounds and to avoid unwanted sounds, otherwise known as noise.(40) 1 9 The level and quality of sound in residential environments potentially effects the health, performance, comfort, and perceived pleasantness of its r eside nts. A health-related issue of environmental noise is that "as people in our society get older, they experience a further reduction in sensitivity at higher frequencies (of sound) . The ensuing loss in hearing ability for higher- freguency music and for women's voices is believed to be due to exposure to a variety of noises of civilization.'' (41) It is suggested that a concern with noise and hearing loss is that the symptoms of these problems are hidden. A person may experience a temporary loss of hearing following exposure to a high dosage of noise; however, without protection and prevention this person may have acquired a permanent hearing loss. ( 42) Although inconclusive, research suggests a possible correlation between exposure to high levels of noise and problems in the nervous and reproductive systems. (43) A noisy environment is said to effect performance levels. Among these possible effects are six factors: (1) steady (white) noises have no performance effects unless greater than .90 decibels (dB); (2) lower-intensity, irregular bursts may have performance effects; (3) higher- freguency sounds have greater effects than those of lower frequencies; performance. (4) noise may increase the variability of Thus, if work is influenced by the speed of machines in the work environment rather than self-paced, 20 performance decrements might take place; (5) noise may effect the quality rather than quantity of performance; and finally (6) complex tasks are more likely to be adversely influenced than simpler tasks. Some of these effects may be transitory and therefore take place only at the beginning of the noise; some effects may take hours to develop. Also, distraction plays a significant role in both these effects and in the resulting performance loss. (44) Discomfort from noise is referred to as annoyance. It is known that relatively pure tones are more annoying than more complex sounds; in addition, sound frequencies most sensitive to the ear such as speech frequencies are annoying. Sound To reduce noise levels stemming from outside the home, outside-wall insulation should be installed. In the case of multi-dwelling units such as apartment complexes, sound reducing partitions should be installed. Within the house itself, noise sources can be controlled through the use of carpets, draperies, and sound-absorptive ceilings. (45) Music is considered a popular sound-pleasantness condition; however, a problems exists in the use of music since people have diverse musical tastes and a concensus may be difficult to attain. (46) To reduce noise levels stemming from outside the home, outside-wall insulation should be installed. In the case of multi-dwelling units such as apartment complexes, sound reducing partitions should be installed. Within the house itself, noise sources can be controlled through the use of carpets, draperies, and sound-absorptive ceilings. (45) The Thermal Environment Humans gain or lose heat from three primary sources 21 of heat transfer within the environment: conduction, convection, and radiation. Conduction is heat transfer through direct contact with materials such as copper pipes and other metals. Convection is heat transfer through media such as moving air. The movement is based on adjacent thermal differences; for example, hot air rises. Radiation is heat transfer from a source through air without an intervening media. A window or wall that is hotter or colder than room air temperature may be heat radiated. There may be severe heat radiation to workers from furnaces or ovens. Evaporation is the mechanism by which humans cool off in hot environments. As persons perspire in an overly hot environment, the sweat evaporates, and the person is then cooled. (47) The thermal environment is composed of four factors which affect human thermal comfort: temperature, humidity, velocity, and radiation sources. Other important factors are insulation, such as clothing, and activity level. Thermal comfort is considered a primary criterion for interior spaces. Safety and performance are also highly relevent factors. (48) Although there are individual differences in conditions at which people report thermal comfort, people are generally comfortable in temperatures in the upper seventies ("F), and are only "slightly cool" or "slightly warm" in temperatures ranging from 68*F (20"C) to 86 • F(30 ° C). Thus, people can be comfortable at a wide range of 22 temperatures. (49) temperatures. (49) Studies of the effects of room temperature on performance suggest that fairly extreme and uncomfortable levels of heat and cold are necessary to have an effect. Some effects of more typical, but warm, room temperatures have resulted in slight performance loss. A study of the effects of temperature on crowding in interior spaces reports that under high temperature and crowded conditions, subjects were less happy and less likely to like another (hypothetical) person. Both effects of high heat and of crowding may intensify any existing feelings, whether positive or negative. (50) Human tolerance for extreme temperatures, under clothed conditions, is analyzed. (See Figure 2) The right hand curve shows that humans can endure (stay alive) at temperatures as high as 90~ F (32°C) indefinitely and temperatures above ll0°F (43°C) for brief periods. The left hand curve shows that humans can stand temperatures as low as so•p (10°C) indefinitely and as low as -25°F (-39°C) for a brief period. It is thought that healthy, appropriately dressed people can tolerate these extreme temperatures; however, there are reports that deaths in hospitals increased when room temperatures went outside the 60-ao• F (16-27° C) range. The center vertical line proposes that comfort occurs at 78°F (25'c) regardless of duration. The far right and left regions are too hot or too cold for the given duration to be safe. The inner 23 Figure 2 Tolerance and Comfort with Temperature and Duration 'WEEl(...-------------.....,..---=---r---- w ..J Ul SRre, BUT WRRM \\.-bLJR.i..._-4:::__~~~~--~~--_..J..----------- -~o -20 O Zo 40 6o 8o loo 12.o DRy BuLB TcMPERA'TllRE , °F (501° R .. H~ Figure 2 Tolerance and Comfort with Temperature and Duration Figure 2 Tolerance and Comfort with Temperature and Duration 'WEEl(...-------------.....,..---=---r---- w ..J Ul SRre, BUT WRRM \\.-bLJR.i..._-4:::__~~~~--~~--_..J..----------- -~o -20 O Zo 40 6o 8o loo 12.o DRy BuLB TcMPERA'TllRE , °F (501° R .. H~ SOURCE: Corwin Bennett, Spaces for People: Human Factors in Design, (New Jersey: Prentice- Hall, Inc., 1977) p.129. 'WEEl(...-------------.....,..---=---r---- w ..J Ul SRre, BUT WRRM \\.-bLJR.i..._-4:::__~~~~--~~--_..J..----------- -~o -20 O Zo 40 6o 8o loo 12.o DRy BuLB TcMPERA'TllRE , °F (501° R .. H~ SOURCE: Corwin Bennett, Spaces for People: Human Factors in Design, (New Jersey: Prentice- Hall, Inc., 1977) p.129. 24 regions are too warm or too cool for comfort. temperatures. (49) It is important to note that the above description does not consider the varying effects of humidity, activity, and acclimiatization which may alter the figures significantly. ( 51) Free Anatomical Movement Residential environments could be enhanced consideration were made of anthropometric factors, measurements of man, and of how man physically fits his environment with the intention of enhancing anatomical movement. Attention should be paid to if the into free body size, seating, and hardware such as handles for doors, lighting, and controls. Factors such as age, evolution, sex differences and cultural differences influence body size. The process of aging is the most significant of these factors as reflected in the growth of the body during the first two decades of life. Over centuries of evolution, people are getting larger; they are taller as well as heavier. Our body size decreases by a fraction of an inch during the course of one day due to the weight of the body on the spine; this results in compression of the discs within the spine. Men and women differ in height: women average 5'3" (159cm) in height, men 5'8" (172cm), thus average adults are 5'5" ( 166cm) tall. There are also body size differences among cultural groups: "Americans are among the largest peoples 25 of the world. We st Europeans are quite large. Orientals are smaller." ( 52) When designing environments for a large, diverse population, the sum of mean estimates alone is inadequate. Designers should attempt to accomodate the greatest percentage of people possible by adjusting the mean figures accordingly. Suppose a console is intended to be low enough for seated people to see over. The mean sitting eye height for a 16- inch (41cm) high seat is about 46 inches (117cm) from the floor. But if we design the console to be 46 inches high, then fully half of the population would not be able to see over it easily! If we set the height equal to that of the smallest 5 percent of the population, that is at 44 inches (112cm), then 95 percent should be able to see over it readily. In some cases it is so inexpensive to accomodate a large fraction of the population that we extend this common 95 percent design goal and design for almost everyone. The standard 6'8'' (203cm) door height is tall enough for more than 99 % of the population. (53) Suppose a console is intended to be low enough for seated people to see over. Free Anatomical Movement The mean sitting eye height for a 16- inch (41cm) high seat is about 46 inches (117cm) from the floor. But if we design the console to be 46 inches high, then fully half of the population would not be able to see over it easily! If we set the height equal to that of the smallest 5 percent of the population, that is at 44 inches (112cm), then 95 percent should be able to see over it readily. In some cases it is so inexpensive to accomodate a large fraction of the population that we extend this common 95 percent design goal and design for almost everyone. The standard 6'8'' (203cm) door height is tall enough for more than 99 % of the population. (53) Useful anthropometric studies have been carried out listing a variety of body measurements. A brief summary of average sizes of various sections of the human body, and the location of these body sections in relation to other sections of the body is available to the planner and designer. (See Table 1 and Figure 3) All linear measures are in inches, with centimeters in parentheses, with the exception of weight which is in pounds; kilograms are in parenthesis. The "50%" numbers, the 50th percentile, are the medians. Half the people are smaller 26 Women Dimtnsion• l'lr 5~ 50'7r 95'7r 99'7c A Weight 9)(42) 104(47) 137<62) 199(91} 236< 107) B St~nding 57(144) 59(149) 63( 159) 67(170) 69( 175) Height C Shoulder 13(34) 14(36) 15(39) 17(44) 18(45) U) Breadth Q) N D Chest 6(15) 6(16) 7(19) 8(21) 9(22) .-1 Depth (/) E Sining 3()(75) 31(78) 33(84) 36(90) 37(93) Height ~ F Sining Eye 26(67) :. 27(69) 29(74) 31(79) 32(82) O 0 Heii;ht l'.Q G Sining 20(52) 21(53) 23(56) 25(60) 26(66) Shoulder Height ~ H Sining 6116) 7(18) 9(23) 11(28) 12(30) 0 Elbow H:ii:ht i:: I Sitting - - - - - 0 Thigh Height ·.-1 J Elbow to - - - - - +.l Finger Tips ;j K Buttocks to 20151) 21(53) 22<57) 211{61) 25(63) .0 Front of Knee ·.-1 H L Dutro.:ks to 16(41) )7(43) 19(48) 21! 53) 22(56) +.l BJck of Knee UJ M Floor-to 14(36) 15(37) 16(41) Ill( 44) 18(46) ·.-1 Sining Height 0 N lJp-.>·ard Rear:h - - - - - frcm Sc~t 0 Forward Reach 28(71) 29(73) 31(79) 34(85) 34(87) from a~ck . 99'7c 6< 107) 9( 175) 8(45) 9(22) 7(93) 2(82) 6(66) 2(30) - - 5(63) 2(56) 8(46) - 4(87) Free Anatomical Movement i i A C d O h di i h i i Fir~I 0 N F H L SOURCE: Corwin Bennett, Spaces for People: Human Factors in Design, (New Je~sey: Prentice- Hall, Inc., 1977)p.33. SOURCE: Corwin Bennett, Spaces for People: Human Factors in Design, (New Je~sey: Prentice- Hall, Inc., 1977)p.33. 28 than these values, half are larger . The other points, the 1st, 5th, 95th, and 99th percentiles, tell what size exceeds only one percent, five percent , 95 percent, or 99 percent of the people. Data in Figure 3 show that sitting eye height is the distance from the seat to eye level: 95 percent of the women have sitting heights of 36 inches (90cm) or less. Seating is a consideration of vital importance from designing for people. As a basic rule, good seating design uses the maximum possible body area in support of the body (54) "Static work" is the term used to describe the difficulty in seat comfort. In the usual dynamic work some part of the body is moving, for example, in walking or lifting. In static work no movement takes place. Such work leads to physical fatigue because of the inadequate exchange of chemical products in the muscles and elsewhere. For example, if the seating is inadequate the lower arms or back must be held by the body in position rather than being supported by chair arms or back. Static work is stressful to particular muscles, as certain muscles must contract to maintain the static position. (55) There are "design do's and don'ts" with regard to seating design. The following list includes ten considerations that should be made when designing seating accomodations for people: 29 1. There is no one healthy human posture. Posture designs should fit the activity taking place while a person is s eated. 2 A person's trunk should be nearly upright . 2 A person's trunk should be nearly upright . 3. A person's back should be supported up to the shoulder blades; this protects the lower back. 3. A person's back should be supported up to the shoulder blades; this protects the lower back. 4. Seats should permit anatomical movement; highly contoured seats meant to fit the body should be avoided. 5. Some upholstery is desirable; over padding which locks the body into one position and restricts movement should be avoided. 6. Proper seat base height is important. If the seat base is too low, the thighs will be raised off the seat base and the buttocks, which should support most of the weight, will only be partially on the seat. If the seat base is much too low it places the trunk and thighs at an acute angle and compresses the internal organs. At this low height it is also difficult to get on and off for the tall, heavy, old, or otherwise disabled. If the seat base is too high the lower legs will dangle at the knees rather than being supported by the feet, producing heavy stress on the underside of the thighs. An adjustable seat base height ranging from 15 inches (37cm) to 18 inches (45cm) is desirable although expensive. 7. Seat bases should be the proper depth. If the seat bases is too short, the weight will be supported by an inadequate portion of the buttocks and thighs if the seat 30 base is too long it will place pressure on the back side of the calves. base is too long it will place pressure on the back side of the calves. Arm rests are necessary for proper body support. 8. Arm rests are necessary for proper body support. 9. Foot rests may be needed if seat height is too great to resting the feet flat on the floor. The foot rest permit should measure at least 2 feet by 2 feet to permit change of position of the feet. 10. For some activities stools are desirable. Stools are preferred to chairs when activity requires the ability to alternately sit and stand frequently. Stools should have back rests for support. (56) A final consideration here for enhancing the anatomical movement of individuals is the importance of designing locations and functions of hardware within the residential environment. Designers need to consider various aspects of hardware including location, size, and force requirements in terms of the human user. Hardware should be safe, efficient, readily operated, comfortable when used, and good looking. The designer must be "concerned, think through how the hardware will be used, and test it. Consider such straightforward ideas as putting a door handle on the opening side of the door instead of in the middle or making a handle fit the hand - not too large or small or ornate, not requiring too much force for many people." {57) A list of hardware needs for consideration in the design of interiors provided and can be adapted to the 31 design of t he reside ntia l e nvironments . (See Tabl e 2) design of t he reside ntia l e nvironments . (See Tabl e 2) design of t he reside ntia l e nvironments . (See Tabl e 2) The Need fo r Safety According to Abraham Mas low, "when psychological needs are satisfied, man wants to kee p and protect what he has. He starts to try to stabi lize his environment for the future". ( 58) The need for safety is characterized by the desire for an understandable, secure, and orderly world. Maslow categorized the manifestations of the safety needs as the needs for "security, stability, dependency, protection, freedom from fear, from anxiety and chaos, need for structure, order, law, limits, and strength in the protector" . A common factor underlying all the above needs is the "need for prediction and control". (59) When these needs are not satisfied, individuals may view the world and others as "unsafe, unjust, inconsistent, or unreliable". (60) Also, peripheral motives may develop which act as coping devices which serve to reduce the anxiety of the individual. However, coping effects are merely temporary and do not cure the situation completely because "they do not eliminate the past threat upon which the anxiety is based". (61) The peripheral motives derived from safety needs and their respective behavioral descriptions are provided. (See Table 3) A. Kaplan provides a useful framework for applying Maslow's safety needs to the work environment which can be 3 2 SOURCE: Corwin Bennett, Spaces for People: Human Factors· in Design, (New Jersey: Prentice- Hall, Inc., 1977) pp. 53-55. Table 2, Cont. l/ulln·1/\'" 0 r ~1 s> a gc lo fil capaci1y (furniture as well as peop!c) Slraight, not curved or chang ing pathway (consider rush, bottlenecks , inlcr·;cct ions) 0 0 Signs, markings to help one find his way Illumi nate 0 0 No large fillets at base of wall that people might step on No slick floors No visual glare at ends of hallway Cabinetry 0 0 Design to fit articles to be stored, not just to fit leftover space Drawer slides for heavy loads-provide stops Watch for door-fold interference, drawer clearance 0 0 Handles that fit, not just pretty or absent or camouflaged Doors that stay open or closed 0 Locks on critical doors or drawers 0 A void deep inaccessible cupboards or drawers Plumbing I Fixtures o Splash o Bumping head o Handles that fit, operate logically, easily, accurately .. Contac,t haznrd remova l o Handrails, handholds • Nonslick surfaces "' Accessible for repair o Do not produce noise , vibration " Consider potential leak problems Bui/;-in Lighr Fixrures I Switches 0 Ri ght location (illuminate task, not observer) o Access for re larnping, cleaning o O ptimize light distribut ion (minimize sh<!dO\V, glare) o Locate switches so they can be found, reCJched EleCT;ical Com·eniences o Adequnte number, located where needed " Accessible not only to normal adul ts but 2lso to children, 1,ovbcelc i1air operntor o M;:ke chiidproof o O ptimize light distribut ion (minimize sh<!dO\V, glare) o Locate switches so they can be found, reCJched Table 2 Conunon Hardware Problems Areas ll'iniluivs o Ease of opening, closing, locking, unlock ing o Ease of cleaning both sides • Ease of seeing th:it the window is closed so that people do not walk through, push furniture through, or foll through o Ease of use as an emergency escape route Doors o · Ease of opening, closing, locking·, unlocking o Clearance for both people and furnishings o Weather-proof o ·Nonsticking, nonrattling o Soundproof o Fireproof o Ventilation through o Seeing that sliding glass door is closed so that people do not walk through o Clearance around in order to get past or get wheelchair into room • Emergency door hardware operable by weakest from a wheelchair o Opens with, not against traffic flow o Door locations to east traffic flow, expedite emergency escape, control noise Stairs o Optimize for user(s), not for leftover space o Accessible for both people and furnishings o Handrails to fit both children and adults, to prevent children from falling through or getting stuck in o Minimize number of continuous steps to conserve energy and minimize stress o Have ramps for wheelchairs SOURCE: Corwin Bennett, Spaces for People: Human Factors· in Design, (New Jersey: Prentice- Hall, Inc., 1977) pp. 53-55. SOURCE: Corwin Bennett, Spaces for People: Human Factors· in Design, (New Jersey: Prentice- Hall, Inc., 1977) pp. 53-55. 33 Table 2, Cont. Table 2, Cont. /lc111 i11g I Cooling S r.11,·111 ,~ / :'1;1t1/Jtllt'lll ------------· /lc111 i11g I Cooling S r.11,·111 ,~ / :'1;1t1/Jtllt'lll ------------· g g ------------·. 0 • 0 Ventilation not dq ;r:.ided when doors arc closed. Continued vcntil:Hion when power fails Undcr ~ t:.indable controls 0 • Automatic ~cnsor~: (s) locllcd where they sense the pertinent conditions Locate vents for optimum performance, not case of installation 0 Vents that can be controlled (reliable, do not break off or not close completely) 0 Watch out for noise produced by system or that passes through system o Space should fit purpose o Space should be convenient, accessible o Space should be illwninated o Consider fire, explosion hazards o Consider closure requirements (ease of, locking, interference) o Storage fixtures, shelving to fit ' 0 Ventilation not dq ;r:.ided when doors arc closed. • Continued vcntil:Hion when power fails 0 Vents that can be controlled (reliable, do not break off or not close completely) o Space should be convenient, accessible o Space should be illwninated o Consider fire, explosion hazards o Consider closure requirements (ease of, locking, interference) o Consider closure requirements (e o Storage fixtures, shelving to fit Table 2, Cont. Table 2, Cont. Table 2, Cont. EleCT;ical Com·eniences o Adequnte number, located where needed " Accessible not only to normal adul ts but 2lso to children, 1,ovbcelc i1air operntor M k hiid f " Accessible not only to normal adul ts but 2lso to children, 1,ovbcelc i1air operntor o M;:ke chiidproof o M;:ke chiidproof 34 SOURCE: Joel Aronoff and John R. Wilson, Personality in the Social Process (New Jersey: Lawrence Erlbaurn Associates, 1985) p.41. Most Common User-Interface Failure Points o People have to locate themselves with respect to parking, bui1ding, en- trance, interior hallway, doors, spaces. o People have to see where they are going (illumination). o People have to manipulate--d.oors, windows, drawers, faucets, thermo- stats, etc. o People have to manipulate--d.oors, windows, drawers, faucets, thermo- stats, etc. • People have to see signs, printed matter, written material, instrument faces, etc. (color, brightness, contrast, format, illwnination). • People have to see signs, printed matter, written material, instrument faces, etc. (color, brightness, contrast, format, illwnination). o People have to maintain balance and posture control, have help to move (handholds, handrails). o People have to move themselves or . furnishings about- clearance (doors, hallways, stairs); stairs. o People have to clean, replace, refurbish, repair. o People need to store things. o People may have to escape in a hurry-routes, more than one exit, emergency illumination, etc. o People may have to escape in a hurry-routes, more than one exit, emergency illumination, etc. o People need peace and quiet, also need to have their fun without bother- ing others. o People need peace and quiet, also need to have their fun without bother- ing others. • People need power conveniences for their electrical add-ons , tools, hob- bies, etc. • People need power conveniences for their electrical add-ons , tools, hob- bies, etc. o People need reasonable thermal environment and good ventilation. o People need reasonable thermal environment and good ventilation. 35 Ta ble 3 Peripheral Motives Derived from Safety Needs Ta ble 3 Peripheral Motives Derived from Safety Needs Peripheral Motive I. Abasement 2. Dependence (succorance) 3. Approval (deference) 4. Order Rejection Contrarience Acquisition Conservance Retention Behavioral Definition To blame oneself; to surrender, apologize, confess, atone, comply, and accept punishment. To seek aid, protection, sympathy, or help. To admire, emulate, cooperate with, yield eagerly to, and willingly serve a leader. To structure events through controlling the arrangement of persons and objects, such as: to exclude or ignore others; to assume a negative or oppositional stance in relation to others; to work for possessions; to collect, repair, or preserve objects; to be miserly in retaining possession of objects. Behavioral Definition to collect, repair, or preserve objects; 36 extended and adapted to an analysis of the residential environment (62) Five aspects of the need f or s afety, or security subneeds, are proposed: territoriality, privacy, orientation, positioning, and anticipation. The first security need, territoriality, is expressed by Kaplan in his framework, as a need which is "human, not animal, by design features of borders, edges and other devices which give definition to space". (63) Territoriality is expressed by others as the need to "possess a defended space as an innate, universal characteristic of humans" and "that our attachment to property is of an ancient biological order ... man has an inherent drive to gain and defend an exclusive property." (64) Geographer J.D. satisfactions which Porteous outlines three territorial the ownership of a house provides: "control over space, personalisation of space as an assertion of identity, and stimulation which is accomplished by defending one's territory." Security, Porteous maintains, is "both physical and psychic and it can be obtained in the home and also in its individual cores, usually bedrooms, boudoirs or studies." (65) Porteous continues by suggesting that ''the rectangular, single-family structure standing on its own yard is that which is preferred in the Western World." (66) The use of fences and other physical barriers such as the planting of trees and shrubs is considered effective in 37 express ing needs fo r t erri t oriality i n that t hey de fine borde r s and give definition to space . ... Ta ble 3 Peripheral Motives Derived from Safety Needs the fence offered protection, made the property look neat, aided in cleaning, clearly delinieated private from public property, revealed residents' concern about their property and trespassing, and suggested that outsiders who invaded could anticipate a hassle with the resident, and would be hard put to justify their entry. Thus a fence provides clearer boundary definitions, facilitates access control, aids in maintenance of the space, and confers stronger proprietary rights upon the resident. (67) Privacy is Kaplan's second security need which he ex- presses as ''the need to be undisturbed by others when desired, provided by the structure of the environment". (68) In her book entitled The Need for Roots, Simone Weil claims that private property is "a vital need of the soul". The soul feels isolated, lost if it is not surrounded by objects which seem to it like an extension of the bodily members. All men have an invincible inclination to appropriate in their own minds anything which over a long, uninterrupted period they have used for their work, pleasure or the necessities of life. It is desirable that the majority of people should own their own house and a little piece of land round it. (69) Privacy, therefore, is a crucial need and substantial effort should be made to plan and design residential environments allowing for the maximum amount of privacy 38 possible . A. Kaplan pr opos e s a third s ecurity need, the need fo r orientation, and r e fers to this as "the need to know one's r e lative position in time and space by environmental cues." (70) the use of Charle s Brockett supports this view in maintaining that "much of Maslow's discussion on the safety needs can be understood as an explanation of the need for the 'frame of reference' -and as indirectly applying to 'physical safety'."(71) Brockett states further that "growth proceeds better when the body is protected from injury and the mind from 'too much' disorientation. As explained by E. Fromm in The Sane Society, the latter need (a frame of reference) refers to the need for a context by which one can understand and deal with the world. He argues that there are two dimensions to this need, dimensions which correspond to the reasoning and feeling capabilities (frames of orientation and devotion, respectively). Ta ble 3 Peripheral Motives Derived from Safety Needs This need (on both dimensions) is created by the awareness of oneself and of the world outside oneself. Such a framework can be adequate for sanity regardless of its validity, he claims, since illusions can be functional. The development of reason is important; however, since by leading to a more accurate picture of reality it can create greater happiness and serenity, and certainly greater potential for growth." (72) D i id ti f th id ti l i t Design considerations for the residential environment which address the "need to know one's relative position in 39 need to emerge is what Maslow refer s to as the need for love and belonging. Once the physiological and safety needs 'are fairly well gra tified' the individual 'will feel sharply the pangs of lonliness, of ostracism, of rejection, of friendlessness, of rootlessness'. The indivi dual's well- being requires that these needs be gratified. (75) Once the physiological and safety needs 'are fairly well gra tified' the individual 'will feel sharply the pangs of lonliness, of ostracism, of rejection, of friendlessness, of rootlessness'. The indivi dual's well- being requires that these needs be gratified. (75) The love and belongingness needs evolve around the desire to experience intimate relationships with other people. Along with this desire come the desires for contact, intimacy, warm and friendly relationships, and to function well in interpersonal situations. (76) There is also the broader need to belong to a wider group. At this stage it is important for an individual to identify with group goals or a place within a group. (77) A common issue underlying this need is the need for intimacy. If a person experiences difficulty in loving relationships, he is in a state of intimacy deprivation. Feelings of anxiety emerge which lead to the development of coping mechanisms to reduce the anxiety. An outline is provided of six mechanisms commonly used to cope with a loss of intimate feelings which permit an individual to be "included, or to participate, in the lives of others without risking the vulnerability of fusion of identity". (See. Table 4) The term "inclusion" is used to describe this general desire . (78) 41 time and space " could include the following: the use of windows in every room to continually inform the resident of time; the placement of clocks, calendars, and seasonal decoration throughout the residential environment. Ta ble 3 Peripheral Motives Derived from Safety Needs The need to know one's position in space could be obtained through the use of area maps as wall hangings, placemats, decorative items, etc. Physical design such as outside porches overlooking yards, open areas and roadways in rural and suburban areas; verandas, and terraces in urban areas. The security need for positioning is fourth on Kaplan's list of security needs and is described as the "disposition of our bodily orientation relative to walls, windows, doors, openings, etc., leading to feelings of safety and security". (73) The need for positioning is closely related to the need for orientation; however, positioning refers more to one's familiarity with the physical layout of an interior environment. The final security need suggested by Kaplan is the need for anticipation: "the need to prepare for the immediate, instantaneous future. The environment, in its design and structure, must meet our perceptual and cultural expectations:(74) The Need for Love and Belongingness Once a persons' need for security is sufficiently satisfied and his environment becomes more stable, the next 40 The fir s t type of behavior or "mode", "passive compliance", refers to the person who achieves participation through passively joining the activities of the group. This type of person wants intimacy; however, he makes no emotional demands and gives no intimacy to others. In the second mode the person "earns the right of participation by overestimating the worth of other members and relates to them through admiration and flattery." This behavior protects the individual from intimacy by creating a distance between himself and other people. The third mode permits the person to participate in interpersonal relationships by entertaining others. In the fourth mode, the person becomes involved by making himself the scapegoat of the group. The fifth mode tests the limits of other's tolerance for aversive behavior to witness the concern for the individual. The sixth and final mode is the "investment of the self in an abstract concept of the group. Through pledging fidelity to the nonpersonal aspect of the group, this individual is able to avoid the personal aspect of intimacy and still achieve membership into a wider entity. Whatever the variant, inclusion is most fundamentally an attempt to find some guarded form of involvement with others so that some degree of intimacy can be achieved". SOURCE: Joel Aronoff and John R. Wilson, Personality in the Social Process (New.Jersey: Lawrence Erlbaum Associates, 1985) p.45. Ta ble 3 Peripheral Motives Derived from Safety Needs (79) Once an individual has sufficiently satisfied his need Once an individual has sufficiently satisfied his need for intimacy, love and belongingness, and the anxiety associated with the deprivation of this need has 43 Table 4 Peripheral Motives Derived from · Love and Belongingness Needs Peripheral Motive 1. Recognition 2. Dominance Behavioral Definition To attempt to command respect by draw- ing the attention of others to one's actions, through the seeking of honors, or by suc- ceeding at extremely difficult feats. To establish one's worth by controlling, persuading, dictating, and directing others. 3. Nurturance (generativity) To establish and maintain one's sense of worth by responsibly caring for the devel- opment of persons, generations, and insti- tutions, .as well as the quality and signifi- cance of achievements and products. 4. Achievement To be competitive in meeting standards of excellence across a wide range of transac- tions with the world. Peripheral Motive 1. Recognition 2. Dominance Behavioral Definitio To attempt to command resp ing the attention of others to o through the seeking of honor ceeding at extremely difficult To establish one's worth by persuading, dictating, and others. 3. Nurturance (generativity) To establish and maintain on worth by responsibly caring f opment of persons, generation tutions, .as well as the quality cance of achievements and pr 4. Achievement To be competitive in meeting excellence across a wide range tions with the world. Behavioral Definition To attempt to command respect by draw- ing the attention of others to one's actions, through the seeking of honors, or by suc- ceeding at extremely difficult feats. To attempt to command respect by draw- ing the attention of others to one's actions, through the seeking of honors, or by suc- ceeding at extremely difficult feats. To establish one's worth by controlling, persuading, dictating, and directing others. To establish and maintain one's sense of worth by responsibly caring for the devel- opment of persons, generations, and insti- tutions, .as well as the quality and signifi- cance of achievements and products. To be competitive in meeting standards of excellence across a wide range of transac- tions with the world. 42 dis sipated, he f eels a s tronge r s ubj ective sense of "rootedness and membership in psychologically meaningful groups". . .. the se people begin to have a wider identification with the species and a greate r understanding of their place in the significant community. Ta ble 3 Peripheral Motives Derived from Safety Needs Through these developments, these wider social referents become an integral part of their personality. These strengths, together with those achieved at prior stages, increase these individuals' sense of well-being by permitting them to enjoy social relations in a more complex and integrated way. (80) The design of residential environments influences the extent to which an individual's need for love and belongingness is satisfied by creating opportunities for interaction and by not inhibiting social satisfaction. (81) A. Kaplan proposes three levels of social interaction for consideration in the design of interiors and site planning which can be extended to the residential environment: 1. The most basic level which fulfills an elemental social need is seeing people and their activities. 2. The intermediate level of social satisfaction is the expression of emotion, giving vent to the feelings of current and recent experiences. 3. The highest level of social interaction is the expression and exchange of ideas and is how the world comes 44 toge ther. (82) Kaplan has provi ded a br oad and seemingly s ound outline for conside rat ions in the design of interior; however, something must said about adequate amounts of interaction, and adequate types of interaction, within the residential environment . A 1978 study conducted by D. McCarthy and S. Saegert entitled "Residential Density, Social Overload, and Social Withdrawal'' proposes the need for adequate amounts of social interaction. (83) The McCarthy and Saegert study compares the effects of residential density on tenants in 14-story apartment buildings and tenants in 3-story walkups within the same low-income housing project. The following is hypothesized: Tenants in the high-rise buildings would come into contact with large numbers of others in the public spaces of their buildings. As these contacts exceed resident's interaction capacity or ability to process relevant incoming social stimuli, tenants would experience social overload. This experience would be manifested by tenants' perceptions of crowding in the building, feelings of less control, safety, and privacy in the immediate residential environment, problematic social relationships among tenants and alientation and dissatisfaction with the residential environment generally. These experiences were not expected to occur for tenants in the low-rise walkups. (84) hypotheses proposed are supported by interview The hypotheses proposed are supported by interview 45 data . Ta ble 3 Peripheral Motives Derived from Safety Needs I t is a l s o r evea l ed tha t high-rise apartme nt r esidents we r e "less socially active beyond the ir building and f elt a greater sense of powerlessness in e ffecting management decisions". (85) data . I t is a l s o r evea l ed tha t high-rise apartme nt r esidents we r e "less socially active beyond the ir building and f elt a greater sense of powerlessness in e ffecting management decisions". (85) The study raises the question about the extent to which building design can alleviate the experience of social overload, even when densities are high . It is suggested that building designers create manageable subgroups of residents which would lessen the probability of social overload. The support for creating "psychological units" within the residential environment is provided. Clearly, all our respondents viewed their buildings as a unit of some kind. For the low-rise apartment residents, this was a unit to which they belonged, in which they felt some sense of trust, social involvment , and responsibility and which served as a base from which to extend their social commitments into the outer world. In contrast, high- rise apartment residents saw the building as a dangerous conglomerate of alien spaces and mainly threatening prople. They made some differentiation between their own floors and the rest of the building but their orientation toward the building did flow over into their feelings about the floor to the extent that they felt less safe in the hall than did low-rise apartment tenants. These tenants made it clear that their major identification was with their own apartments. They neither identified with nor took responsibility for the people and spaces of the building or the project as a whole. In fact, withdrawal from external social contacts characterized their responses to a wide range of questions. (86) 46 These fi ndi ngs s upport the suggestion that designer s serious ly consider the importance of creating environmental units with which residents can identify. Need for Self-Esteem Once an individual has sufficiently satisfied his need for physiological factors, security, and love and belongingness, a fourth need emerges called the need for self-esteem. According to Maslow, people desire a ''stable, firmly based, usually high evaluation of themselves". Ta ble 3 Peripheral Motives Derived from Safety Needs The need for self-esteem is characterized by two dimensions: self- esteem or the "desire for strength, for achievement, for adequacy, for mastery, and competence,... and for independence and freedom"; and the esteem of others through reputation, prestige, dominance, recognition, attention, dignity, or appreciation. (87) Maslow makes a qualitative distinction between self- esteem based on the opinion of others and that based on their deserved respect: "it is useful to ' distinquish the actual co~petence and achievement that is based on their will power, determination, and responsibility, from that which comes naturally and easily out of one's own true inner nature, one's constitution, one's biological fate or destiny', or as Horney puts it, out of one's Real Self rather than out of the idealized pseudo-self". (88) {n 47 indi vidual is stressed and e l abora t ed on by G. F . Kawash and G.W. Scherf. indi vidual is stressed and e l abora t ed on by G. F . Kawash and G.W. Scherf. There is probably no pers ona lit y t rait more significant in the context of total psychological functioni ng than self-esteem, a characteristic that has been related, either empirically or theoretically, to much vital phenomena as depression, satisfactory levels of functioning in interpersonal relations, and material acceptance of children. A low self-esteem is almost certainly an impediment to intimate interpersonal functioning. The continual self- depreciation, lack of confidence, and uncertainties would be a source of irritation to, say, a marital partner who could not empathize with these behaviors. For people to be married for a substantial period of time, either compatible levels of self-esteem would be necessary or else some relationship between them would develop whereby an acceptance of differences (and consequently thereof) would be worked out. (89) When an individual satisfies his need for self-esteem, he begins to feel that "challenges can be met with confidence, that required skills exist or can be attained, and that one's abilities can be relied upon." (90) Individuals begin to welcome responsibility for their actions as ways of proving their competence and the world is considered an arena that provides desirable opportunities to exhibit one's competence and skill. SOURCE: Joel Aronoff and John R. Wilson , Personality in the Social Process (New Jersey: Lawrence Erlbaum Associates, 1985) p.47. Ta ble 3 Peripheral Motives Derived from Safety Needs When experiences fail to provide rewards such as competence, the individual is deprived of his self-esteem and the concomitant feelings that he has "not been fully 48 effective, cannot rely on his or her abilities, and thus is searching for experience that will develop a strong sense of self-worth". (91) Maslow explains that when an individual becomes frustrated in attempts to gain self-esteem, he experiences "feelings of inferiority, of weakness, and of helplessness" feelings which can cause either "basic discouragement" or neurotic tendencies. (92) Anxiety develops and the individual becomes frustrated through need deprivation. As a result, the individual develops coping devices through which he seeks partial gratification. These devices are peripheral motives which, as in the previous cases, do not completely fulfill the need since they do not address the underlying source of anxiety. Four major peripheral motives are suggested which are concerned with establishing or maintaining a strong degree of positive self-evaluation: recognition, dominance, nurturance (generativity) and achievement. (See Table 5) A. Kaplan renames the need for self-esteem as "Ego Needs" and refers to Maslow's statement that "there are the needs that related to one's self-esteem - needs for self- independence, achievement, competence, confidence, knowledge". Kaplan continues by proposing that II the environmental design achievement of this principle which higher level is most permits control - the individual's ability to have control over his environment; Four major peripheral motives are suggested which are concerned with establishing or maintaining a strong degree of positive self-evaluation: recognition, dominance, nurturance (generativity) and achievement. (See Table 5) A. Kaplan renames the need for self-esteem as "Ego Needs" and refers to Maslow's statement that "there are the needs that related to one's self-esteem - needs for self- independence, achievement, competence, confidence, knowledge". Kaplan continues by proposing that II the environmental design achievement of this principle which higher level is most permits control - the individual's ability to have control over his environment; 49 Table 5 Periphe ral Mot ive s De rived from Esteem Needs Table 5 Periphe ral Mot ive s De rived from Esteem Needs Peripheral Motive Inclusion f!ehavioral Definition To participate in the lives of others through; a. passive compliance; b. overestimation and flattery of others' virtues; c. being entertaining; d. being a scapegoat; e. provoking others to test the limits of their tolerance; f. pledging fidelity to an abstract concept of the group. 50 the c apacity to modi fy t he e nvi r onment in or der to achie ve personal short and long ter m goals and objectives. Control is effective through the us e of control devices: on-off switche s , etc., they descr i be adjustment through manual manipulation. Arrangeme nt of furniture, equipment, etc. is made through the selection and change of environmental surfacing, material and color ... Control is achieved by having alternatives and options in order to be able to choose between alternative states. Environment conditions must be selected. All levels of needs must be satisfied by choosing routes and pathways . Environmental control and alternatives lead to coping and personal feelings of being free in contrast to a lack of control leading to feelings of frustration and impotence . " (93) the c apacity to modi fy t he e nvi r onment in or der to achie ve personal short and long ter m goals and objectives. Control is effective through the us e of control devices: on-off switche s , etc., they descr i be adjustment through manual manipulation. Arrangeme nt of furniture, equipment, etc. is made through the selection and change of environmental surfacing, material and color ... Control is achieved by having alternatives and options in order to be able to choose between alternative states. Environment conditions must be selected. All levels of needs must be satisfied by choosing routes and pathways . Environmental control and alternatives lead to coping and personal feelings of being free in contrast to a lack of control leading to feelings of frustration and impotence . " (93) The importance of a sense of control in one's house as a component of self-esteem is also supported by J. Table 5 Periphe ral Mot ive s De rived from Esteem Needs Agnew who states that although he finds conflicting meanings and a variety of individual experiences, there is a "shared emphasis 'on the importance of the individual's increasing search for a realm of personal control in a world where he or she generally feels impotent ~ . " ( 94) Agnew maintains that the single-family detached house is a sufficient symbol of self-sufficiency and personal autonomy because of its "greater isolation and insulation from others". (95) Agnew entitled refers to a study conducted by R.M. Rakoff "Ideology in Everyday Life: The Meaning of the 51 House'' which focuses on informant's attitudes toward the owned house relative to personal control: "first, having control over one's own private space gave people a feeling of freedom from the control and intrusion of others ... Second, and more importantly, people feel that by being in control of their own private space, they had the power and opportunity to make something of themselves to be more of an individual; to achieve a kind of self- fulfillment". (96) It is further suggested by A. Rapoport that a sense of "perceived control, mastery, and competence can be achieved not only through the choice of a particular environment but also through professional activities, social achievement and other means as well as, or instead of, through dwelling personalisation: the link and explanatory variable is lifestyle." ( 97) Rapoport predicts that personalisation of the residential environment is important as a means of indicating self-identity among individuals who cannot achieve a sense of mastery and self-identity through their occupation, and profession. (98) The social status of a group should be considered in designing residential environments due to some existing differences in the ways these groups identify with their home and the way this identification is made known to others. In support of this, Rapoport describes a study which In support of this, Rapoport describes a study which 52 compares the extent to which r e sidents of two areas of Milwaukee asserted personalisation of their homes. One area is the South Side, a blue-collar area, and the other area is the Upper East Side, a professional and academic area. It is reported that the blue-collar residents asserted an extremely high degree of personalisation for the external residence, whereas residents of the upper- class area did not assert any personalisation. Table 5 Periphe ral Mot ive s De rived from Esteem Needs Rapoport proposes that the reason for this is that the group norm of the upper-class group is the acceptance of stately old houses within the Upper East Side. Thus it is "the fact of living in that area, of having chosen it, which is the environmental way of establishing identity." Self-identity is achieved for this group more in terms of professional and academic achievements and recognition than through personalisation of the home. Suburban-status homes are also analyzed in terms of residents' attempts to demonstrate identity personalization of their homes. through In their analysis of the various functions of the Canadian middle-class suburban house, Seeley, Sim and Loosley describe the house as a stage which has been designed at great expense to be impressive to visitors and reflect the social standing of the owners. The hall or reception room, the living- room, dining-room and recreation room are the rooms that receive the most attention in terms of decorating and housekeeping. They state that the use of picture windows in the front overlooking the street rather than in the back with a view of the garden 53 illustrates this 'staging or display orientation'. Through these windows, the mildly curious passing observer may identify where the drapes permit, the owner of a chandelier, or a striking red brocade chair. (99) In a study of a New York suburban community, presentation of the self extends from the interior " ... to the facade of the house, the front garden and the neighborhood ... The social characteristics of families are judged by others on the basis of their residential landscapes with attention to such small details as mailboxes ... and such details can also be used as an effective objective measure of certain social characteristics". (100) 54 54 Self-Actualization Self-Actualization The fifth and final need felt by an individual is the need for self actualization: this is one's need for full exercise of one's ability of awareness and understanding after one's earlier needs have been expressed and satisfied. Maslow refers to this need of the individual as "the capstone of all his other needs, man wants to realize the full range of his individual potential as a human being". (101) A. Kaplan reports on his study of Maslow's need hierarchy interpreted for the work environment: This level is an environmental issue only in that all other levels must usually be arrived at first. To become self-fulfilled in Maslow's terms calls for growth, self-development and self- actualization. This is an inner human consciousness which is, in part, derived from a comprehension and internalization of the roles and meanings of all interwoven environments and environmental energies and the total identification with them. (102) This level is an environmental issue only in that all other levels must usually be arrived at first. To become self-fulfilled in Maslow's terms calls for growth, self-development and self- actualization. This is an inner human consciousness which is, in part, derived from a comprehension and internalization of the roles and meanings of all interwoven environments and environmental energies and the total identification with them. (102) Similarily , in the residential environment the final need of self-actualization is not transferred to design criteria but rather achieved through the fulfillment of the 55 previous needs within the self-actualizing process. previous needs within the self-actualizing process. previous needs within the self-actualizing process. Individuals who have reached this level have been referred to as "Self-Actualizers"; they are people who "have developed or are developing to their full capacities. Characteristically, these people have a superior perception of reality, increased acceptance of self, and increases in spontaneity, detachment, autonomy, and creativity." (103) In a comparison of self-actualizing and non-self- actualizing individuals, the personality dimensions of time usage and autonomy have been measured using Strostrom's Personal Orientation Inventory (POI). J.A. Goldman and P.V. Olczak administered the POI to study participants and report the following regarding time usage and autonomy of the self-actualizer's personality. Individuals who have reached this level have been referred to as "Self-Actualizers"; they are people who "have developed or are developing to their full capacities. Self-Actualization Characteristically, these people have a superior perception of reality, increased acceptance of self, and increases in spontaneity, detachment, autonomy, and creativity." (103) In a comparison of self-actualizing and non-self- actualizing individuals, the personality dimensions of time usage and autonomy have been measured using Strostrom's Personal Orientation Inventory (POI). J.A. Goldman and P.V. Olczak administered the POI to study participants and report the following regarding time usage and autonomy of the self-actualizer's personality. The self actualized person is time competent and thus appears to live more fully in the here-and-now. He appears to be less burdened by guilts, regrets, and resentments from the past than is the non-self-actualized person, and his aspirations are tied meaningfully to present working goals. In other words, a self-actualized individual is one who is more efficient in his use of time than a non-self-actualized individual. terms The autonomy dimension of personality is discussed in of one's inner-directed and other-directed orientation. terms The autonomy dimension of personality is discussed in of one's inner-directed and other-directed orientation. The autonomy dimension of personality is discussed in The inner-directed person is guided by a small number of principles initiated by internalized parental values that are adhered to rigidly. The other- 56 directed pers on receives guidance from others and i s directed by his contemporaries. Approval from others becomes his highest goal, and he tends to invest all power in the actual imaginary approving groups. Manipulation in the form of pleasing others and insuring constant acceptance becomes his primary method of relating. An Autonomous person is independent of extreme other-directedness, which results in a liberation from social pressures and social expectancies. (104) Underlying the precondition for self-actualization is awareness: "insight into oneself and the outer world, including the culture. Whatever restricts such awareness, then, will undoubtedly diminish the opportunities for self- actualization - or at least adversely influence its course." (105) By becoming aware of oneself and one's environment, one can better understand the ways the environment affects him. With this understanding comes a sense of control over the environment. Individuals can manipulate the design of their residential environments to meet their needs, and therefore create an atmosphere which supports the process of self-actualization. 57 Conclusion The thesis proposed in the present paper can be integrated into the comprehensive-rational planning process. This is a five-step continuing process consisting of: inventory, goal articulation, development of alternatives, analysis and selection of alternative; implementation, and evaluation. In the inventory stage of the planning process, planners and designers should learn as much as possible about the people who will inhabit the residential area they are designing in the pre-construction stage. well as subjective sources of information residents should be investigated. Objective as about the Objective characteristics of the residents could include such indicators as age, ethnic background, socio-economic status, profession, and so forth. Subjective characteristics of the residents could include attitudinal surveys and personal interviews. In addition to learning as much as possible about the future residents, planners and designers should 58 i nve ntory the physical, s ocial, cultura l charact eris tics of r eside ntial sit e i s planned. economic, political, and the region where the It is important for the pla nne r and designer to know about characteristics such as climate conditions, regional economic trends, town and city political admi nistrations and their policies, and local and regional socio-economic conditions. The information obtained in the inventory stage of the planning process should be integrated and compiled into a profile of potential residents. This resident profile should be referred to continually and systematically updated. In the second stage of the planning process, planners and designers should articulate the goals of their clients. A special emphasis should be made to consider the needs of future residents as a primary goal in light of the information presented in this thesis. The development of alternative design plans in the third planning stage should be based upon the needs of the residents as outlined in this thesis. In the fourth planning stage, the alternative which best meets the needs of the residents should be selected. The plans for implementing the selected alternative is through the actual construction of the residential development and area is the fifth stage. Once the construction is completed and residents have begun to move into the residential development, planners and designers should institute an 5 9 evaluation program to continually monitor the physical and social quality of the residential area. It is important to emphasize that the planning process is an ongoing process and does not end once the development has been constructed. Conclusion Once the planner has completed the evaluation stage of the process, it is vitally important to return back to the inventory stage and follow through the process again, and perhaps again, in a constant effort to improve what has been built. The information obtained in the evaluation stage of the planning process as well as any information obtained throughout the process should be considered in the second planning process. The value of the comprehensive -rational planning process is that it allows for changes discovered through the inventory stage to be considered. In addition, the process is self-correcting, thus allowing for any discrepancies discovered in the evaluation stage to be rectified in the following process. Abraham Maslow's Hierarchy of Prepotent Needs model provides planners and designers with a unique and model for understanding basic human needs which well-integrated into this planning process. useful can be There are deep-rooted and personal meanings attached to the home :a "man's castle is his home". The amount of consideration made in residential environments nature of the home. the planning and should reflect the Every effort should design of meaningful be made to 60 improve beginning the quality of the with the most basic res idential elements of environment residential satisfaction, the extent to which user needs are met. 61 61 (12) Angus Campbell, Philip E. Converse, and FOOTNOTES (1) Abraham Maslow, Motivation and Personality (New York: Harper & Brothers, 1954)pp. 1-411. (2) James s. Duncan,ed., Housing and Identity (New York: Holmes & Meier Publishers, Inc., 1982),p.1. (3) Robert M. Rakoff, "Ideology in Everyday Life: The Meaning of the House," Politics and Society 7 (1977):85. ( 4 ) Ibid. , p. 8 5 . (5) Amos Rapoport, "Identity and Environment: A Cross- Cultural Perspective," in Housing and Identity: Cross-Cultural Perspectives, ed. J.S. Duncan (New York: Holmes & Meier Publishers, Inc., 1982),p.21. (6) Robert M. Rakoff, "Ideology in Everyday Life: The Meaning of the House," Politics and Society 7 ( 1977): 85-86. (7) James s . Duncan, "From Container of Women to Status Symbol: the Impact of Social Structure on the Meaning of the House," in Housing and Identity: Cross-Cultural Perspectives, ed. James s. Duncan (New York: Holmes & Meier Publishers, Inc., 1982)pp.36-37 . (8) Amos Rapoport, "Identity and Environment: A Cross- Cultural Perspective, " in Housing and Identity: Cross-Cultural Perspectives, ed. James s. Duncan (New York: Homes & Meier Publishers, Inc., 1982) p.21. (8) Amos Rapoport, "Identity and Environment: A Cross- Cultural Perspective, " in Housing and Identity: Cross-Cultural Perspectives, ed. James s. Duncan (New York: Homes & Meier Publishers, Inc., 1982) p.21. (9) Robert M. Rakoff, "Ideology in Everyday Life: The Meaning of the House," Politics and Society 7 (1977):94. (9) Robert M. Rakoff, "Ideology in Everyday Life: The Meaning of the House," Politics and Society 7 (1977):94. (10) Ibid . , p.94. (12) Angus Campbell, Philip E. Converse, and 62 Willard L. Rodgers, The Quality of American Life: Perceptions, Evaluations, and Satisfactions (New- York: Russell Sage Foundation, 1976)p.265. Willard L. Rodgers, The Quality of American Life: Perceptions, Evaluations, and Satisfactions (New- York: Russell Sage Foundation, 1976)p.265. (13) Sue Weidema nn and James R. Anderson, "Residents' Perceptions of Satisfaction and Safety: A Basis for Cha nge i n Multifamily Housing," Environment and Be havior 14(b) (November 1982):698. (14) Ibid., p.695. (14) Ibid., p.695. (15) Ibid., p.698. (16) Ibid., p.697. (17) Kenneth H. Craik and Ervin H. Zube, eds., Perceiving Environmental Quality: Research and Applications (New York: Plenum Press, 1976)pp.132-133. (18) Angus Campbell, Phillip E. Converse, and Willard L. Rodgers, The Quality of American Life: Perceptions, Evaluations, and Satisfactions (New York: Russell Sage Foundation, 1976),pp.264-265. (19) Kenneth H. Craik and Ervin H. Zube, eds., Perceiving Environmental Quality: Research and Applications (New York: Plenum Press, 1976)pp.150-151. (20) Ibid., pp.150-151. FOOTNOTES (20) Ibid., pp.150-151. (21) Ibid., p.134. (21) Ibid., p.134. (22) Angus Campbell, Phillip E. Converse, and Willard L. Rodgers, The Quality of American Life: Perceptions, Evaluations, and Satisfactions (New York: Russell Sage Foundation, 1976),pp.9-10. (23) Charles Hampden-Turner, Maps of the Mind: Charts and Concepts of the Mind and its Labyrinths, (New York: Collier Books, MacMillan Publishing, Co., 1982)p.118. (24) Charles »rockett, ''Toward a Clarification of the Need Hierarchy Theory: Some Extensions of Maslow's Conceptualization," Interpersonal Development 6(2) (1975-76):77. (25) Ibid., p.78. (26) Ibid., p.77. (27) Archie Kaplan, ''Maslow Interpreted for the Work Environment," Man-Environment systems 6(4) (July 1976):246. 63 (28) David Le , t e r e t a l., "Mas low's Hierarchy of Need s and Psychological Health," Journal of General Psychol.!?_gy 109(1) (July 1983):83-85. (29) Archie Kaplan, "Maslow I nterpreted for the Work Environment," Ma n-Environment Systems 6(4) (July 1976):247. ( 30) Corwin Bennett, Spaces for People: Human Factors Jersey: Prentice-Hall, Inc., 1977)p.87. in Design, (New (31) Ibid., p.88. (31) Ibid., p.88. ( 32) Ibid., pp.88-89 . ( 32) Ibid., pp.88-89 . ( 33) Ibid., p.92. ( 33) Ibid., p.92. (34) Ibid., p. 91. ( 35) Ibid., p.100. (36) Ibid., p.102. (37) Ibid., p.104. (38) Ibid., pp.106-107. (39) Ibid., p.109. (40) Ibid., p.113. (41) Ibid., p.115. (42) Ibid., p.117. (43) Ibid., p.118. (44) Ibid., pp.119-120. (45) Ibid., p.123. (46) Ibid., pp.124-125. (47) Ibid., p.128. (48) Ibid., p.128. (49) Ibid., p.131. ( 50) Ibid., pp.130-131. ( 51) Ibid., pp.129-130. ( 52) Ibid., p.29. 64 ( 5 3) Ibid., p.30. (54) Ibid., p.35. ( 55) Ibid., p.38. ( 55) Ibid., p.38. ( 56) Ibid., pp.38-42. (57) Ibid., p.52. (58) Archie Kaplan, "Maslow Interpreted for the Work Environment," Man-Environment Systems 6(4) (July 1976):247. (59) Joel Aronoff and John R. Wilson, Personality in the Social Process (New Jersey: Lawrence Erlbaum Associates, 1985) p.20. (60) Ibid., p.20. (60) Ibid., p.20. (61) Ibid., p.40. (61) Ibid., p.40. (62) Archie Kaplan, "Maslow Interpreted for the Work Environment," Man-Environment Systems 6(4) (July 1976):246. (63) Ibid., p.246. (64) Nancy G. Duncan, "Home Ownership and Social Theory," in Housing and Identity: Cross-Cultural Perspectives, ed. James S. Duncan (New York: Holmes & Meier Publishers, Inc., 1982)pp.112-113. (65) Ibid., pp.112-113. (66) Ibid., pp.112-113. (67) Kathleen H. Dockett, Sidney Brower, and Ralph B. FOOTNOTES Taylor, "Territorial Cognitions of Threat and Defense in the urban Residential Environment," Papers in the Social Sciences: ~Journal of the College of Liberal and Fine Arts, University of the District of Columbia 1(1981):31. (68) Archie Kaplan, "Maslow Interpreted for the Work Environment," Man-Environment Systems 6(4) (July 1976):246. (68) Archie Kaplan, "Maslow Interpreted for the Work Environment," Man-Environment Systems 6(4) (July 1976):246. (69) Nancy G. Duncan, "Home Ownership and Social Theory," in Housing and Identity: Cross-Cultural Perspectives, ed. James s. Duncan (New York: Holmes & Meier Publishers, Inc., 1982)p.112. 65 (70) Arc hie Kaplan, "Maslow Interpreted for the Work Environment," Man-Environment Systems 6(4) (July 1976):247. (71) Charles Brockett, "Toward a Clarification of the Need Hierarchy Theory: Some Extensions of Maslow's Conceptualization," Interpersonal Development 6(2) (1975-76):82. (72) Ibid., p.82. (73) Archie Kaplan, "Maslow Interpreted for the Work Environment," Man-Environment Systems 6(4) (July 1976):247. (74) Ibid., p.247. (75) Charles Brockett, "Toward a Clarification of the Need Hierarchy Theory: Some Extensions of Maslow's Conceptualization," Interpersonal Development 6(2) ( 1975-76): 43-44. (76) Joel Aronoff and John R. Wilson, Personality in the Social Process (New Jersey: Lawrence Erlbaum Associates, 1985) p.21. (77) Ibid., p.21. (77) Ibid., p.21. (78) Ibid., pp.44-45. (78) Ibid., pp.44-45. (79) Ibid., pp.44-46. (80) Ibid., p.22. (81) Archie Kaplan, "Maslow Interpreted for the Work Environment," Man-Environment Systems 6(4) (July 1976):247. (82) Ibid., p.247. (83) Dennis McCarthy and Susan sa:egert, "Residential Density, Social overload, and Social Withdrawal," Human Ecology 6(3) (September 1978). (83) Dennis McCarthy and Susan sa:egert, "Residential Density, Social overload, and Social Withdrawal," Human Ecology 6(3) (September 1978). (84) Ibid., p.253. (85) Ibid., p.253. (86) Ibid., p.270. (87) Charles Brockett, "Toward a Clarification of the Need Hierarchy Theory: Some Extensions of Maslow's Conceptualization," Interpersonal Development 6(2) ( 1975-76): 79. (87) Charles Brockett, "Toward a Clarification of the Need Hierarchy Theory: Some Extensions of Maslow's Conceptualization," Interpersonal Development 6(2) ( 1975-76): 79. 66 (88) Ibid., p.79. (89) George F. Kawash and Gerhard w. Scherf, "Self- Esteem, Loc us of Control, and Approval Motivation in Married Couples," Journal of Clinical Psychology 31(4) (October 1975):715. (90) Joel Aronoff and John R. Wilson, Personality in the Social Process (New Jersey: Lawrence Erlbaum Associates, 1985) p.22. (91) Ibid., p.23. (92) Charles Brockett, "Toward a Clarification of the Need Hierarchy Theory: Some Extensions of Maslow's Conceptualization," Interpersonal Development 6(2) (1975-76):79. (93) Archie Kaplan, "Maslow Interpreted for the Work Environment," Man-Environment Systems 6(4) (July 1976):247. (105) Charles Brockett, "Toward a Clarification of the Need Hierarchy Theory: Some Extensions of Maslow's Conceptualization," Interpersonal Development 6(2) (1975-76):87. FOOTNOTES (94) John Agnew, "Home Ownership and Identity in Capitalist Societies," in Housing and Identity: Cross-Cultural Perspectives, ed. James s. Duncan (New York: Holmes & Meier Publishers, Inc., 1982)p.76. (95) Ibid., p.76. (95) Ibid., p.76. (96) Ibid., p.76. (96) Ibid., p.76. (97) Amos Rapoport, "Identity and Environment: A Cross- Cultural Perspective," in Housing and Identity: Cross-Cultural Perspectives, ed. James s. Duncan (New York: Holmes & Meier Publishers, Inc., 1982)p.22. (98) Ibid., p.22. (99) Nancy G. Duncan, "Home ownership and Social Theory," in Housing and Identity: Cross-Cultural Perspectives, ed. James s. Duncan (New York: . Holmes & Meier Publishers, Inc., 1982)p.120. (100) Ibid., pp.120-121. (101) Archie Kaplan, "Maslow Interpreted for the Work Environment," Man-Environment Systems 6(4) (July 1976):247. (102) Ibid., p.247. (103) Jeffrey A. Goldman and Paul v. Olczak, "Self- Actualization and the Act of Volunteering: Further 67 Evidence for the Persona l Or i e ntation Inventor y: Further Evide nce for t he Pe r s onal Orientation Inventory," ~ournal of Clinical Psychology 31(2) (October 1975):287. Evidence for the Persona l Or i e ntation Inventor y: Further Evide nce for t he Pe r s onal Orientation Inventory," ~ournal of Clinical Psychology 31(2) (October 1975):287. (104) Ibid., p . 228. (104) Ibid., p . 228. (105) Charles Brockett, "Toward a Clarification of the Need Hierarchy Theory: Some Extensions of Maslow's Conceptualization," Interpersonal Development 6(2) (1975-76):87. 68 68
https://openalex.org/W2082983455	https://link.springer.com/content/pdf/10.1007/BF03353683.pdf	English	null	Nanomaterials for Cardiac Tissue Engineering Application	Nano-micro letters	2,011	cc-by	4,399	1Division of Cardiology, Xinhua Hospital School of Medicine, Shanghai Jiaotong University, Shanghai 200092, China 2Key Laboratory for Thin Film and Microfabrication of Ministry of Education, Research Institute of Micro/Nanometer Science and Technology, Shanghai Jiaotong University, Shanghai 200240, China *Corresponding author. E-mail: zhangyachen65@yahoo.com.cn; wangying@sjtu.edu.cn Nanomaterials for Cardiac Tissue Engineering Application Yachen Zhang1,∗, Yong Tang1, Ying Wang2,∗, Liying Zhang2 Yachen Zhang1,∗, Yong Tang1, Ying Wang2,∗, Liying Zhang2 (Received 10 December 2011; accepted 20 December 2011; published online 30 December 2011.) Abstract: In recent years, the emerging cardiac tissue engineering provides a new therapeutic method for heart diseases. And in the tissue engineering, the scaﬀold material which can mimic the structure of the extracellular matrix properly is a key factor. The rapid expansion of nano-scaﬀolds during the past ten years has led to new perspectives and advances in biomedical research as well as in clinical practice. Here we search articles published in recent years extensively on cardiac tissue engineering scaﬀold materials and nanotechnology. And we review the traditional scaﬀold materials and the advances of the nano-scaﬀolds in cardiac tissue engineering. A thorough understanding of the nano-scaﬀolds would enable us to better exploit technologies to research the ideal scaﬀold material, and promote the cardiac tissue engineering using in the clinical practice as soon as possible. Keywords: Cardiac tissue engineering; Nano-scaﬀolds; Nanomaterials eywords: Cardiac tissue engineering; Nano-scaﬀolds; Nanomaterials Citation: Yachen Zhang, Yong Tang, Ying Wang and Liying Zhang, “Nanomaterials for Cardiac Tissue Engineering Application”, Nano-Micro Lett. 3 (4), 270-277 (2011). http://dx.doi.org/10.3786/nml.v3i4. p270-277 www.nmletters.org Nano-Micro Lett. 3 (4), 270-277 (2011)/ http://dx.doi.org/10.3786/nml.v3i4.p270-277 Nano-Micro Lett. 3 (4), 270-277 (2011)/ http://dx.doi.org/10.3786/nml.v3i4.p270-277 Nano-Micro Lett. 3 (4), 270-277 (2011)/ http://dx.doi.org/10.3786/nml.v3i4.p270-277 Traditional scaﬀold materials of good biocompatibility and cheap price, the biolog- ical material became one of the earliest applications of the scaﬀold materials in cardiac tissue engineer- ing. In the laboratory, with collagen as the basis, researchers have fabricated the three-dimensional my- ocardial model which contains a variety of extracellu- lar matrix proteins and growth factors successfully, and by using of the model, they cultivate regeneration my- ocardial cells which show good diﬀerentiation like the normal myocardial cells in vivo environment [6-8]. In Figure 1 the high degree of integrity and stability of the bioartiﬁcial myocardial tissue patch and cells dis- tribution was observed. As a natural extracellular com- ponent, ﬁbrin has ability to conduct signal between myocardial cells. In addition, diﬀerent concentrations of ﬁbrin can inﬂuence tissue density and mechanical strength, and using this characteristic can make the implantation tissue to get a better spatial distribution [9,10]. Recently, the cardiac tissue engineering scaﬀold materials made of hyaluronic acid and alginate should also be a worthwhile research direction.Since hyaluronic acid and alginate are not widely used in cardiac tissue engineering by now, but the function of promoting pro- liferation has been proved in laboratory [11-13]. The scaﬀold materials which serve as temporary 3D substrates, provide a proper microenvironment for seed- ing cells, and they have been shown to actively regulate cellular responses including attachment, proliferation, diﬀerentiation and matrix deposition [3-5]. The ECM- mimicking microenvironment is the place of getting nu- trition, waste excretion, gas exchange and metabolism for seeding cells. In a regeneration strategy, the scaf- fold materials should be biodegraded gradually in or- der to promote new tissue formation by providing new adequate space. Traditional scaﬀold materials include biological scaﬀold materials and synthetic scaﬀold ma- terials. Introduction The basic strategy of the tissue engineering is the construction of a biocompatible scaﬀold to replace, re- generates or repairs damaged cells or tissues [2]. In car- diac tissue engineering, the ideal scaﬀold should mimic the structure of the extracellular matrix (ECM), which is very important for the proliferation and diﬀerenti- ation of the seeding cells. So, to seek the bionic my- ocardial extracellular matrix material in the myocar- dial tissue engineering for the cultured myocardial cells is a key factor for the translation from the tissue en- gineering into clinical practice. Now the common used scaﬀold materials include traditional scaﬀold material, nanometer scaﬀold material, and composited scaﬀold material. The nano-scaﬀolds have many unique advan- tages in the ﬁld of cardiac tissue engineering. In this paper the research advances of the scaﬀold materials used in cardiac tissue engineering will be repoted for its wide perspective in myocardial cell regeneration. As the world’s ﬁrst major chronic diseasescoronary heart disease causes huge costs per year. The cost of this disease in America was alarming more than $500 billion last year [1]. With the development of med- ical treatment, the demands of the patients who are suﬀering the serious diseases are not only sustaining life, but also improving the quality of life. The tissue- based and cell-based strategies have come to the fore- front as viable alternatives for the treatment of heart disease. One of these novel approaches is cardiac tis- sue engineering. The aim of cardiac tissue engineering is to understand the relationships of myocardial struc- ture and function, design and construct cardiac struc- ture and function, cultivate the new myocardial cells to replace the lost/dysfunctional cells and recover the normal function of the heart. Nano-Micro Lett. 3 (4), 270-277 (2011)/ http://dx.doi.org/10.3786/nml.v3i4.p270-277 Nano-scaﬀolds Nanotechnology is deﬁned by the size of a material (generally 1-100 nm) or manipulation on the molecu- lar level, and the 3 D space of the nano-scaﬀold should be at least one dimensional in nanometer level. The electrospinning process produced polymer ﬁbers in the nanometer range with an approximate diameter of 100 nm (Fig. 3). The rapid expansion of nano-scaﬀolds during the past ten years has led to new perspectives and advances in biomedical research as well as in clin- ical practice. Nano-scaled materials have been widely applied to the ﬁelds of regenerative medicine, includ- ing tissue engineering (TE), cell therapy, diagnosis and drug and gene delivery. Biological scaﬀold materials Biological scaﬀold materials include ﬁbrin, collagen, hyaluronic acid and sodium alginate and so on. These natural polymers retained the normal grid structure, and they have a good biocompatibility, and they are beneﬁcial to cell adhesion, proliferation and diﬀeren- tiation. The biological material is widespread, and the price is relatively cheap. Base on the advantages (a) (b) (c) (d) 10 nm 100 μm 100 nm 1 μm Fig. 1 Celluar seeding of the bioartiﬁcial myocardial tissue patch: (a) Arrangement of cells in 3D collagen structure; (b) Junction between cellular body and collagen ﬁbril, demonstrated by electron microscopy (x8000); (c) Collagen ﬁbrils are embedded in amorphous collagen matrix (x63000); (d) Cells attach along collagen ﬁbrils and are distributed homogenously even in deeper layers of the bioartiﬁcial myocardial tissue patch (MF-20 stain) [4]. (a) 1 μm (a) (d) 100 μm (c) 10 nm (c) Fig. 1 Celluar seeding of the bioartiﬁcial myocardial tissue patch: (a) Arrangement of cells in 3D collagen structure; (b) Junction between cellular body and collagen ﬁbril, demonstrated by electron microscopy (x8000); (c) Collagen ﬁbrils are embedded in amorphous collagen matrix (x63000); (d) Cells attach along collagen ﬁbrils and are distributed homogenously even in deeper layers of the bioartiﬁcial myocardial tissue patch (MF-20 stain) [4]. 271 Nano-Micro Lett. 3 (4), 270-277 (2011)/ http://dx.doi.org/10.3786/nml.v3i4.p270-277 Natural tissue or cell derived ECM, is currently be- ing tried for cardiac tissue engineering [14-16]. The ori- gin of ECM needs to be considered, as diﬀerent tissues exhibit diﬀerent ECM compositions and ultrastructure, which may aﬀect the formation of the desired tissue. So suitable ECM for cardiac regeneration may be the one that originates from heart tissue. It provides the ap- propriate cell guidance and diﬀerentiation signals, but this material is still far from mature [17]. tional cellular interactions and other biological charac- teristics. But the disadvantages of synthetic material can be improved with adhesion peptides or designed to release biological molecules [23,24]. Synthetic material Synthetic materials include polyesterselastomers and so on. The polyesters such as polylactic acid (PLA) and polyglycolic acid (PGA) are common used for my- ocardial tissue engineering. The reasons why they are popular are mostly depended on the good biocompat- ible of their degradation products, their mechanical properties and simple to manufacture. The pore size distribution of the polymer foam is analyzed with the public domain NIH Image program. Figure 2 is an ex- ample of this image analysis procedure, and it provide a better understanding of the meaning of pore size cal- culated by this method [18]. But at the same time, the degradation products may cause inﬂammatory re- sponse [19]. What’s more, the bulk degradation kinetic of the polyesters may lead to a sudden loss of struc- tural integrity, and this is bad for the target tissue. The elastomers are synthetic mimicals of natural rub- ber. A 3D cardiac construct made out of polyurethane shows good cell adhesion, and in-vitro or in-vivo eval- uations on tissue inﬂammation [20-21]. But to our dis- appointed, the polyurethane release a toxic byproduct- diisocyanate [19], and this disadvantage limits its clin- ical use. Nano-scaﬀold used in cardiac tissue engineering The microscopic and submicroscopic structure of the scaﬀold surface has very important inﬂuence in adhe- sion and growth of the myocardial cells [25]. The nano- scaﬀold has a better speciﬁc eﬀect of bulk and sur- face advantages, which is much superior than millime- ter and micrometer scaﬀold materials. The collagen ﬁbers with diameters in the nanometer and submicron range are the major component of the ECM, so fabri- cating the nanoscaled scaﬀold becomes the pursuits of the researchers. While some studies have found that the smallest ﬁbers (near 100 nm) produced by electro- spining are superior [26,27], others have concluded that slightly larger, submicron ﬁbers (near 400 nm) oﬀer the best performance [28]. Although there is a debate about which nanoscaled materials will show better, there is no doubt nano-diameter ﬁber scaﬀolds provide a signif- icant increase in functional surface area compared with Compared with natural materials, synthetic material has its advantages and disadvantages [22]. Synthetic material have a wide range of properties that may be obtained and customized with respect to mechanics, chemistry, and degradation, but may be limited in func- (a) (b) (c) Fig. 2 Thresholding process of image analysis of LDI-glycerol-PEG-AA polymer foam: (a) Initial SEM image; (b) Thresh- olding SEM image; (c) Labeled pores of an SEM image of LDI-glycerol-PEG-AA polymer foam [24]. (a) (a) (b) (c) Fig. 2 Thresholding process of image analysis of LDI-glycerol-PEG-AA polymer foam: (a) Initial SEM image; (b) Thresh- olding SEM image; (c) Labeled pores of an SEM image of LDI-glycerol-PEG-AA polymer foam [24]. 272 Nano-Micro Lett. 3 (4), 270-277 (2011)/ http://dx.doi.org/10.3786/nml.v3i4.p270-277 Nano-Micro Lett. 3 (4), 270-277 (2011)/ http://dx.doi.org/10.3786/nml.v3i4.p270-2 (a) (b) Fig. 3 Scanning electron microscope images of poly(DL-lactide-co-glycolide) nanoﬁbers at 5000x magniﬁcation (a) and 30 000x magniﬁcation (b). The polymer ﬁber diameter was slightly variable with a mean diameter of approximately 100 nm [29]. (a) (b) (b) Fig. 3 Scanning electron microscope images of poly(DL-lactide-co-glycolide) nanoﬁbers at 5000x magniﬁcation (a) and 30 000x magniﬁcation (b). The polymer ﬁber diameter was slightly variable with a mean diameter of approximately 100 nm [29]. (a) (b) 50μm 1μm Fig. 4 Scanning electron micrographs of a PLLA nanoﬁbrous scaﬀold prepared from 2.5% PLLA/THF solution at a phase separation temperature of 8 ℃: (a) 500% (b) 20 000% [31]. (b) Fig. Nano-scaﬀold used in cardiac tissue engineering 4 Scanning electron micrographs of a PLLA nanoﬁbrous scaﬀold prepared from 2.5% PLLA/THF solution at a phase separation temperature of 8 ℃: (a) 500% (b) 20 000% [31]. fabricating nanoﬁbrous biomaterials, including phase separation, self-assembly, electrospinning and so on. Phase separation techniques have been used to pre- pare porous polymer membranes for puriﬁcation and separation purposes. In laboratory, researchers have generated nanoﬁbrous structures by manipulating the phase separation process (Fig. 4) [31-33]. But the out- comes of the phase-separation technique is not perfect, the generated nanoﬁbrous materials are lack of inter- connected macropores, which are critical for cell seed- ing and recruiting, mass transfer, vascularization and tissue organization [34]. So the phase-separation tech- nique needs to be improved to overcome this defect. Self-assembly is a kind of technology to organize indi- vidual molecules into a well-deﬁned and stable hierar- chical structure with preprogrammed non-covalent in- teractions [35-40]. Self-assembly have its unique advan- tages. For example, the molecules concerned interact at conventional materials with no roughness at the nanoscale. So the nano-scaﬀold is much more better for cell adhesion because of greater surface. In contrast with traditional scaﬀold materials, the 3-D nanoﬁbrous scaﬀolds provide a superior microen- vironment for promoting cell functions. Since nanoﬁ- brous scaﬀolds have nanometer pore sizes, cells are unable to penetrate by themselves, so the seeding cells must be incorporated into the scaﬀold during fabrica- tion to ensure proper cell distribution [29]. We can use this characteristic to guide cell distribution in order to make the regenerated tissues be more closer to normal cardiac tissue structure [30]. Technologies for generating nanoﬁbrous bioma- terials Technologies for generating nanoﬁbrous bioma- terials Technologies for generating nanoﬁbrous bioma- terials At present there is several major technologies for 273 Nano-Micro Lett. 3 (4), 270-277 (2011)/ http://dx.doi.org/10.3786/nml.v3i4.p270-277 spun poly(caprolactone) (PCL) nanoﬁber matrix and native ECM in rat cornea (Fig. 6). With the advances of the technology, it is hopeful to generate nanoscaled scaﬀolds by using electrospinning in the future [44,45]. an atomic level driven by physical or chemical aﬃnity self-assembly can increase the sensitivity and speciﬁcity. And because the atomic level interactions between com- ponents are retained throughout the expansion process, so we can retain nanoscale properties even in bulk ma- terials (Fig. 5) [41]. But just as the phase separation, self-assembly also has some limitations, such as self- assembled nanoﬁbrous scaﬀolds are limited to biological molecules in the form of hydrogels and the degradation of the scaﬀolds have not been systematically addressed. In comparison with phase separation and self-assembly, the technology of electrospinning is simple, economical and it can generate porous scaﬀolds with submicron di- ameter [42-43]. What’s more, the electrospinning can be used in both synthetic and natural biology material scaﬀolds. Electrospinning is a well-established process that has been used to produce ultraﬁne ﬁbers and it has been popular in the ﬁeld of tissue engineering. There is studies demonstrated a vivid similarity between electro- Fig. 6 Similarity between native ECM protein structure and electrospun polymeric nanoﬁber matrix: (a) Fibroblasts cultured on collagen ﬁbrils of rat cornea [42]; (b) Endothelial cells cultured on electrospun PCL nanoﬁber matrix [43]. Nanocomposites in cardiac tissue engineering Recently, the function of nanocomposites in cardiac tissue engineering causes a hot discussion among the re- searchers. The key limitation of porous matrix used for cardiac tissue engineering is that their pore walls limit the interaction of cells, and delay electrical signal prop- agation [46]. The 3D nanocomposites of gold nanowires within macroporous alginate scaﬀolds have been devel- oped to bridge the non-conducting pore walls, and this can increase electrical signal propagation throughout the cell-seeded scaﬀold, and enhance the organization of functioning tissue [47]. The functional mechanism of the nanowires is unclearly now. Nanowires may create (a) (a) (a) (b) (a) (b) (b (b) (b) b) b (b) (b) (b) (b) (b) (b) (b) (b (b) b (b) (b) b) b (b) b) b) b) b ( ) ( ) (b) (b) b) Fig. 5 Examples of static self-assembly: (a) An array of millimeter-sized polymeric plates assembled at a wa- ter/perﬂuorodecalin interface by capillary interactions [39]. (b) A 3D aggregate of micrometer plates assemble by capillary forces [40]. (b) (b) (b (b) (b) b) b (b) (b) (b) (b) (b) (b) (b) (b (b) b (b) (b) b) b (b) b) b) b) b ( ) ( ) (b) (b) b) (b) (b) (b (b) (b) b) b (b) (b) (b) (b) (b) (b) (b) (b (b) b (b) (b) b) b (b) b) b) b) b ( ) ( ) (b) (b) b) (a) (a) Fig. 5 Examples of static self-assembly: (a) An array of millimeter-sized polymeric plates assembled at a wa- ter/perﬂuorodecalin interface by capillary interactions [39]. (b) A 3D aggregate of micrometer plates assemble by capillary forces [40]. 10 μm (a) (a) (a) (a) (a) (a) (a) (a) (a (a) (a) (a) (a) (a (a) (a) (a) (a) (a) (a (a) (a) 10 μm (a) (a) (a) (a) (a) (a) (a) (a) (a (a) (a) (a) (a) (a (a) (a) (a) (a) (a) (a (a) (a) (b) 50 15 kV ×500 0000 (b) (b) (b) (b) (b) b) b) b) b) b) 50 μm 15 kV ×500 0000 50 μm 15 kV 0000 ×500 10 μm 274 Nano-Micro Lett. 3 (4), 270-277 (2011)/ http://dx.doi.org/10.3786/nml.v3i4.p270-277 the disadvantage of the limited interaction between cells. So fabricating novel conductive, biodegradable composites became the purpose of our study. And many articles have reported that the nanocomposites can be a promising application in cardiac tissue engi- neering. Nanocomposites in cardiac tissue engineering Carbon nanoﬁber composites (CNM), which are conductive, have the ability to transform non- conductive polymers to conductive and they can mimic natural proteins like collagen [52,53]. And CNF pos- sess nanoscale geometries which imitate the extracellu- lar matrix of heart tissue, can improve cytocompatibil- ity of pure PLGA [54]. conductive bridges across the scaﬀold materials, con- necting adjacent pores and/or cell bundles. Alterna- tively, the nanowires may enhance the expression of the electrical coupling protein connexin43. Cx-43 has been shown to regulate cell-cell communication, inﬂu- ence electrical coupling, and promote contractile behav- ior [48,49]. And Cx-43 can be upregulated under stim- ulated conditions, so the nanowires can be a promising candidate for cardiac tissue engineering. Although poly(lactic-co-glycolic acid) (PLGA), one of the polymer, has desirable biodegradability and bio- compatibility properties [50,51], also fails to overcome A B Fig. 7 Connexin 43 gap junction protein was found between cardiomyocytes in the nanowire-containing scaﬀolds. Nuclei are coloured in blue, B is ampliﬁcation of A [47]. B B Fig. 7 Connexin 43 gap junction protein was found between cardiomyocytes in the nanowire-containing scaﬀolds. Nuclei are coloured in blue, B is ampliﬁcation of A [47]. Conclusion nethon, S. Dai, G. De Simone, T. B. Ferguson, E. Ford, K. Furie, C. Gillespie, A. Go, K. Greenlund, N. Haase, S. Hailpern, P. M. Ho, V. Howard, B. Kissela, S. Kittner, D. Lackland, L. Lisabeth, A. Marelli, M. M. McDermott, J. Meigs, D. Mozaﬀarian, M. Mus- solino, G. Nichol, V. L. Roger, W. Rosamond, R. Sacco, P. Sorlie, R. Staﬀord, T. Thom, S. Wasserthiel- Smoller, N. D. Wong and J. Wylie-Rosett, Circula- tion 121, 948 (2010). http://dx.doi.org/10.1161/ CIRCULATIONAHA.109.192666 nethon, S. Dai, G. De Simone, T. B. Ferguson, E. Ford, K. Furie, C. Gillespie, A. Go, K. Greenlund, N. Haase, S. Hailpern, P. M. Ho, V. Howard, B. Kissela, S. Kittner, D. Lackland, L. Lisabeth, A. Marelli, M. M. McDermott, J. Meigs, D. Mozaﬀarian, M. Mus- solino, G. Nichol, V. L. Roger, W. Rosamond, R. Sacco, P. Sorlie, R. Staﬀord, T. Thom, S. Wasserthiel- Smoller, N. D. Wong and J. Wylie-Rosett, Circula- tion 121, 948 (2010). http://dx.doi.org/10.1161/ CIRCULATIONAHA.109.192666 Although in the experimental stage, the cardiac tis- sue engineering has been made great progress, the real clinical application still has a long time to go. 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https://openalex.org/W2107918320	https://repository.ubn.ru.nl//bitstream/handle/2066/138121/138121.pdf	English	null	Glucose-6-Phosphate Dehydrogenase Status and Risk of Hemolysis in Plasmodium falciparum-Infected African Children Receiving Single-Dose Primaquine	Antimicrobial agents and chemotherapy	2,014	cc-by	2,966	Version of the following full text: Publisher’s version Downloaded from: http://hdl.handle.net/2066/138121 Download date: 2024-10-24 Version of the following full text: Publisher’s version Downloaded from: http://hdl.handle.net/2066/138121 Download date: 2024-10-24 Glucose-6-phosphate dehydrogenase status and risk of hemolysis in Plasmodium falciparum- infected African children receiving single-dose primaquine Eziefula, A.C.; Pett, H. van; Grignard, L.; Opus, S.; Kiggundu, M.; Kamya, M.R.; Yeung, S.; Staedke, S.G.; Bousema, T.; Drakeley, C. 2014, Article / Letter to editor (Antimicrobial Agents and Chemotherapy, 58, 8, (2014), pp. 4971-3) Doi link to publisher: https://doi.org/10.1128/AAC.02889-14 Glucose-6-Phosphate Dehydrogenase Status and Risk of Hemolysis in Plasmodium falciparum-Infected African Children Receiving Single- Dose Primaquine Alice C. Eziefula,a Helmi Pett,b Lynn Grignard,a Salome Opus,c Moses Kiggundu,c Moses R. Kamya,c,d Shunmay Yeung,a Sarah G. Staedke,a Teun Bousema,a,b Chris Drakeleya Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdoma; Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, The Netherlandsb; Infectious Diseases Research Collaboration, Kampala, Ugandac; Department of Medicine, Makerere University College of Health Sciences, Kampala, Ugandad Alice C. Eziefula,a Helmi Pett,b Lynn Grignard,a Salome Opus,c Moses Kiggundu,c Moses R. Kamya,c,d Shunmay Yeung,a Sarah G. Staedke,a Teun Bousema,a,b Chris Drakeleya Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdoma; Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, The Netherlandsb; Infectious Diseases Research Collaboration, Kampala, Ugandac; Department of Medicine, Makerere University College of Health Sciences, Kampala, Ugandad on May 1, 2017 by UNIV http://aac.asm.org/ Downloaded from Sarah G. Staedke, Teun Bousema, Chris Drakeley Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdoma; Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, The Netherlandsb; Infectious Diseases Research Collaboration, Kampala, Ugandac; Department of Medicine, Makerere University College of Health Sciences Kampala Ugandad on May 1, 2017 by UNIVERSITEITSBIBLIOTHEEK http://aac.asm.org/ Downloaded from Glucose-6-phosphate dehydrogenase (G6PD) enzyme function and genotype were determined in Ugandan children with uncom- plicated falciparum malaria enrolled in a primaquine trial after exclusion of severe G6PD deﬁciency by ﬂuorescent spot test. G6PD A heterozygotes and hemizygotes/homozygotes experienced dose-dependent lower hemoglobin concentrations after treatment. No severe anemia was observed. D eclines in malaria due to Plasmodium falciparum have been documented in a number of settings where malaria is en- demic. It is debated whether scaling-up of conventional malaria control will sustain these declines or achieve elimination unless augmented by tools that speciﬁcally reduce transmission. Pri- maquine is the only currently available drug that actively clears mature P. falciparum gametocytes and prevents malaria transmis- sion to mosquitoes (1). The wide-scale use of primaquine is ham- pered by its hemolytic effect in people with glucose-6-phosphate dehydrogenase (G6PD) deﬁciency. The mutation deﬁciency alters G6PD enzyme function (2), exposing red blood cells to oxidative stress and resultant hemolysis in the presence of a stressor, such as primaquine (3, 4). Primaquine-induced hemolysis is dose related (1, 5, 6). While testing for G6PD deﬁciency is widely recom- mended prior to the radical treatment of Plasmodium vivax with 14 days of primaquine, P. Glucose-6-Phosphate Dehydrogenase Status and Risk of Hemolysis in Plasmodium falciparum-Infected African Children Receiving Single- Dose Primaquine falciparum transmission may be consid- erably reduced by a single, low dose of primaquine (1, 7) and may avoid the necessity to screen for G6PD deﬁciency. We determined G6PD enzyme function and the presence of the most common African G6PD mutation (G6PD A; 202A/376G) in a cohort of Ugandan children treated with low-dose primaquine for clearing P. falciparum gametocytes. This was a randomized, double- blinded placebo controlled trial with four parallel arms. Ugandan children 1 to 10 years old with uncomplicated P. falciparum ma- laria, hemoglobin concentration (Hb) of 8 g/dl, and normal G6PD enzyme function based on a ﬂuorescent spot test (FST; R&D Diagnostics, Agia Paraskevi, Greece) were enrolled and ran- domized to treatment with artemether lumefantrine (AL) alone or with a single dose of primaquine at 0.1, 0.4, or 0.75 mg/kg of body weight on the last day of AL treatment (7, 8). Genotyping of G6PD 202A and G6PD 376G was performed (9, 10). Hb was measured on days 0, 1, 2, 3, 7, 10, 14, 21, and 28 after enrollment by HemoCue 201 (Angelholm, Sweden) and expressed as absolute and relative change compared to baseline values. These values were normally distributed, presented using mean values and stan- dard deviations, and analyzed using linear regression models. Be- cause the age distribution of the red blood cell population inﬂu- ences the severity of drug-induced hemolysis (11), we adjusted all G6PD enzyme function based on a ﬂuorescent spot test (FST; R&D Diagnostics, Agia Paraskevi, Greece) were enrolled and ran- domized to treatment with artemether lumefantrine (AL) alone or with a single dose of primaquine at 0.1, 0.4, or 0.75 mg/kg of body weight on the last day of AL treatment (7, 8). Genotyping of G6PD 202A and G6PD 376G was performed (9, 10). Hb was measured on days 0, 1, 2, 3, 7, 10, 14, 21, and 28 after enrollment by HemoCue 201 (Angelholm, Sweden) and expressed as absolute and relative change compared to baseline values. These values were normally distributed, presented using mean values and stan- dard deviations, and analyzed using linear regression models. 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). Glucose-6-Phosphate Dehydrogenase Status and Risk of Hemolysis in Plasmodium falciparum-Infected African Children Receiving Single- Dose Primaquine Be- cause the age distribution of the red blood cell population inﬂu- ences the severity of drug-induced hemolysis (11), we adjusted all D on May 1, 2017 by UNIVERSITEITSBIBLIOTHEEK sm.org/ y 1, 2017 by UNIVERSITEITSBIBLIOTHEEK Received 27 March 2014 Returned for modiﬁcation 30 April 2014 Accepted 3 June 2014 Published ahead of print 9 June 2014 Address correspondence to Teun Bousema. teun.bousema@lshtm.ac.uk. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.02889-14. Copyright © 2014, Eziefula et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license. doi:10.1128/AAC.02889-14 TABLE 1 Baseline characteristics Characteristic Value by G6PD 202 A genotype Wild type Heterozygous P value for difference from wild type Homozygous/hemizygous P value for difference from wild type No. of participants (% study population) 373 (80.9) 61 (13.2) 27 (5.9) % female (no. of females/total no. of participants) 46.7 (174/373) 100.0 (61/61) 0.001 3.7 (1/27) 0.001 Mean (SD) age in yrs 5.0 (2.6) 4.8 (2.3) 0.61 4.9 (2.4) 0.86 Mean (SD) baseline Hb concn in g/dl 11.2 (1.5) 11.4 (1.4) 0.20 10.9 (1.4) 0.38 % 376G genotype (no. of participants with genotype/total no.) Heterozygous 18.6 (69/371) 78.7 (48/61) 0.0 (0/27) Homozygous 12.9 (48/371) 21.3 (13/61) 0.001 100.0 (27/27) 0.001 August 2014 Volume 58 Number 8 Antimicrobial Agents and Chemotherapy p. 4971–4973 aac.asm.org 4971 Eziefula et al. TABLE 2 G6PD 202 A genotype and hemoglobin levelsa TABLE 2 G6PD 202 A genotype and hemoglobin levelsa TABLE 2 G6PD 202 A genotype and hemoglobin levels Characteristic Value by treatment arm 0.75 mg/kg primaquine 0.4 mg/kg primaquine 0.1 mg/kg primaquine Placebo No. on May 1, 2017 by UNIVERSITEITSBIBLIOTHEEK http://aac.asm.org/ Downloaded from on May 1, 2017 by UNIVERSITEITSBIBLIOTHEEK http://aac.asm.org/ Downloaded from on May 1, 2017 by UNIVERSITEITSBIBLIOTHEE http://aac.asm.org/ ed from a On day 7 after initiation of treatment with artemether-lumefantrine (AL) plus placebo or AL plus different doses of primaquine. All primaquine or placebo treatment was administered together with six doses of AL; primaquine/placebo was given on day 2 of treatment, together with dose 5 of AL. P values are compared to G6PD-normal individuals, adjusted for baseline Hb concentration. ith artemether-lumefantrine (AL) plus placebo or AL plus different doses of primaquine. All primaquine or placebo treatment was L; primaquine/placebo was given on day 2 of treatment, together with dose 5 of AL. P values are compared to G6PD-normal individuals, on May 1, 2017 by UNIVERSITEITSBIBLIOTHEEK m.org/ gous individuals (P 0.020). For all individuals, Hb concentra- tions normalized during follow-up. The current ﬁndings provide important data on the hemolytic effect of single-, low-dose prim- aquine. Our results show that the predominant test for G6PD deﬁciency screening, the FST (13), failed to identify a substantial proportion of individuals who were genotypically G6PD deﬁcient, particularly female heterozygotes, who experienced signiﬁcant re- ductions in hemoglobin following higher doses of primaquine. The observation that some G6PD-deﬁcient individuals were FST normal is unsurprising since the test may be insufﬁciently sensi- tive to detect mild G6PD deﬁciency (13), but there are few supportive published data. We observed statistically signiﬁcant decreases in Hb following single-dose primaquine in these G6PD- deﬁcient individuals. A hemolytic effect of a single dose of 0.75 mg/kg primaquine base has been reported before (6); our study shows that a reduction in Hb concentrations is also evident after a single dose of 0.4 mg/kg but not 0.1 mg/kg. Moreover, reductions in Hb were transient, with no participant experiencing clinical symptoms suggestive of anemia and none requiring related clini- cal care. Although these ﬁndings are notable, a major limitation of the study is that individuals who were determined G6PD deﬁcient based on the FST were excluded from the study (n 32), thereby plausibly removing those most severely deﬁcient and thereby those with the highest risk of primaquine-induced hemolysis. There is therefore a need for conﬁrmatory trials to formally assess primaquine safety in G6PD-deﬁcient individuals, in particular with the World Health Organization recommended dose of 0.25 mg/kg. Such studies will have to take into account interindividual differences in primaquine metabolism that determine primaquine efﬁcacy in P. on May 1, 2017 by UNIVERSITEITSBIBLIOTHEEK http://aac.asm.org/ Downloaded from vivax (14) and potentially also safety. comparisons for baseline Hb concentration. All trial participants (n 468) were G6PD normal by FST. DNA was available for 461 individuals of whom 27 (5.9%) were homozygous/hemizygous, 61 were heterozygous (13.2%), and 373 (80.9%) were normal for the G6PD variant A (wild type [WT]). All individuals with the 202A mutation also had the 376G mutation, and individuals were classiﬁed based on the 202A mutation (Table 1). G6PD 202 A heterozygous individuals experienced a mean reduction in Hb concentration on day 7 after treatment of 1.08 g/dl (standard de- viation [SD], 1.14; P 0.048) in the 0.75-mg/kg treatment arm and 0.99 g/dl (SD, 1.48; P 0.054) in the 0.4-mg/kg treatment arm (Table 2). Homozygous/hemizygous individuals in the 0.75- mg/kg and 0.4-mg/kg arms also experienced a reduction in abso- lute Hb concentration on day 7, although this was statistically signiﬁcant in the 0.4-mg/kg arm only (P 0.043). When changes in Hb concentration on day 7 were expressed as a proportion of baseline Hb concentration, the same trend was observed with sta- tistically signiﬁcant decreases in the 0.75-mg/kg arm for heterozy- gous individuals and in the 0.4-mg/kg arm for homozygous/hem- izygous individuals. No statistically signiﬁcant changes in absolute or relative Hb concentrations were observed for heterozygous or homozygous/hemizygous individuals in the 0.1-mg/kg arm or placebo arm (Table 2). We found no explanation for the numer- ically large, but statistically nonsigniﬁcant, reduction in Hb con- centration in homozygous/hemizygous individuals on day 7 after receiving AL without primaquine. A previous study found no he- molysis after AL in homozygous/hemizygous individuals (12), and we conclude our observation may be a spurious ﬁnding and related to our small sample size. We observed no statistically sig- niﬁcant associations between G6PD genotype and absolute or rel- ative Hb concentrations in any treatment arm on days 3 and 10 after initiation of treatment (see the supplemental material). Six- ty-nine individuals experienced a reduction of 2 g/dl in the ﬁrst 2 weeks of follow-up: 13.7% (51/373) of the WT individuals, 26.2% (16/61; P 0.031) of the heterozygous individuals, and 7.4% (2/27; P 0.48) of the G6PD 202 A homozygous/hemizy- y 1, 2017 by UNIVERSITEITSBIBLIOTHEEK Antimicrobial Agents and Chemotherapy Glucose-6-Phosphate Dehydrogenase Status and Risk of Hemolysis in Plasmodium falciparum-Infected African Children Receiving Single- Dose Primaquine of study participants G6PD normal 98 90 93 92 G6PD heterozygous 14 13 16 18 G6PD hemizygous/homozygous 4 10 6 7 Mean absolute change (SD) in Hb on day 7 G6PD normal, in g/dl 0.41 (0.95) 0.25 (1.22) 0.30 (1.07) 0.11 (1.33) G6PD heterozygous, in g/dl 1.08 (1.14) 0.99 (1.48) 0.07 (0.98) 0.49 (1.40) P value 0.048 0.054 0.35 0.28 G6PD hemizygous/homozygous, in g/liter 1.10 (1.34) 0.48 (0.76) 0.07 (1.21) 1.02 (0.81) P value 0.21 0.043 0.91 0.22 % relative change (SD) in Hb on day 7, in g/dl G6PD normal 3.25 (8.60) 1.28 (11.24) 2.16 (9.71) 0.23 (11.34) G6PD heterozygous 9.38 (10.4) 7.79 (12.57) 0.01 (8.56) 3.26 (12.26) P value 0.044 0.073 0.34 0.33 G6PD hemizygous/homozygous 7.97 (12.40) 4.29 (7.70) 0.05 (11.45) 8.59 (7.28) P value 0.36 0.028 0.93 0.16 a On day 7 after initiation of treatment with artemether-lumefantrine (AL) plus placebo or AL plus different doses of primaquine. All primaquine or placebo treatment was administered together with six doses of AL; primaquine/placebo was given on day 2 of treatment, together with dose 5 of AL. P values are compared to G6PD-normal individuals, adjusted for baseline Hb concentration. REFERENCES 1. White NJ. 2013. Primaquine to prevent transmission of falciparum ma- laria. Lancet Infect. Dis. 13:175–181. http://dx.doi.org/10.1016/S1473 -3099(12)70198-6. 2. Alving AS, Carson PE, Flanagan CL, Ickes CE. 1956. Enzymatic deﬁ- ciency in primaquine-sensitive erythrocytes. Science 124:484–485. http: //dx.doi.org/10.1126/science.124.3220.484. 10. Hirono A, Beutler E. 1988. Molecular cloning and nucleotide sequence of cDNA for human glucose-6-phosphate dehydrogenase variant A(-). Proc. Natl. Acad. Sci. U. S. A. 85:3951–3954. http://dx.doi.org/10.1073/pnas.85 .11.3951. on May 1, 2017 by UNIVERSITEIT http://aac.asm.org/ Downloaded from on May 1, 2017 by UNIVERSITEITSBIBLIOTHEEK http://aac.asm.org/ Downloaded from g 3. Beutler E, Yeh M, Fairbanks VF. 1962. The normal human female as a mosaic of X-chromosome activity: studies using the gene for C-6-PD- deﬁciency as a marker. Proc. Natl. Acad. Sci. U. S. A. 48:9–16. http://dx .doi.org/10.1073/pnas.48.1.9. 11. Alving AS, Johnson CF, Tarlov AR, Brewer GJ, Kellermeyer RW, Carson PE. 1960. Mitigation of the haemolytic effect of primaquine and enhancement of its action against exoerythrocytic forms of the Chesson strain of Plasmodium vivax by intermittent regimens of drug administra- tion: a preliminary report. Bull. World Health Organ. 22:621–631. 4. Beutler E. 1959. The hemolytic effect of primaquine and related com- pounds: a review. Blood 14:103–139. on May 1, 2017 by UNIVERSITEITSBIBLIOTHE http://aac.asm.org/ d from 5. Alving AS, Johnson CF, Tarlov AR, Brewer GJ, Kellermeyer RW, Carson PE. 1960. Mitigation of the haemolytic effect of primaquine and enhancement of its action against exoerythrocytic forms of the Chesson strain of Plasmodium vivax by intermittent regimens of drug administra- tion: a preliminary report. Bull. World Health Organ. 22:621–631. p y p g 12. Premji Z, Umeh RE, Owusu-Agyei S, Esamai F, Ezedinachi EU, Oguche S, Borrmann S, Sowunmi A, Duparc S, Kirby PL, Pamba A, Kellam L, Guiguemdé R, Greenwood B, Ward SA, Winstanley PA. 2009. Chlor- proguanil-dapsone-artesunate versus artemether-lumefantrine: a ran- domized, double-blind phase III trial in African children and adolescents with uncomplicated Plasmodium falciparum malaria. PLoS One 4:e6682. http://dx.doi.org/10.1371/journal.pone.0006682. 6. Shekalaghe SA, ter Braak R, Daou M, Kavishe R, van den Bijllaardt W, van den Bosch S, Koenderink JB, Luty AJ, Whitty CJ, Drakeley C, Sauerwein RW, Bousema T. 2010. In Tanzania, hemolysis after a single dose of primaquine coadministered with an artemisinin is not restricted to glucose-6-phosphate dehydrogenase-deﬁcient (G6PD A) individuals. Antimicrob. Agents Chemother. 54:1762–1768. http://dx.doi.org/10.1128 /AAC.01135-09. 13. ACKNOWLEDGMENTS This study was funded primarily by a Wellcome Trust Bloomsbury Clin- ical Fellowship to A.C.E. (090558), supplemented by funds from the Bill & Melinda Gates Foundation to C.D. and T.B. (OPP1034789). We thank the parents and guardians and study participants for their We thank the parents and guardians and study participants for their 4972 Antimicrobial Agents and Chemotherapy 4972 aac.asm.org aac.asm.org G6PD Status and Single-Dose Primaquine Bousema T, Drakeley C. 2013. Study protocol for a randomised con- trolled double-blinded trial of the dose-dependent efﬁcacy and safety of primaquine for clearance of gametocytes in children with uncomplicated falciparum malaria in Uganda. BMJ Open 3(3):piie002759. http://dx .doi.org/10.1136/bmjopen-2013-002759. Bousema T, Drakeley C. 2013. Study protocol for a randomised con- trolled double-blinded trial of the dose-dependent efﬁcacy and safety of primaquine for clearance of gametocytes in children with uncomplicated falciparum malaria in Uganda. BMJ Open 3(3):piie002759. http://dx .doi.org/10.1136/bmjopen-2013-002759. patience and commitment and all members of the study team (notably Asiphas Owaraganise, Grace Gabagaya, Salome Opus, Samuel Okiror, Hamuza Isabirye, Josephine Zawedde, Abubaker Kawuba, Christobel Achen). 9. Fanello CI, Karema C, Avellino P, Bancone G, Uwimana A, Lee SJ, d’Alessandro U, Modiano D. 2008. High risk of severe anaemia after chlorproguanil-dapsoneartesunate antimalarial treatment in patients with G6PD (A-) deﬁciency. PLoS One 3:e4031. http://dx.doi.org/10.1371 /journal.pone.0004031. August 2014 Volume 58 Number 8 REFERENCES Domingo GJ, Satyagraha AW, Anvikar A, Baird K, Bancone G, Bansil P, Carter N, Cheng Q, Culpepper J, Eziefula C, Fukuda M, Green J, Hwang J, Lacerda M, McGray S, Menard D, Nosten F, Nuchprayoon I, Oo NN, Bualombai P, Pumpradit W, Qian K, Recht J, Roca A, Satimai W, Sovannaroth S, Vestergaard LS, Von Seidlein L. 2013. G6PD testing in support of treatment and elimination of malaria: recommendations for evaluation of G6PD tests. Malar. J. 12:391. http://dx.doi.org/10.1186/1475 -2875-12-391. on May 1, 2017 by UNIVERSITEITSBIBLIOTHEEK m.org/ 7. Eziefula AC, Bousema T, Yeung S, Kamya M, Owaraganise A, Gabagaya G, Bradley J, Grignard L, Lanke KH, Wanzira H, Mpimbaza A, Nsobya S, White NJ, Webb EL, Staedke SG, Drakeley C. 2014. Single dose primaquine for clearance of Plasmodium falciparum gametocytes in chil- dren with uncomplicated malaria in Uganda: a randomised, controlled, double-blind, dose-ranging trial. Lancet Infect. Dis. 14:130–139. http://dx .doi.org/10.1016/S1473-3099(13)70268-8. 14. Bennett JW, Pybus BS, Yadava A, Tosh D, Sousa JC, McCarthy WF, Deye G, Melendez V, Ockenhouse CF. 2013. Primaquine failure and cytochrome P-450 2D6 in Plasmodium vivax malaria. N. Engl. J. Med. 369:1381–1382. http://dx.doi.org/10.1056/NEJMc1301936. y 1, 2017 by UNIVERSITEITSBIBLIOTHEEK 8. Eziefula AC, Staedke SG, Yeung S, Webb E, Kamya M, White NJ, August 2014 Volume 58 Number 8 aac.asm.org 4973
https://openalex.org/W2752900162	https://www.pure.ed.ac.uk/ws/files/45000133/20171012_Garden_Manuscript.pdf	English	null	Exploiting multimetallic catalysts to access polymer materials from CO<sub>2</sub>	Green materials	2,017	cc-by	4,399	General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Edinburgh Research Explorer Short Communication (Perspective) Keywords: improved catalysts, carbon dioxide, green polymers, polymer materials ice \| science ICE Publishing: All rights reserved Short Communication (Perspective) Keywords: improved catalysts, carbon dioxide, green polymers, polymer materials ICE Publishing: All rights reserved Short Communication (Perspective) ice \| science ICE Publishing: All rights reserved Exploiting Multimetallic Catalysts to Access Polymer Materials from CO2 Jennifer A. Garden* Christina Miller Research Fellow, University of Edinburgh, Edinburgh, UK Jennifer A. Garden* Christina Miller Researc Polycarbonates and polyurethanes derived from CO2 have the potential to consume a greenhouse gas and convert it into value added polymer products. To be commercially viable, these processes are dependent on the development of highly efficient polymerization catalysts, which are often multimetallic. One emerging method to improve catalyst efficiency is through the design of co-operative, mixed-metal catalysts. Such heterometallic, homogenous catalysts have shown significantly improved performance metrics for CO2/epoxide ring-opening copolymerization, in comparison to their homometallic analogues. This perspective highlights the recently realized reactivity enhancements achieved using multimetallic catalysts, along with the material properties and scope of CO2-derived polymers. Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 Short Communication (Perspective) Keywords: improved catalysts, carbon dioxide, green polymers, polymer materials \| science ICE Publishing: All rights reserved Catalyst values of 624 h-1 achieved at 120 °C and 50 bar of CO2 pressure. Heterometallic 4 showed significantly improved performance metrics for CO2/ cyclohexene oxide (CHO) ROCOP, in comparison to the homometallic analogues, either alone or in combination. Notably, 4 is active at atmospheric pressure, and the productivities (turnover numbers, TON = 344) and activities (turnover frequencies, TOF = 34 h-1) are five times higher than a 1:1 mixture of the homometallic di-Mg and di-Zn analogues. values of 624 h-1 achieved at 120 °C and 50 bar of CO2 pressure. Heterometallic 4 showed significantly improved performance metrics for CO2/ cyclohexene oxide (CHO) ROCOP, in comparison to the homometallic analogues, either alone or in combination. Notably, 4 is active at atmospheric pressure, and the productivities (turnover numbers, TON = 344) and activities (turnover frequencies, TOF = 34 h-1) are five times higher than a 1:1 mixture of the homometallic di-Mg and di-Zn analogues. The presence of a catalyst underpins the successful synthesis of polycarbonates from CO2/epoxide ROCOP, where some of the most active are homogeneous homometallic metal complexes based on salen,22 porphyrin,23 β-diiminate24 or di-phenolate25 ligand scaffolds.26-29 Careful homogeneous catalyst design has led to improved activities and selectivities, along with high CO2 uptake (>99%) to form nearly perfect polycarbonate linkages. A breakthrough in the mechanistic understanding came from the realization that epoxide opening and enchainment could occur through a bimetallic mechanism, as discerned using dimeric β- diiminate zinc acetate catalysts bearing two zinc centres in close proximity.30 This finding inspired the development of multimetallic catalysts comprising two metals in a dinucleating ligand scaffold,31-34 which have shown exceptional activities24, 35 and some of which are active at just 1 bar of CO2 pressure.36-39 However, until recently, the development of homogeneous heterometallic catalysts has been neglected, despite reports that ternary mixed-metal systems display cooperative heterometallic effects, and have demonstrated good activities for CO2/epoxide ROCOP.40-48 A major contributor to this lack of investigation is the synthetic challenge associated with the synthesis and characterization of heterometallic catalysts, which often includes the synthesis, purification and manipulation of highly air- and moisture-sensitive starting materials. Scheme 2. “Chain shuttling” mechanism proposed for CO2/epoxide copolymerization by a multimetallic catalyst.49,50 Scheme 2. “Chain shuttling” mechanism proposed for CO2/epoxide copolymerization by a multimetallic catalyst.49,50 The concept of heterometallic reactivity enhancement has since been extended to homogeneous rare-earth metal complexes. Catalyst Rare- earth metal catalysts are attractive for CO2/epoxide ROCOP, as the oxyphilic and Lewis acidic nature of rare-earth metals can facilitate epoxide activation and CO2 insertion. Yao et al. recently reported the synthesis of two novel heterometallic catalysts for CO2/CHO ROCOP, based on Nd2Zn and Y2Zn complexes supported by di-phenolate ligands.51 These novel complexes display high catalyst activities with TOF values up to 267 h-1 at 70 °C and 0.7 MPa. Furthermore, these systems give impressive, albeit lowered, catalyst activities at ambient temperature (TOF = 82 h-1, 25 °C, 7 bar of CO2 pressure). These heterometallic catalysts were successfully applied to synthesize high molecular weight poly(cyclohexene carbonate) (Mn = 296 kg mol-1). In contrast, the homometallic analogues are completely inactive towards CO2/CHO ROCOP. These recent studies highlight how heterometallic systems can offer new potential within ROCOP, and provide an interesting direction for future catalyst exploration. Detailed kinetic and mechanistic studies based on a multimetallic catalyst, supported by a macrocyclic ligand scaffold (Scheme 2, RHS, 1), have indicated that the polymerization occurs through a “chain shuttling” mechanism, where the growing polymer chain switches between two metal centres (Scheme 2, LHS).49 These insights suggested that the two metal centres play different roles in the propagation. Whilst one metal binds the epoxide, to position it in close proximity and activate it towards nucleophilic attack, it is the second metal carbonate bond which ring opens the epoxide to propagate the polymerization. These studies hinted that a heterometallic catalyst could exhibit even greater activity than the homometallic systems. This hypothesis was further supported by preliminary experimental studies, where a mixed catalyst system comprising di-Mg, di-Zn and MgZn acetate complexes (Scheme 2, 3), gave double the activity of a 1:1 mixture of the di- Zn (Scheme 2, 1) and di-Mg (Scheme 2, 2) acetate catalyst systems.50 1. Introduction The commercial success of producing sustainable polymer materials is crucially dependent on a suitable and efficient catalyst system; some of the most active are multimetallic complexes. The emerging theme of mixed-metal (heterometallic) cooperativity is an attractive means to improve catalyst activity and selectivity.4 This concept is particularly appealing, from both an economic and environmental perspective, when the metals involved are abundant, low-toxicity main-group and first-row transition metals. When two different metals are held in close proximity within a molecular environment, cooperative effects are often observed, which can give unusual and sometimes unexpected reactivities that cannot be accessed using either homometallic component in isolation.4-10 Elegant homogeneous heterometallic catalysts have been designed for a range of polymerizations, which outperform their homometallic analogues in terms of activity and selectivity (Figure 1).11-21 However, until recently, heterometallic ROCOP catalyst development was lagging behind that of olefin polymerization and the ROP of lactones. Reforming the current plastics industry to incorporate renewable resources, reduce our reliance on petrochemical feedstocks and diminish our impact on the environment presents a major challenge for the immediate future. Although this is a significant task, progress has already been achieved with some commercial polymer materials now produced either partially or wholly from renewable sources.1 One of the best-established commercial examples is bio-degradable polylactic acid, produced through the ring-opening polymerization (ROP) of bioderived lactide. Another particularly promising strategy is the ring-opening copolymerization (ROCOP) of CO2 and epoxides, to form polycarbonates (Scheme 1). In today’s climate, the use of CO2 as an alternative C1 feedstock is rapidly gathering attention, as CO2 is abundant, cheap, non-toxic and a waste product from many industrial processes. The CO2/epoxide ROCOP process is currently on the brink of commercialization, and the resultant telechelic polycarbonates can subsequently be used to synthesize polyurethanes, through reaction with a diisocyanate (Scheme 1). Polyurethane materials have many applications, including construction materials, high performance coatings and furniture.2 Assessment of the life cycle analysis of polyurethane materials shows that incorporating 20% of CO2 content can reduce the overall greenhouse gas emissions of the polymer synthesis by up to 19%.3 Figure 1. Polymer materials accessed using high efficiency heterometallic catalysts. Scheme 1. Synthesis of polycarbonates and polyurethanes from CO2/epoxide ring-opening copolymerization. Figure 1. Polymer materials accessed using high efficiency heterometallic catalysts. Scheme 1. Synthesis of polycarbonates and polyurethanes from CO2/epoxide ring-opening copolymerization. 3. CO2-derived Polycarbonates and their Material Properties The commercially available MOF, HKUST-1 ([Cu3(btc)2(H2O)3], where btc = benzene-1,3,5- tricarboxylate), was used to capture CO2 under aerobic conditions at atmospheric pressure.63 The captured CO2 was thermally released and directly copolymerized with propylene oxide or cis- 2-butylene oxide, in the presence of a Co(III) salen catalyst, to form poly(propylene carbonate) or poly(butylene carbonate), respectively. Importantly, the polymer yields obtained and the material properties observed were near-equivalent, when the CO2 source was the MOF filled under aerobic conditions, or a pressurized CO2 cylinder in the absence of HKUST-1. Studies using CO2 captured from a CCS demonstrator plant attached to a UK power station showed that a homogeneous di-Mg catalyst (Scheme 2, 2) could successfully catalyze CO2/CHO ROCOP.36 The catalyst performance was almost equivalent using this “impure” CO2 source in comparison to “pure”, research grade CO2. Further studies revealed that this catalyst system demonstrated a high level of tolerance towards added impurities which are often present in captured CO2, including air, water, CO, N2 and amines. These findings indicate that CO2/epoxide ROCOP has real world potential to convert waste CO2 to useful polymer materials. There is much scope to derivatize such polycarbonate materials, through reaction with diisocyanates to form polyurethanes,64 “switch chemistry” to form block copolymers,65, 66 or post-polymerization derivatization of functional groups on the epoxide, such as olefins59 or trimethoxysilane.67 Within academic research, CHO is often selected as the benchmark epoxide for catalyst development, on account of its lower toxicity and ease of handling compared to other epoxides. Characterization studies of high molecular weight poly(cyclohexene carbonate) (PCHC) with a broad distribution (Mw = 252 kg mol-1, Đ = 6) have revealed it to be a brittle material, which displayed an elongation at break of just 1-2% and a Tg value of 115 °C.55 Industrial interest has largely focused on CO2/propylene oxide ROCOP, where the poly(propylene carbonate) products have environmental benefits, including biodegradability and benign decomposition emissions. Poly(propylene carbonate) materials are of high commercial relevance, thanks to their suitability for many applications including adhesives, coatings and binders.22, 56 However, the range of applications is limited by the low softening temperature of this material (Tg = 40 °C). 3. CO2-derived Polycarbonates and their Material Properties The ROCOP of CO2/epoxides is an attractive route to synthesize polycarbonates which can contain up to 50% CO2 by mass. The synthesis of CO2-derived polycarbonates is considered to be safer than that of conventional polycarbonate materials, which are often produced through the condensation polymerization of bisphenol- A (BPA) with phosgene, which is a highly toxic nerve gas.52 It is worth noting that an alternative route, based on a melt process using BPA and diphenyl carbonate, is also applied industrially.53 However, it is important to note that the material properties of In 2015, a pure, homogeneous, heterometallic MgZn catalyst was reported (Scheme 2, 4).18 The preparation of this complex presents a significant synthetic challenge: to incorporate two labile metals of similar properties within a symmetrical ligand scaffold, whilst avoiding redistribution of the two metal centres to form the homometallic analogues (5 and 6). This heterometallic catalyst gave efficient, well-controlled polymerizations, with TOF CO2-derived polycarbonates differ from conventional polycarbonates. The amorphous polymers derived from BPA/phosgene copolymerization display high impact strength, heat resistance, toughness and transparency, and are used in a range of applications including medical, optical and electronic devices.54 CO2-derived polycarbonates give markedly different material properties, which vary according to the molecular weight and the epoxide co-monomer selected. CO2-derived polycarbonates differ from conventional polycarbonates. The amorphous polymers derived from BPA/phosgene copolymerization display high impact strength, heat resistance, toughness and transparency, and are used in a range of applications including medical, optical and electronic devices.54 CO2-derived polycarbonates give markedly different material properties, which vary according to the molecular weight and the epoxide co-monomer selected. alternative to BPA-based polycarbonates.61, 62 An alternative renewable epoxide, 1,4-cyclohexadiene oxide, has been derived from the mono-epoxidation of cyclohexa-1,4-diene from fatty acid methyl esters found in plant oils.59 Subsequent ROCOP with CO2 produced renewable, unsaturated polycarbonates, with the same Tg value as the saturated analogue, PCHC (Tg = 115 °C). Through rapid recent advances in CO2/epoxide ROCOP, a range of polycarbonate materials have been synthesized, with different material properties. To make these processes truly sustainable, the CO2 source must be carefully considered. High energy compressions of CO2 gas should be avoided, and the reaction system must be tolerant towards impurities found in industrial waste CO2. Promising advances have recently been made in this area. The application of metal-organic frameworks (MOFs), to capture, store and release CO2 into CO2/epoxide ROCOPs, has been reported by Darensbourg et al. 3. CO2-derived Polycarbonates and their Material Properties Studies of a homologous series of CO2-derived polycarbonates have shown that poly(propylene carbonate), poly(pentene carbonate), poly(hexene carbonate, poly(octene carbonate) and poly(cyclohexene carbonate) are all amorphous materials, and display initial decomposition temperatures (Td) under 300 °C, as determined using TGA and DSC analysis.57 The Td values increase in line with increasing hydrocarbon content of the polymer, with the exception of PCHC, which displays the highest initial decomposition temperature (Td = 290 °C) on account of the stiff cyclohexyl rings incorporated into the polymer chain. To access completely bio-based poly(carbonates) from CO2/epoxide ROCOP, additional research has focused on the use of renewable epoxides, such as limonene oxide58 and 1,4- cyclohexadiene oxide.59 Limonene oxide is derived from limonene, an alicyclic terpene which is annually produced on a scale exceeding 70, 000 tons from waste orange peel.60 High molecular weight (>100 kg mol-1) poly(limonene carbonate) has successfully been synthesized through CO2/limonene oxide ROCOP, using a β-diiminate zinc acetate catalyst.52 This bio- based polycarbonate displayed attractive material properties, including excellent thermal resistance (Tg = 130 °C), thermal stability (up to 240 °C), hardness and optical transparency. Indeed, recent advances have shown that post-polymerization modification of poly(limonene carbonate) can offer rigid polymers, with tunable Tg values up to 180 °C, as a potential References 1. Zhu Y, Romain C and Williams CK (2016), Sustainable polymers from renewable resources. 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Dalton Transactions 43(11): 4268-4286. 9. 4. Conclusion Whilst homogeneous heterometallic catalyst design for CO2/epoxide ROCOP is still in its infancy, recent findings suggest that it has the potential to grow into a fruitful area of high activity catalyst development. Not only have enhanced activities been reported using cooperative heterometallic catalysts but good activities have been achieved under atmospheric pressure and ambient temperature reaction conditions. Thanks to efforts across a range of fields, including catalyst design, engineering and materials, CO2-derived polymers synthesized through CO2/epoxide ROCOP are now on the edge of commercialization. This process provides an exciting opportunity to expand and exploit multimetallic catalyst development for a range of CO2- derived materials in the immediate future, to contribute to a more sustainable world. Acknowledgements J. A. 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https://openalex.org/W4256036270	https://journals.plos.org/plosone/article/file?type=printable&id=10.1371/journal.pone.0044313	English	null	Correction: Characterization of a Novel Phenol Hydroxylase in Indoles Biotranformation from a Strain Arthrobacter sp. W1	PloS one	2,012	cc-by	10,500	Received May 10, 2012; Accepted August 1, 2012; Published September 13, 2012 Copyright: 2012 Qu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Natural Science Foundation of China (No. 51078054; No. 14 21176040). The fu design, data collection and analysis, decision to publish, or preparation of the manuscript. supported by the National Natural Science Foundation of China (No. 51078054; No. 14 21176040). The funders had no role in study nd analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E mail: qyy@dlut edu cn * E-mail: qyy@dlut.edu.cn . These authors contributed equally to this work. During the history of microbial indigo production, the most representative study was performed by Ensley et al. in 1983, which proved that recombinant Escherichia coli expressing naphthalene dioxygenase (NDO) from Pseudomonas putida PpG7 could result in indigo formation [3]. Since then, a number of wild indigo- producing microorganisms induced by aromatic hydrocarbons and recombinant E. coli strains harboring monooxygenases (MOs) or dioxygenases (DOs) have been proved to transform indole to indigo or oxidized indoles [2–4,11,14–23]. The generally accepted pathway encoded by DOs is initiated by the oxidation of indole at the C-2 and C-3 positions to obtain cis-indole-2,3-dihydro-2,3-diol, which is then dehydrated to indoxyl, and the dimerization of two molecules of indoxyl leads to the production of indigo [3]. The transformation pathway encoded by MOs was firstly given by Mermod et al. in 1986 [24], which can be described as indole is directly hydroxylated at the C-3 position of the pyrrole ring forming indoxyl, and then undergoes dimerization to form indigo likewise. However, there has been no report on any kind of phenol hydroxylase (PH) in Arthrobacter strains as biocatalyst for indigoids biosynthesis from indoles. Therefore, the use of PH in the area of Characterization of a Novel Phenol Hydroxylase in Indoles Biotranformation from a Strain Arthrobacter sp. W1 Yuanyuan Qu1., Shengnan Shi2., Hao Zhou1., Qiao Ma1., Xinliang Li1, Xuwang Zhang1, Jiti Zhou1 1 Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Dalian, China, 2 State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin, China Abstract Background: Indigoids, as popular dyes, can be produced by microbial strains or enzymes catalysis. However, the new valuable products with their transformation mechanisms, especially inter-conversion among the intermediates and products have not been clearly identified yet. Therefore, it is necessary to investigate novel microbial catalytic processes for indigoids production systematically. Findings: A phenol hydroxylase gene cluster (4,606 bp) from Arthrobacter sp. W1 (PHw1) was obtained. This cluster contains six components in the order of KLMNOP, which exhibit relatively low sequence identities (37–72%) with known genes. It was suggested that indole and all the tested indole derivatives except for 3-methylindole were transformed to various substituted indigoid pigments, and the predominant color products derived from indoles were identified by spectrum analysis. One new purple product from indole, 2-(7-oxo-1H-indol-6(7H)-ylidene) indolin-3-one, should be proposed as the dimerization of isatin and 7-hydroxylindole at the C-2 and C-6 positions. Tunnel entrance and docking studies were used to predict the important amino acids for indoles biotransformation, which were further proved by site-directed mutagenesis. Conclusions/Significance: We showed that the phenol hydroxylase from genus Arthrobacter could transform indoles to indigoids with new chemical compounds being produced. Our work should show high insights into understanding the mechanism of indigoids bio-production. Citation: Qu Y, Shi S, Zhou H, Ma Q, Li X, et al. (2012) Characterization of a Novel Phenol Hydroxylase in Indoles Biotranformation from a Strain Arthrobacter sp. W1. PLoS ONE 7(9): e44313. doi:10.1371/journal.pone.0044313 Editor: Paulo Lee Ho, Instituto Butantan, Brazil Received May 10, 2012; Accepted August 1, 2012; Published September 13, 2012 Received May 10, 2012; Accepted August 1, 2012; Published September 13, 2012 Introduction Indigo, as one of the oldest dyes, is still used for printing and dyeing worldwide, which is primarily produced by chemical synthesis in modern textile industries [1]. However, researchers have been trying to seek more competitive and greener alternatives for commercial production, which has rejuvenated an interest in the microbial production of indigo [2–5]. Compared with indigo biosynthesis, there have been only sporadic reports on investigating the biotransformation of indole derivatives to form indigoid pigments [6,7], which can be probably used as chief precursors in dyeing industries, and can also serve as anticancer compounds for therapeutic application [8,9]. Recently, the intermediates derived from indole biotrans- formation have been identified such as isatic acid, 7-hydro- xyindole, isoindigo, 2-oxindole, 6-hydroxyindole, dioxindole and indoxyl [10–13]. But the inter-conversion among these com- pounds needs further investigation and the new types of pigments from indoles should be purified and studied systematically. September 2012 \| Volume 7 \| Issue 9 \| e44313 1 PLOS ONE \| www.plosone.org Biotransformation of Indoles by Phenol Hydroxylase Figure 1. SDS-PAGE analysis of protein samples of PH_IND from E. coli BL21 (DE3). Line 1. Whole cells of pET-28a(+); Line 2. Cell extracts of pET-28a(+); Line 3. Precipitation of pET-28a(+); Line 4. Whole cells of strain PH_IND; Line 5. Cell extracts of strain PH_IND; Line 6. Precipitation of strain PH_IND; M. Protein markers. Arrows show the positions of the six ORFs. ORF1. 10.4 kDa; ORF2. 37.6 kDa; ORF3. 10.5 kDa; ORF4. 59.2 kDa; ORF5. 13.5 kDa; ORF6. 38.6 kDa. SDS-PAGE was performed with 5% and 15% acrylamide concentrations for the concentrating and separating gels, respectively. doi:10.1371/journal.pone.0044313.g001 biocatalysis and fine chemical production needs to be thoroughly exploited. As a member of bacterial multi component monoxygenases (BMMs), phenol hydroxylase (EC 1.14.13.7) was firstly identified in 1990 in Pseudomonas sp. CF600 responsible for phenol and (di)methylphenol degradation [25–27]. It was reported to catalyze the regiospecific hydroxylation of a number of substituted phenols at the ortho position with respect to the hydroxyl moiety [28]. Generally, PH is commonly composed of six polypeptides in the order of KLMNOP, which can be designated as PHK, PH(LNO)2, PHM and PHP [25,29]. PHP is a NADH-oxidoreductase responsible for supplying electrons to the diiron cluster housed in the active site. The hydroxylase (200–255 kDa) is composed of three polypeptides (L, N, O) organized in a dimeric form (LNO)2. PHN is responsible for binding various substrates. Cloning and expression of the PH gene cluster from strain W1 The primers (Raw TEST-F/Raw TEST-R) based on the conserved region of PH were used to amplify the genes encoding the large subunit of PH. A 685 bp homological gene was amplified from genomic DNA of strain W1. Thermal asymmetric interlaced PCR (TAIL-PCR, entails consecutive reactions with nested sequence-specific primers and a shorter arbitrary degenerate primer), a genome walking method, was adopted to amplify 59 and 39-terminal flanking regions [34]. After TAIL-PCR for the 59- terminal flanking region, four ORFs (ORF1, ORF2, ORF3 and ORF4) were obtained. However, the length of the 59-terminal flanking region obtained could not meet the requirements. Thus, the 59-terminal flanking region was amplified again using special primers and then the full length was obtained. The 39-terminal flanking region was amplified by three steps of TAIL-PCR, and the nucleotide sequence obtained was used to design the specific primers for further TAIL-PCR processes. After three steps of TAIL-PCR, two complete ORFs were obtained. As is well known, the N component (a subunit) harbors a non- heme carboxylate-bridged diiron center for catalysis and plays a key role in the selectivity of substrate. Therefore, it is essential to analyze and obtain the information of conserved residues in the N component. It was suggested that two typical EXRH motifs were found conserved at positions 138–141 and 233–236 (in strain Pseudomonas sp. OX1), which were assigned to the ligands of the catalytic diiron (Figure S2) [29]. Operon organization and sequence alignment of PHs Operon organization and sequence alignment of PHs The complete organization of the respective operon and conversed amino acids of various PHs in each multi component system were analyzed by BLAST. According to the results, all the PHs exhibited similar organization with six operons (Figure S2). Six ORFs from strain W1 showed relatively low sequence identities with those of several well-studied bacteria, such as Ralstonia eutropha E2, Pseudomonas sp. CF600, Pseudomonas putida H and Comamonas testosteroni R5 [27]. The most conserved ORF was the a subunit of oxygenase (orf4, N component in strain W1) with 64%–72% sequence identity. Whereas, the lowest sequence identity existed in the c subunit of oxygenase (orf5, O component in strain W1), which showed 37%–42% identity with other corresponding components (Figure S2, Table S1). Introduction PHM (10– 16 kDa) is a regulatory protein, devoid of any cofactor or metal ion, which is essential for efficient catalysis. And PHK is responsible for assembling iron at the active site [30]. However, which residues play the key role in binding substrates of PH are still unclear. Therefore, it is essential to obtain information on the active site of the PHN, which will help improve our knowledge of enzyme-ligand interactions. It has been shown that molecular informatics technology is useful in revealing the configuration of the substrate-enzyme complex, and also exhibits significant abilities in predicting the catalytic potential of enzymes [31–33]. Figure 1. SDS-PAGE analysis of protein samples of PH_IND from E. coli BL21 (DE3). Line 1. Whole cells of pET-28a(+); Line 2. Cell extracts of pET-28a(+); Line 3. Precipitation of pET-28a(+); Line 4. Whole cells of strain PH_IND; Line 5. Cell extracts of strain PH_IND; Line 6. Precipitation of strain PH_IND; M. Protein markers. Arrows show the positions of the six ORFs. ORF1. 10.4 kDa; ORF2. 37.6 kDa; ORF3. 10.5 kDa; ORF4. 59.2 kDa; ORF5. 13.5 kDa; ORF6. 38.6 kDa. SDS-PAGE was performed with 5% and 15% acrylamide concentrations for the concentrating and separating gels, respectively. doi:10.1371/journal.pone.0044313.g001 with phenol were 2.137 s21 and 5.429 mM, respectively. The kcat/ Km of PH_IND (0.394 s21/mM21) was lower than that of phenol hydroxylase from Pseudomonas stutzeri OX1 [35]. with phenol were 2.137 s21 and 5.429 mM, respectively. The kcat/ Km of PH_IND (0.394 s21/mM21) was lower than that of phenol hydroxylase from Pseudomonas stutzeri OX1 [35]. The aim of this study was to investigate the biotransformation of indoles to indigoids by a novel phenol hydroxylase from Arthrobacter sp. W1. Homology modeling and molecular docking were applied to analyze substrate specificity and interactions between indoles and PH. The color products were identified by HPLC-MS and NMR analysis. Our work should show high insights into the potential for commercial indigoids production by PH from genus Arthrobacter, which will pave the way to novel avenues in green chemistry. rmation of indole and its derivatives by whole To investigate the substrate range of strain PH_IND, in vitro assays were performed with indole and 14 kinds of derivatives. During the transformation process, the control group (host cells E. coli BL21 (DE3)) showed no activities toward all the substrates. Color formation could be observed with almost all the substrates (Table S2), however, the color exhibited differently from the same substrate compared with the previous literatures [6–8,12,20,36]. Therefore, considering the type and position of the substituted group, 6 kinds of indole derivatives were selected for further PH genes were successfully expressed (designated as PH_IND) under the transcriptional control of a strong promoter T7, and the SDS-PAGE analysis revealed the presence of six ORFs for PH_IND (Figure 1). The effects of pH, salt concentration, temperature and metal ions on the activities of crude PH_IND were shown in Figure S1, which presented the optimal range of pH 6.0–8.0, salt concentration 0.5–1.0% and temperature 40– 60uC. Under the optimal conditions, the kcat and Km of PH_IND PLOS ONE \| www.plosone.org September 2012 \| Volume 7 \| Issue 9 \| e44313 2 Biotransformation of Indoles by Phenol Hydroxylase Table 1. Characteristics of indigoids produced by whole cells of strain PH_IND. rmation of indole and its derivatives by whole Substrate Transformation yield (%) Products NMR analysis of products Indole 98.80 Indigo 1H NMR (400 MHz, d6-Me2SO) d = 6.95 (t, 2H, J = 8 Hz), 7.33 (d, 2H, J = 8 Hz), 7.51 (t, 2H, J = 7.6 Hz), 7.61 (d, 2H, J = 8 Hz), 10.50 (s, 2H, NH) 2-(7-oxo-1H-indol-6(7H)-ylidene) indolin-3-one 1H NMR (500 MHz, d6-Me2SO) d = 6.27 (t, 1H, J = 1.9 Hz, H-29), 6.81 (d, 1H, J = 9.5 Hz, H-49), 7.07 (t, 1H, J1 = 7.4 Hz, J2 = 7.3 Hz, H-5), 7.23 (t, 1H, J = 2.5 Hz, H-39), 7.44 (d, 1H, J = 7.9 Hz, H-7), 7.57 (t, 1H, J1 = 7.4 Hz, J2 = 7.8 Hz, H-6), 7.62 (d, 1H, J = 7.5 Hz, H-4), 7.95 (d, 1H, J = 9.4 Hz, H-59), 11.99 (s, 1H, NH), 12.45 (s, 1H, NH9) 13C NMR (100 MHz, d6-Me2SO): d = 105.5 (C-29), 112.2 (C-3a9), 114.2 (C-7), 118.7 (C-59), 119.5 (C-49), 119.8 (C-2), 122.7 (C-5), 124.6 (C-4), 127.6 (C-39), 127.8 (C-7a9), 130.8 (C-69), 136.9 (C-6), 140.1 (C-3a), 151.7 (C-7a), 175.8 (C-79), 190.3 (C-3) 4-Methylindole 95.61 4,49-dimethylindigo 1H NMR (400 MHz, d6-Me2SO) d = 2.46 (s, 6H, CH3), 6.77 (d, 2H, J = 7.2 Hz), 6.95 (t, 2H, J = 8.4 Hz), 7.20 (d, 2H, J = 7.2 Hz), 11.04 (s, 2H, NH) 5-Methylindole 94.82 5,59-dimethylindigo 1H NMR (400 MHz, d6-Me2SO) d = 2.36 (s, 6H, CH3), 6.89 (d, 2H, J = 7.6 Hz),7.26 (dd, 2H, J1 = 7.6, J2 = 2 Hz), 7.70 (d, 2H, J = 8 Hz), 10.93 (s, 2H, NH) 7-Methylindole 89.34 7,79-dimethylindigo 1H NMR (400 MHz, d6-Me2SO) d = 2.46 (s, 6H, CH3), 7.00 (t, 2H, J = 8 Hz), 7.16 (d, 2H, J = 7.6 Hz), 7.52 (d, 2H, J = 8 Hz), 11.46 (s, 2H, NH) 4-Chloroindole 78.66 4,49-dichloroindigo 1H NMR (400 MHz, d6-Me2SO) d = 7.05 (d, 2H, J = 7.2 Hz), 7.38 (d, 2H, J = 7.2 Hz), 7.71 (dd, 2H, J1 = 8, J2 = 2.4 Hz), 11.46 (s, 2H, NH) 7-Chloroindole 78.89 5,59-dichloroindigo 1H NMR (400 MHz, d6-Me2SO) d = 7.00 (t, 2H, J = 7.6 Hz), 7.15 (d, 2H, J = 7.6 Hz), 7.52 (d, 2H, J = 8 Hz), 11.45 (s, 2H, NH) 5-Methoxyindole 91.25 5,59-dimethoxyindigo 1H NMR (400 MHz, d6-Me2SO) d = 3.77 (s, 6H, OCH3), 7.08 (d, 2H, J = 8 Hz),7.15 (dd, 2H, J1 = 8.8, J2 = 2.4 Hz), 7.26 (d, 2H, J = 8.8 Hz), 10.27 (s, 2H, NH) Transformation yield (%) was calculated as the reduced indoles concentration to the initial indoles concentration in the reaction systems. rmation of indole and its derivatives by whole doi:10.1371/journal.pone.0044313.t001 Table 1. Characteristics of indigoids produced by whole cells of strain PH_IND. NMR analysis of products 1H NMR (400 MHz, d6-Me2SO) d = 2.36 (s, 6H, CH3), 6.89 (d, 2H, J = 7.6 Hz),7.26 (dd, 2H, J1 = 7.6, J2 = 2 Hz), 7.70 (d, 2H, J = 8 Hz), 10.93 (s, 2H, NH) 1H NMR (400 MHz, d6-Me2SO) d = 2.46 (s, 6H, CH3), 7.00 (t, 2H, J = 8 Hz), 7.16 (d, 2H, J = 7.6 Hz), 7.52 (d, 2H, J = 8 Hz), 11.46 (s, 2H, NH) rmation yield (%) was calculated as the reduced indoles concentration to the initial indoles concentration in the reaction systems. 371/journal.pone.0044313.t001 yield (%) was calculated as the reduced indoles concentration to the initial indoles concentration in the reaction systems. l 0044313 t001 light blue product showed a molecular ion of m/z 291 ([M+]) (Figure S3). The 1H NMR spectra exhibited a symmetric structure like indigo (Table 1, Figure S4). Thus, the light blue product from 4-methylindole was presumed to be 4,49-dimethylindigo (Table 1). investigation in this study. It was suggested that strain PH_IND was able to catalyze the formation of dyestuffs from indole, 4-, 5- and 7-methylindole, 4- and 7-chloroindole and 5-methoxyindole (Table 1). The substrates were classified into 4 groups, which were described in detail as follows. Biotransformation of indole (group 1): When indole was incubated with strain PH_IND, three colored products were observed by TLC with Rf values of 0.70, 0.31 and 0.09, respectively (Figure 2, Sample 2). The molecular ion and retention time (tr) of the pink product (Rf = 0.09) was at m/z 263 ([M+]) and 10.1 min (data not shown), which was identical to the authentic standard of indirubin. The HPLC retention time (tr) of the light blue product (Rf = 0.70) was 9.6 min and its mass spectrum was primarily characterized by a molecular ion at m/z 263 ([M+]) (Figure S3), and its 1H NMR spectrum was consistent with the authentic standard of indigo (Table 1, Figure S4). According to the different polarity, the purple product (Rf = 0.31) derived from indole was neither indigo nor indirubin, although the molecular mass was the same. The spectroscopic data of this product were listed in Table 1. rmation of indole and its derivatives by whole 1H NMR and 13C NMR clearly showed an unsymmetrical structure of a pyrrole ring (hydrogen at C-2 and C- 3 positions) connecting with a benzene ring (at C-6 and C-7 positions of the benzene ring) by a double bond. Therefore, the structure of purple product was proposed as 2-(7-oxo-1H-indol- 6(7H)-ylidene) indolin-3-one, which was further certified by 1H-1H COSY, 1H-13C HSQC and 1H-13C HMBC (Figure S5). Figure 2. Identification of transformation products by TLC. The transformation samples were extracted with equal volume of ethyl acetate and concentrated by N2. 200 mL of the extracts were applied to the TLC plates (silica gel 60 F254), and then the TLC plates were resolved with a solvent mixture of dichloromethane-methanol (50:1, v/ v). The samples were designated as following: 1. Indigo (standard); 2. Products of indole transformation; 3. Products of 4-methylindole transformation; 4. Products of 5-methylindole transformation; 5. Products of 7-methylindole transformation; 6. Products of 5-methox- yindole transformation; 7. Products of 4-chloroindole transformation; 8. Products of 7-chloroindole transformation. The products with different Rf values were indicated in three regions by the arrows. doi:10.1371/journal.pone.0044313.g002 Figure 2. Identification of transformation products by TLC. The transformation samples were extracted with equal volume of ethyl acetate and concentrated by N2. 200 mL of the extracts were applied to the TLC plates (silica gel 60 F254), and then the TLC plates were resolved with a solvent mixture of dichloromethane-methanol (50:1, v/ v). The samples were designated as following: 1. Indigo (standard); 2. Products of indole transformation; 3. Products of 4-methylindole transformation; 4. Products of 5-methylindole transformation; 5. Products of 7-methylindole transformation; 6. Products of 5-methox- yindole transformation; 7. Products of 4-chloroindole transformation; 8. Products of 7-chloroindole transformation. The products with different Rf values were indicated in three regions by the arrows. doi:10.1371/journal.pone.0044313.g002 Figure 2. Identification of transformation products by TLC. The transformation samples were extracted with equal volume of ethyl acetate and concentrated by N2. 200 mL of the extracts were applied to the TLC plates (silica gel 60 F254), and then the TLC plates were resolved with a solvent mixture of dichloromethane-methanol (50:1, v/ v). The samples were designated as following: 1. Indigo (standard); 2. Products of indole transformation; 3. Products of 4-methylindole transformation; 4. Products of 5-methylindole transformation; 5. Products of 7-methylindole transformation; 6. Products of 5-methox- yindole transformation; 7. Products of 4-chloroindole transformation; 8. Products of 7-chloroindole transformation. rmation of indole and its derivatives by whole The light blue color was identified as 4,49-dichloroindigo (Table 1), and the other purple product was also presumed to be a 2-(7-oxo-1H- indol-6(7H)-ylidene) indolin-3-one like compound according to the analysis above. The mass spectra of the purified product showed a base peak at m/z 331 ([M+]) with an expected dichloride ratio of 9:6:1 for a peak at m/z 331, 332 and 333 (Figure S3). The 1H NMR analysis (Table 1 and Figure S4) was similar to that of 4,49- dichloroindigo reported previously [12]. Biotransformation of 7- chloroindole led to the production of 7,79-dichloroindigo as a single color compound, according to the Rf value of 0.76 (Figure 2, Sample 8), HPLC-MS and 1H NMR analysis (Figure S3, Figure S4 and Table 1). The mass spectra were characterized by a molecular ion at m/z 331 ([M+]). cavity volume into consideration, PH_IND seemed to be the efficient candidate for chemicals production. efficient candidate for chemicals production. With the modeled 3D structure of PHNW1, indole analogues were docked into the active site cavity. The most favorable binding conformations predicted by AutoDock vina were shown in Figure 4. The residues around the active site were shown in Table 3. Several interesting points could be implicated from the enzyme-substrate complex. Firstly, all the indole analogues could be classified into two-types: located far away from the diiron center (3-methylindole) (Figure 4H), and located near the diiron center (the other analogues) (Figure 4A–G). Secondly, the substrates, except for 3-methylindole, interacted with a number of residues including Leu-204, Phe-205 and Val-102 by hydrophobic and aromatic stacking, which might stabilize the enzyme-substrate complex. Meanwhile, several indole analogues could be stabilized by forming hydrogen bonds with the adjacent residues. 7- Chloroindole and 7-methylindole formed a hydrogen bond with Glu-136 (Figure 4D), 4-chloroindole and 4-methylindole formed hydrogen bond with Glu-231 (Figure 4B, E), while indole formed two hydrogen bonds with Glu-197 and Glu-136 (Figure 4A). Biotransformation of 5-methoxyindole (group 4): The biotrans- formation of 5-methoxyindole yielded one main color product (Rf = 0.51) (Figure 2, Sample 6). The mass spectrum of the product was determined by a molecular ion at m/z 321 ([M+]). The 1H NMR shifts and assignments were showed in Table 1 and Figure S4, which were consistent with the report by Guengerich et al. [8]. Thus, the color product was identified as 5, 59-dimethoxyindigo. rmation of indole and its derivatives by whole The products with different Rf values were indicated in three regions by the arrows. doi:10.1371/journal.pone.0044313.g002 Biotransformation of 4-, 5- and 7-methylindole (group 2): TLC analysis of 4-methylindole transformation products revealed the presence of two main color products with Rf values of 0.76 and 0.31, respectively (Figure 2, Sample 3). HPLC-MS analysis of the PLOS ONE \| www.plosone.org September 2012 \| Volume 7 \| Issue 9 \| e44313 3 Biotransformation of Indoles by Phenol Hydroxylase Figure 3. Homology modeling and identified substrate tunnel. A. The residues involved in coordinating dinuclear iron. These residues were labeled in cyan, and the dinuclear iron sites were shown in sphere; B. Tunnel identified in the homology modeling of PHN component from Arthrobacter sp. W1 using CAVER. The white sticks represent five formed residues, i.e. Thr-201, Asn-202, Phe-205, Glu-231 and Met-235. The tunnel is labeled as yellow surface and the residues formed entrance are in purple. doi:10.1371/journal.pone.0044313.g003 The other purple product was located in the same region with that derived from indole with a similar Rf value (Figure 2, Sample 3, region 2), which implicated it should possess the similar structure to 2-(7-oxo-1H-indol-6(7H)-ylidene) indolin-3-one. However, only one color product derived from 5-methylindole was determined as 5,59-dimethylindigo with m/z 291 ([M+]) and a Rf value of 0.62 (Figure 2, Sample 4 and Figure S3). The 1H NMR shifts and assignments were shown in Table 1 and Figure S4. One color product formed from 7-methylindole (Rf = 0.61) (Figure 2, Sample 5). HPLC-MS analysis of the product of 7-methylindole showed a molecular ion m/z of 291 ([M+]) (Figure S3). 1H NMR of the 7- methylindole product was exhibited in Table 1 and Figure S4. According to TLC, HPLC-MS and 1H NMR analysis, the single product of 7-methylindole was identified as 7,79-dimethylindigo. Figure 3. Homology modeling and identified substrate tunnel. A. The residues involved in coordinating dinuclear iron. These residues were labeled in cyan, and the dinuclear iron sites were shown in sphere; B. Tunnel identified in the homology modeling of PHN component from Arthrobacter sp. W1 using CAVER. The white sticks represent five formed residues, i.e. Thr-201, Asn-202, Phe-205, Glu-231 and Met-235. The tunnel is labeled as yellow surface and the residues formed entrance are in purple. doi:10.1371/journal.pone.0044313.g003 Biotransformation of 4- and 7-chloroindole (group 3): Biotrans- formation of 4-chloroindole yielded two color products with Rf values of 0.74 and 0.30, respectively (Figure 2, Sample 7). rmation of indole and its derivatives by whole It was also found that the location of light blue and purple products from 5-methoxyindole showed in Sample 6 were a little different from Sample 2, Sample 3 and Sample 7 (Figure 2) due to the methoxy group at the C-5 position. two hydrogen bonds with Glu-197 and Glu-136 (Figure 4A). The distance between C-7 of the benzene ring and Fe1of the active site was 2.96 A˚ for indole, 3.37 A˚ for 4-chloroindole, 3.39 A˚ for 4-methylindole, 3.49 A˚ for 5-methoxyindole, 4.43 A˚ for 5- methylindole, 3.72 A˚ for 7-methylindole and 3.70 A˚ for 7- chlorolindole. Whereas, C-7 of 3-methylindole was 17.5 A˚ away from Fe1of the active site, which indicated that it couldn’t be transformed by strain PH_IND. It was shown that indole and its derivatives were oriented in a different way (Figure 4). When indole was substituted at 4 or 5 positions, the orientations of substrates were adjusted to reduce the distance between C-7 and Fe1 of diiron (Figure 4B, 4E, 4G) [12]. Consequently, it proved that indole and its derivatives could be catalyzed by strain PH_IND to some extent, and also the C-7 position seemed more easily to be attacked by strain PH_IND than other C atoms in the six- membered ring. Homology modeling of the catalytic domain of PH Homology modeling of the catalytic domain of PH In order to explain the different conversion behavior of indole derives, three-dimensional structure of the N-component from PHW1 was constructed based on a homology template. Structural alignment of the PHW1 model to the template PHOX1 exhibited 0.16 A˚ of RMSD of the 488 residues, confirming that the fold was essentially the same. The quality validation of the model was performed by ERRAT, Verify_3D and PROCHECK analysis. As a typical feature of phenol hydroxylase, the dinuclear iron (II) sites were coordinated by four carboxylates and two histidines (Fe1 was coordinated by His-139, Glu-106 and Glu-136, Fe2 was coordi- nated by His-234, Glu-231 and Glu-197) (Figure 3A). In order to find the most direct route of small molecules entering the active site pocket, tunnel analysis of PHNW1 was performed by CAVER. The tunnel entrance was about 6.00 A˚ in diameter, which was located above the diiron center and formed by the side chains of Thr-201, Asn-202, Phe-205, Glu-231 and Met-235 (Figure 3B). Comparisons of the active sites in various oxygenases were shown in Table 2. It was suggested that the cavity of PHNOX1 is 587.1 A˚ 3, which is larger than that of NDO, MMO and P450. As a homologous protein, PHNW1 (508.4 A˚ 3) exhibited the similar cavity volume with PHNOX1. Therefore, taking entrance size and Critical residues identification by site-directed mutation Critical residues identification by site-directed mutation In order to verify the importance of gating and iron binding residues identified by homology modeling, Asn-202 and His-139 were taken for mutagenesis studies, which were located at the entrance of active site pocket and diiron center (Fe1), respectively. The Asn-202 was mutated to Phe-202 with a bulky side chain, September 2012 \| Volume 7 \| Issue 9 \| e44313 PLOS ONE \| www.plosone.org 4 Biotransformation of Indoles by Phenol Hydroxylase Table 2. Comparisons of active sites in various oxygenases. Form of iron Enzyme PDB ID Size of tunnel entrance (A˚ ) Volume of cavity (A˚ 3) Reference Non-heme Fe (II) PH Pseudomonas stutzeri OX1 2INP 6.0 587.1* (27) TMO Pseudomonas mendocina KR1 3DHG 4.4 750.8* (27) MMO Methylococcus capsulatus 1XU3 2.0 246.6* (27) NDO Pseudomonas putida NCIB9816-4 1EG9 4.6 480.0 (37) Heme Fe (II) P450 2A6 Human microsomal 1Z10 ,1.4 440.0 (12) PH: phenol hydroxylase; TMO: toluene 4-monooxygenase; MMO: methane monooxygenase; NDO: naphthalene 1,2-dioxygenase. The distance of Val-260 CG1 and His-295 NE2. **Calculated by CASTp server. doi:10.1371/journal.pone.0044313.t002 y(OBzl) indole to form a color product (4-OBzl-2-(49-OBzl-19,79- dihydro-79-oxo-69H-indol-69-ylidene) indolin-3-one) [12]. which can reduce the size of entrance from 6.00 A˚ to 4.47 A˚ . Meanwhile, the His-139 was mutated to Ala-139, which could not coordinate with iron. SDS-PAGE of crude cell extracts from two mutants was shown in Figure S6. It was suggested that both mutants lost the catalytic activity of transforming indole to indigo (Figure S7), which indicated that Asn-202 and His-139 were the critical residues for indole transformation. In order to investigate the relationship between the function (substrate specificity) and structure (of active site and substrate- enzyme complex), molecular simulation was performed. The surface residues involved in the formation of tunnel, which named Thr-201, Asn-202, Phe-205, Glu-231 and Met-235, were conserved among different PHs. The pore diameter (,6.00 A˚ ) was large enough to accommodate indole and substituted indoles (Table 2). However, the analogous pore was absent in some crystal structures of BMMs due to the shifting of Asn-214 (Asn-202 in PHNW1), which suggested that the size of substrate entrance to the diiron center was the determinant for substrate selectivity of BMMs [32]. In the previous literature, cytochrome P450 2A6 was modified by random and site-directed mutagenesis and the mutant I300V could transform bulky substituted indoles [12]. Discussion In this study, we designed the experiments to show that PH obtained from Arthrobacter sp. W1 can be used to produce dyestuffs from indole and its derivatives. The indigo productivity of 44.75 mg L21 h21 by whole cells of strain PH_IND was obtained in this study. As previously reported, a recombinant E. coil OST3410 carrying the PH gene from Acinetobacter sp. ST-550 produced 52 mg mL21 of indigo in the presence of diphenyl- methane at 24 h [14]. Cultures of E. coli JM101 that expressed HbpAind during growth in LB medium had an indigo productivity of 5 mg L21 h21 [20]. Recombinant E. coli HB101 expressed the naphthalene dioxygenase gene produced 1 mg L21 h21 of indigo [3]. Therefore, strain PH_IND seemed to be more efficient than other recombinant strains. However, the indigo yield is not the only standard to evaluate the function of indigo-producing microorganisms, because the transformation depends on various variables, such as inducers, solvents and culture conditions. As reported previously, E. coli strains expressing mPH from Pseudo- monas sp. KL28 and KL33 could catalyze the production of dyestuffs and hydroxyl indoles from indole derivatives [6]. In comparison with mPHKL28 and mPHKL33, strain PH_IND can oxidize indole derivatives to a larger library of new and complex indigoids. The recombinant E. coli expressing various enzymes responsible for indoles transformation were summarized in Table S2. By contrast, strain PH_IND exhibited strong catalytic function, which could hydroxylate 14 kinds of indoles. p However, not only the entrance size and cavity volume of active pocket, but also the suitable orientation of substrate would affect the specificity of enzyme. To our best knowledge, there is no crystal structure of PH-indole (or indole derivatives) complex, so docking studies can be applied to obtain the binding information of different substrates. Firstly, the lowest energy binding confor- mation of 3-methylindole was significantly different from others, which indicated that it was hard to be transformed. Secondly, the bound mode depended on the position and type of the substituent, which meant the substituent on different position of indole should affect the affinity between ligands and protein. It was previously reported that when indole was docked into the active cavity of naphthalene 1,2-dioxygenase (NDO), indole was oriented as the five-membered ring pointing inward, while the six-membered ring pointing towards the entrance of the substrate channel [37]. Critical residues identification by site-directed mutation The mutant provides an additional 83 A˚ 3 space of active site, which is large enough to allow the bulky substituted indoles binding in a suitable conformation [12]. As shown in Table 2, the cavity of PH_OX1 is 587.1 A˚ 3, which is larger than that of NDO, MMO and P450, which suggested potential versatile substrate spectra of PH_OX1. Therefore, taking entrance size and cavity volume for consider- ation, PH is an efficient candidate for chemicals production. September 2012 \| Volume 7 \| Issue 9 \| e44313 Discussion However, both five-membered ring and six-membered ring in this study were oriented in the same way to the diiron active site, which were different from the binding mode of NDO. All the docking analyses can supply useful information to predict the potential performance of PH_IND for indoles oxidation. As for indole transformation by strain PH_IND, three color products (indigo, indirubin and 2-(7-oxo-1H-indol-6(7H)-ylidene) indolin-3-one) were identified (Figure 2). 2-(7-oxo-1H-indol-6(7H)- ylidene) indolin-3-one appeared to be a novel pigment, because no such structure was reported by oxidizing indole using any kind of oxygenase. In the recent reports, some novel indigoid pigments have been identified and produced, which should be used as dyestuffs and be of therapeutic values [11,12]. Due to their similar chemical structures, this new product would be potential as the precursor for both dyeing and pharmaceutical production. The similar structure was also identified by the mutant (N297Q/ I300V) of cytochrome P450 2A6, which oxidized 4-benzylox- Indole belongs to heteroaromatic with p-election densities on carbon atoms of the pyrrole ring greater than those of the benzene ring, and it maintains an unbroken benzene ring and bears a positive charge on nitrogen and a negative charge on the C-3 PLOS ONE \| www.plosone.org September 2012 \| Volume 7 \| Issue 9 \| e44313 5 Biotransformation of Indoles by Phenol Hydroxylase Figure 4. Interactions between PHNW1 component and indole derivatives. Orientations of docked indoles in the active site of PHNW1 component: A. Indole; B. 4-Methylindole; C. 5-Methylindole; D. 7-Methylindole; E. 4-Chloroindole; F. 7-Chloroindole; G. 5-Methyoxyindole; H. 3 Methylindole. Atom designation: carbon atom, blue; hydrogen atom, white; chlorine atom, green; oxygen atom, red. Orange spheres represent fo diiron, of which located above is designated as Fe1; green represent for tunnel entrance residues Asn-202 and Phe-205; other important residues are shown in grey. doi:10.1371/journal.pone.0044313.g004 Figure 4. Interactions between PHNW1 component and indole derivatives. Orientations of docked indoles in the active site of PHNW1 component: A. Indole; B. 4-Methylindole; C. 5-Methylindole; D. 7-Methylindole; E. 4-Chloroindole; F. 7-Chloroindole; G. 5-Methyoxyindole; H. 3- Methylindole. Atom designation: carbon atom, blue; hydrogen atom, white; chlorine atom, green; oxygen atom, red. Orange spheres represent for diiron, of which located above is designated as Fe1; green represent for tunnel entrance residues Asn-202 and Phe-205; other important residues are shown in grey. doi:10 1371/journal pone 0044313 g004 g y doi:10.1371/journal.pone.0044313.g004 Table 3. Residues involved in binding different indoles. Table 3. Cloning, sequence analysis, and expression of PH gene cluster DNA manipulations were carried out according to standard operating procedures [40]. The primers used in this study were listed in Table S3. Genomic DNA of strain W1 was extracted using Mini BEST Bacterial Genomic DNA Extraction Kit (TaKaRa Co. Ltd. Dalian, China). The primers (Raw TEST-F/ Raw TEST-R) based on the conserved region of PH were used to amplify the target genes. Conditions for PCR were as follows: 98uC for 1 min, followed by 30 cycles of 98uC for 10 s, 55uC for 15 s, 72uC for 30 s, and a final extension step of 10 min at 72uC. The PCR products were recovered by TaKaRa Agarose Gel DNA Purification Kit Ver. 2.0 and sequenced by TaKaRa Co. Ltd. (Dalian, China). Three nested PCR procedures were applied to amplify the upstream and downstream regions of PH by TAIL- PCR using TaKaRa Genome Walking Kit (TaKaRa Co. Ltd. Dalian, China). The fragments containing the entire coding sequences were isolated and cloned into the compatible sites of pMD18-T simple vector for sequencing. According to the analysis mentioned above, the overview of the proposed pathways by various oxygenases is depicted in Figure 5. Firstly, indole can be attacked at C-2 and C-3 positions to form cis-indole-2,3-dihydrodiol and indole oxide by dioxygenase and styrene oxygenase, respectively [3,22]. Then it undergoes the C-3 oxidation pathway, in which indigo and indirubin should be formed as the main color products. From Figure 5, it is obvious that the versatile products should be contributed to monooxy- genase (including P450). There are three pathways by different MOs, which are named C-3, C-2 and C-7 oxidation pathways. From the results of this study and previous reports, C-3 often occurs with the C-2 and C-7 oxidation pathways. In other words, most MOs can hydroxylate indoles at the C-3 position, which leads to form indigo and indirubin. Whereas, some MOs can attack both C-3 and C-2 positions, thus the products should be composed of indigo, indirubin and isoindigo [2,5,11,19,24]. In the previous studies, both C-2 and C-3 oxidation pathways are generally accepted and well studied [8,23,38]. The C-7 oxidation pathway is previously reported by another Pseudomonas PH yielding 7-hydrxyindole from indole [6] and by P450, which produced one new product, i.e. 4-OBzl-2-(49-OBzl-19,79-dihydro- 79-oxo-69H-indol-69-ylidene)indolin-3-one from 4-OBzl-indole [12]. Other than this, this is the first report on the formation of such compound. Biotransformation of Indoles by Phenol Hydroxylase atom, which means that C-3 is highly reactive toward electro- philic reagents [1]. All in all, the potential position of PH attacking substrates is determined not only by indole molecular characteristics (e.g. charge, substitute group and electronic density), but also by the distance between C atoms of the six- membered ring (especially C-7) and the active site Fe1. The site- directed mutagenesis can explain well that the substrate tunnel and residue involved in coordinating iron are truly essential for catalysis. Chemicals Indole, indirubin, indigo, 3-, 4-, 5- and 7-methylindole, 5- methoxyindole, 4- and 7-chloroindole, kanamycin and isopropyl- b-D-thiogalactopyranoside (IPTG) were purchased from J&K Scientific Ltd. (China). All other reagents and solvents were obtained from general commercial suppliers and used without further purification. Genome Walking Kit, Mini BEST Plasmid and DNA Purification Kit, DNA Ligation Kit, and Primer STARH HS DNA Polymerase, Sca I, Hind III, Aat II and Sal I were all purchased from TaKaRa Co. Ltd. (Dalian, China), and used according to the instructions of the manufacturer. It is no accident that the purple compound is found from transformation of indole, 4-methylindole, 4-cholorlindole and 5- methoxyindole. It reveals that C-7 of the six-membered ring locates the nearest to the active site Fe1, whereas other C atoms are far away from the active site. As previously reported, C-3 of the five-membered ring was the most active position, which could be firstly attacked by the oxygenase [3,11]. In addition, the position and type of substituted group is a secondary factor to be considered. We presume that the distance between C-7 and Fe1 is the predominant factor to form the purple compounds. According to the docking studies, we find that although the position of the methyl group and methoxy group are both at 5 positions, there is no purple product formed during the process of 5-methylindole transformation. This can be explained by the orientation of the two molecules are distinctly different from each other (Figure 4). Also, the distances between C-7 and Fe1 for 5-methylindole and 5- methoxyindole are 4.43 A˚ and 3.49 A˚ , respectively. Considering both the orientation tendency and the distance between C-7 and Fe1, 5-methylindole should not be transformed to a purple product. Cloning, sequence analysis, and expression of PH gene cluster After sequencing, the genes were subcloned into the pET-28a(+) by Sac I and Hind III yielding the recombined plasmid pET- 28a(+)/PH. Subsequently, the resulting plasmids were introduced into E. coli BL21 (DE3) for expression, and the recombinant strain harboring the PH genes was designated as PH_IND. Analysis of potential ORFs and comparison of amino acid sequences (or nucleotide sequences) were performed with the ORF finder and BLAST programs on the National Center for Biotechnology Information website. Multiple-sequence alignment was performed by Clustal 61.8. In conclusion, we identified the phenol hydroxylase gene cluster responsible for indigoids bio-production and the color transfor- mation products were indentified. Meanwhile, the catalytic mechanisms of PH_IND were proposed as the dimerization of isatin and 7-hydroxylindole at the C-2 and C-6 positions leading to form a novel purple product. Also, docking studies supported the experiments well and predicted some important residues, which is conductive to perform site-directed mutagenesis to expand the substrate specificities. Discussion Residues involved in binding different indoles. Table 3. Residues involved in binding different indoles. Substrate Residues within 4 A˚ Indole Val102 Leu105 Glu106 Ala109 Gln132 Glu136 Phe196 Glu197 Leu204 4-Methylindole Glu106 His139 Gln143 Glu197 Thr201 Leu204 Phe205 Phe223 Ser227 Glu231 His234 5-Methylindole Val102 Leu105 Glu106 Ala109 Phe177 Phe196 Glu197 Leu204 Glu231 7-Methylindole Phe98 Val102 Glu106 Glu143 Phe196 Glu197 Thr201 Leu204 Phe205 Glu231 4-Chloroindole Glu106 His139 Gln143 Glu197 Thr201 Leu204 Phe205 Met209 Phe223 Glu231 His234 Ser237 7-Chloroindole Phe98 Val102 Glu106 Glu143 Phe196 Glu197 Thr201 Leu204 Phe205 5-Methyoxyindole Phe98 Glu106 Gln143 Phe196 Glu197 Thr201 Leu204 Phe205 Phe208 Met209 Phe223 Ser 227 Glu231 doi:10.1371/journal.pone.0044313.t003 September 2012 \| Volume 7 \| Issue 9 \| e44313 September 2012 \| Volume 7 \| Issue 9 \| e44313 PLOS ONE \| www.plosone.org 6 Biotransformation of Indoles by Phenol Hydroxylase Biotransformation of Indoles by Phenol Hydroxylase Bacterial strains, plasmids and growth conditions The bacterial strains and plasmids used in this study were listed in Table 4. The vector plasmids, pMD18-T and pET-28a(+), were purchased from TaKaRa Co. Ltd. (Dalian, China) and Novagen (USA), respectively. pET-28a(+) containing PHKLMNOP cloned from strain W1 was constructed in this study. Strain W1 was cultured as previously described [39]. E. coli strains were grown in lysogeny broth (LB) medium containing kanamycin (Kan, 30 mg/ mL) at 37uC and 150 r/min. For PH gene expression, E. coli BL21 (DE3) harboring the appropriate pET-28a(+) hybrid plasmid was induced by addition of IPTG (1 mM). Construction of site-directed mutants Mutants were produced by site-directed mutagenesis by the method of Tang et al [41]. The mutants with mutation position Asn-202 and His-139 were obtained using plasmid pET-28a(+)/ PH_IND as the template by three-step PCR. The mutagenic primers were listed in Table S4. The resulting PCR products (1.1 kb) were completely sequenced, digested with Aat II and Sal I, PLOS ONE \| www.plosone.org September 2012 \| Volume 7 \| Issue 9 \| e44313 7 Biotransformation of Indoles by Phenol Hydroxylase Figure 5. Summary of transformation pathways of indole by various oxygenases. DO. dioxygenase; SO. styrene oxygenase; MO. monooxygenase. R represents for substitute group i.e. methyl-, chloro-, methyoxy-, etc. The pathways catalyzed by strain PH_IND could be proposed by C-3 oxidation and C-7 oxidation pathways. The indole is firstly hydroxylated at the C-3 positions to form indoxyl (3-hydroxyindole) by strain PH_IND, which undergoes further oxidation to form isatin as well as the indigoids precursors. Finally, two molecules of indoxyl polymerize to form indigo, and indoxyl can form indirubin with isatin. And the new compound is formed by 7-hydroxyindole and isatin. doi:10.1371/journal.pone.0044313.g005 Figure 5. Summary of transformation pathways of indole by various oxygenases. DO. dioxygenase; SO. styrene oxygenase; MO. monooxygenase. R represents for substitute group i.e. methyl-, chloro-, methyoxy-, etc. The pathways catalyzed by strain PH_IND could be proposed by C-3 oxidation and C-7 oxidation pathways. The indole is firstly hydroxylated at the C-3 positions to form indoxyl (3-hydroxyindole) by strain PH_IND, which undergoes further oxidation to form isatin as well as the indigoids precursors. Finally, two molecules of indoxyl polymerize to form indigo, and indoxyl can form indirubin with isatin. And the new compound is formed by 7-hydroxyindole and isatin. doi:10.1371/journal.pone.0044313.g005 and cloned into the plasmid pET-28a(+)/PH digested with the same enzymes. The resulting plasmid were designated as pET- 28a(+)/PH -Asn-202 and pET-28a(+)/PH -His-139. For protein expression, the recombinant plasmids were introduced into E. coli BL21 (DE3), and the recombinant strains were designated as PH_IND-Asn-202 and PH_IND-His-139, respectively. Table 4. Bacterial strains and plasmids used in this study. Strains or plasmids Characteristics Source Strains Arthrobacter sp. W1 Wild type, CGMCC 4763, phenol+, indole+, benzoic acid+, salicylic acid+, aniline+, catechol+, o (m, p)-cresol+, hydroquinone+, o (m, p)-aminophenol+, m (p)-nitroaniline+ (39) E. coli JM109 recA1, endA1, gyrA96, thi-1, hsdR17 (rk 2mk +), e142 (mcrA2), supE44, relA1, D (lac-proAB)/F9 [traD36, proAB+, lac Iq, lacZDM15] TaKaRa E. Supporting Information Figure S1 Characteristics of crude PH_IND. A. The effects of pH on the enzyme activity. Assay mixtures contained different pH (5.0–11.0), 2.5 mM NADH, 0.02 mg protein and 200 mg/L phenol at 20uC; B. The effects of salt concentration on the enzyme activity. Assay mixtures contained 50 mM Tris-HCl (pH 8.0), 2.5 mM NADH, 0.02 mg protein and different concentrations of NaCl (0.5–5%) at 20uC; C. The effects of metal ions on the enzyme activity. Assay mixtures contained 50 mM Tris-HCl (pH 8.0), 2.5 mM NADH, 0.02 mg protein and 200 mg/L phenol at 20uC with 1 mM of each metal ions; D. The effects of temperature on the enzyme activity. Assay mixtures contained 50 mM Tris-HCl (pH 8.0), 2.5 mM NADH, 0.02 mg protein and 200 mg/L phenol at different temperature (20–70uC). (PDF) For thin layer chromatography (TLC) analysis, the ethyl acetate samples were removed with stream nitrogen. The resulting dried pigment cells were dissolved in dichloromethane to a volume of 500 mL. The thickness of the TLC plate was 0.15,0.2 mm (silica gel 60 F254), on which 200 mL of sample was added by several times, and then the TLC plates were resolved with a solvent mixture of dichloromethane-methanol (50:1, v/v). Following TLC separation of the indigoid pigments described above, the separated pigment-containing samples were isolated from the TLC plate and the pigments were extracted from the silica matrix with dichloromethane. The solvents were evaporated, and the pigments were suspended in d6-Me2SO for liquid chromatography and mass spectrometry (HPLC-MS) analysis (Hewlett Packard 1100 MSD, America). The HPLC was operated as: RX-C18 column (150 mm62.1 mm Agilent, CA, USA); 65% (v/v) CH3OH (in H2O containing 0.1% formic acid) for 10 min, followed by a 65–75% (v/v) CH3OH linear gradient over 20 min; flow rate, 0.2 mL/min; column temperature, 25uC; UV detection, 242 nm. MS was equipped with a standard API-1 atmospheric pressure chemical ionization (APCI) source in the positive or negative ion mode. N2 was used as a sheath gas (50 p.s.i.), vaporizer temperature was set to 350uC, and the corona current was maintained at 5 mA. The capillary was set at 220uC and 25 V (or 225 V in the negative mode). The tube lens voltage was set to 80 V (or to 296 V in the negative mode). The collision-induced dissociation was set to 230 V in tandem mass spectrometry experiments. Biotransformation of Indoles by Phenol Hydroxylase All the NMR experiments were performed on a Varian INOVA-500 MHz spectrometer at 298 K (Bruker Avance 500 Hz, Switzerland). Detailed structural information for purple product from indole was obtained by three separate two- dimensional (1H-1H COSY, 1H-13C HSQC and 1H-13C HMBC) NMR analysis. All the NMR experiments were performed on a Varian INOVA-500 MHz spectrometer at 298 K (Bruker Avance 500 Hz, Switzerland). Detailed structural information for purple product from indole was obtained by three separate two- dimensional (1H-1H COSY, 1H-13C HSQC and 1H-13C HMBC) NMR analysis. Biotransformation and products identification The nucleotide sequence of the phenol hydroxylase gene cluster (4606 bp) of Arthrobacter sp. W1 has been submitted to GenBank with the accession number FJ610336. All biotransformation reactions were performed in 50-mL flasks at 30uC and 150 r/min. The control group was set using host cells of E. coli BL21 (DE3). For whole cell preparation, cells were harvested by centrifugation (8000 r/min, 15 min), washed with phosphate sodium buffer (0.1 M, pH 7.0), and resuspended in the same buffer to OD600 = 2.0. The reaction mixtures consisted of 20 mL cell suspensions, 1 mM glucose and 0.2 g/L indole derivatives using dimethyl sulfoxide (DMF) as a cosolvent. After completion of the reactions (20 h), the mixtures were extracted with an equal volume of ethyl acetate. Modeling of PHNW1 and substrates docking The initial three-dimensional model of the PHNW1 component was built by homology-modeling method [42]. The crystal structure of PH from Pseudomonas sp. OX1 solved by Sazinsky et al. (PDB ID: 2INP) was chosen as the template based on the result of protein sequence blast (BLASTP) [29]. The general simulation method and model quality validation were similar to the previous report [42]. Modeller 9v8 was used to construct the homology modeling of PHN W1 containing diiron atoms. To find interactions between active site and protein surface, the most possible tunnel was identified using CAVER [43]. The effects of pH (50 mM Na2HPO4/NaH2PO4 (pH 6.0–7.0) and Tris-HCl (pH 8.0–11.0)), temperature (20–70uC), phenol concentration (50–3000 mM), salt concentration (0.5%–5%) and metal ions on enzyme activities were determined. Molecular docking studies performed by AutoDock Vina 1.1.1 were used to identify the important interactions between PHN and indole derivatives [44]. The coordinate files of ligands were prepared by PRODRG server. Ca of Leu105 was defined as the center of the 40640640 grid box. PyMOL was used to visualize the docking results and measure the distance between two atoms. The proteins of strain PH_IND and its mutants were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with 5% and 15% acrylamide for the concentrating and separating gels, respectively. Enzyme assay Enzyme activities of the cell extracts were determined at room temperature by UV/Vis spectrophotometer. The cell pellets were disrupted by sonication (225 W at 4uC for 30 min, Ultrasonic processor CPX 750), and then the cell debris was removed by centrifugation at 22000 r/min for 20 min at 4uC. The supernatant was immediately used for the assays of enzyme activities. For phenol hydroxylase activity, assay mixtures contained 50 mM Tris-HCl (pH 8.0), 2.5 mM NADH, 0.02 mg protein and 200 mg/ L phenol. Each assay was started with the addition of NADH to the reaction mixture containing fresh crude extracts. Activity of phenol hydroxylase was measured by the consumption of NADH at 340 nm. One unit of activity was defined as the amount of enzyme that caused the oxidation of 1 mM NADH per min in the presence of phenol. The kinetic parameters (kcat, Km and kcat/Km) were determined using whole cells as described previously [35]. Construction of site-directed mutants coli BL21 (DE3) F2, ompT, hsdSB (rB 2mB 2), gal (l c I 857, ind1, Sam7, nin5, lacUV5-T7gene1), dcm (DE3) TaKaRa Plasmids pMD18-T Vector used for cloning TaKaRa pET-28a (+) Vector used for expressing Novagan pET-28a (+)/PH The recombinant plasmid containing PH genes This study doi:10.1371/journal.pone.0044313.t004 September 2012 \| Volume 7 \| Issue 9 \| e44313 PLOS ONE \| www.plosone.org 8 References 1. Huxtable RJ (2001) The mutability of blue. MolInterv1: 141–144 13. Gillam EM, Notley LM, Cai H, De Voss JJ, Guengerich FP (2000) Oxidation of indole by cytochrome P450 enzymes. Biochemistry 39: 13817–13824. 2. Choi HS, Kim JK, Cho EH, Kim YC, Kim JI, et al. (2003) A novel flavin- containing monooxygenase from Methylophaga sp. strain SK1 and its indigo synthesis in Escherichia coli. Biochem Biophys Res Commun 306: 930–936. 14. Doukyu N, Toyoda K, Aono R (2003) Indigo production by Escherichia coli carrying the phenol hydroxylase gene from Acinetobacter sp. strain ST-550 in a water-organic solvent two-phase system. Appl Microbiol Biotechnol 60: 720– 725. 3. Ensley BD, Ratzkin BJ, Ossulund TD, Simon MJ, Wackett LP, et al. (1983) Expression of naphthalene oxidation genes in Escherichia coli results in the biosynthesis of indigo. Science 222: 167–169. 15. Doukyu N, Arai T, Aono R (1998) Effects of organic solvents on indigo formation by Pseudomonas sp. strain ST-200 grown with high levels of indole. Biosci Biotechnol Biochem 62: 1075–1080. y g 4. Gillam EM, Aguinaldo AM, Notley LM, Kim D, Mundkowski RG, et al. (1999) Formation of indigo by recombinant mammalian cytochrome P450. Biochem Biophys Res Commun 265: 469–472. 16. Drewlo S, Bramer CO, Madkour M, Mayer F, Steinbuchel A (2001) Cloning and expression of a Ralstonia eutropha HF39 gene mediating indigo formation in Escherichia coli. Appl Environ Microbiol 67: 1964–1969. 5. Qu YY, Pi WQ, Ma F, Zhou JT, Zhang XW (2010) Influence and optimization of growth substrates on indigo formation by a novel isolate Acinetobacter sp. PP-2. Bioresour Technol 101: 4527–4532. 17. Hart S, Koch KR, Woods DR (1992) Identification of indigo-related pigments produced by Escherichia coli containing a cloned Rhodococcus gene. J Gen Microbiol 138: 211–216. 6. Kim JY, Kim JK, Lee SO, Kim CK, Lee K (2005) Multi component phenol hydroxylase-catalysed formation of hydroxyindoles and dyestuffs from indole and its derivatives. Lett Appl Microbiol 41: 163–168. 18. O’Connor KE, Hartmans S (1998) Indigo formation by aromatic hydrocarbon- degrading bacteria. Biotechnol Lett 20: 219–233. 7. Kim JY, Lee K, Kim Y, Kim CK, Lee K (2003) Production of dyestuffs from indole derivatives by naphthalene dioxygenase and toluene dioxygenase. Lett Appl Microbiol 36: 343–348. 19. Rui L, Reardon KF, Wood TK (2005) Protein engineering of toluene ortho- monooxygenase of Burkholderia cepacia G4 for regionspecific hydroxylation of indole to from various indigoid compounds. Appl Microbiol Biotechnol 66: 422– 429. 8. Table S3 Oligonucleotide primers used in this study. (PDF) Figure S5 Identification of new purple product derived from indole. A. Mass spectra; B. 1H NMR spectra; C. 13C NMR spectra; D. 1H-1H COSY; E. 1H-13C HSQC; F. 1H-13C HMBC. The conditions for each spectrum were the same with those described above. (PDF) Table S4 Primers used for site-directed mutagenesis. (PDF) Author Contributions Conceived and designed the experiments: YQ SS HZ QM. Performed the experiments: SS HZ QM XZ. Analyzed the data: YQ SS HZ QM. Conceived and designed the experiments: YQ SS HZ QM. Performed the experiments: SS HZ QM XZ. Analyzed the data: YQ SS HZ QM. Contributed reagents/materials/analysis tools: XL HZ XZ JZ. Wrote the paper: YQ SS HZ QM. Figure S6 SDS-PAGE analysis of protein samples of strain PH_IND and its mutants. Line 1. Cell extracts of strain PH_IND; Line 2. Cell extracts of strain PH_IND-Asn-202; Line 3. Contributed reagents/materials/analysis tools: XL HZ XZ JZ. Wrote the paper: YQ SS HZ QM. (PDF) Table S1 Amino acid identity between PHW1 and other binuclear iron hydroxylases. (PDF) Table S2 Production of dyestuffs from indole deriva- tives by Escherichia coli expressing different oxygenas- es. (PDF) Table S3 Oligonucleotide primers used in this study. (PDF) Supporting Information Figure S2 Gene cluster analysis and primary structure alignment of PHN component. A. PH gene cluster from strain W1 and related strains Comamonas testosteroni R5, Ralstoniaeu- tropha E2, Pseudomonas sp. CF600, Pseudomonas putida H. B. Structural alignment of primary structure of PHNs from Pseudomonas sp. OX1, Pseudomonas sp. CF600, Acinetobacter radio- resistens S13, Arthrobacter sp. W1, Ralstonia eutropha E2 and Bacillus thermoleovorans sp. A2. The top line shows the secondary structure of PHN of Pseudomonas sp. OX1. The conserved residues are shown in red background. Figure S3 Mass spectra of purified indigoid products formed from indoles by strain PH_IND. A. Products of indole transformation; B. Products of 4-methylindole transforma- tion; C. Products of 5-methylindole transformation; D. Products of 7-methylindole transformation; E. Products of 4-chloroindole transformation; F. Products of 7-chloroindole transformation; G. September 2012 \| Volume 7 \| Issue 9 \| e44313 PLOS ONE \| www.plosone.org 9 Biotransformation of Indoles by Phenol Hydroxylase Products of 5-methoxyindole transformation. HPLC-MS condi- tions were as follows: HPLC, 65% (v/v) CH3OH (in H2O containing 0.1% formic acid) for 10 min, followed by a 65–75% (v/v) CH3OH linear gradient over 20 min; MS was equipped with a standard API-1 atmospheric pressure chemical ionization (APCI) source in the positive or negative ion mode. N2 was used as a sheath gas (50 p.s.i.), vaporizer temperature was set to 350uC, and the corona current was maintained at 5 mA. The capillary was set at 220uC and 25 V (or 225 V in the negative mode). The tube lens voltage was set to 80 V (or to 296 V in the negative mode). The collision-induced dissociation was set to 230 V in tandem mass spectrometry experiments. (PDF) Products of 5-methoxyindole transformation. HPLC-MS condi- tions were as follows: HPLC, 65% (v/v) CH3OH (in H2O containing 0.1% formic acid) for 10 min, followed by a 65–75% (v/v) CH3OH linear gradient over 20 min; MS was equipped with a standard API-1 atmospheric pressure chemical ionization (APCI) source in the positive or negative ion mode. N2 was used as a sheath gas (50 p.s.i.), vaporizer temperature was set to 350uC, and the corona current was maintained at 5 mA. The capillary was set at 220uC and 25 V (or 225 V in the negative mode). The tube lens voltage was set to 80 V (or to 296 V in the negative mode). Supporting Information The collision-induced dissociation was set to 230 V in tandem mass spectrometry experiments. (PDF) Cell extracts of strain PH_IND-His-139; M. Protein markers. Arrows show the positions of the six ORFs. ORF1. 10.4 kDa; ORF2. 37.6 kDa; ORF3. 10.5 kDa; ORF4. 59.2 kDa; ORF5. 13.5 kDa; ORF6. 38.6 kDa. SDS-PAGE was performed on 5% and 15% acrylamide concentrations for the concentrating and separating gels, respectively. (PDF) Figure S7 Biotransformation of indole by strain PH_IND and its mutans. A. Indole biotransformation by strain PH_IND; B. Indole biotransformation by strain PH_IND-Asn-202; C. Indole biotransformation by strain PH_IND-His-139. The left bottles were the control groups at 0 h; the right bottles were the test groups at 12 h. (PDF) Figure S4 1H NMR spectra of purified indigoid prod- ucts formed from indoles by strain PH_IND. A. Products of indole transformation; B. Products of 4-methylindole transforma- tion; C. Products of 5-methylindole transformation; D. Products of 7-methylindole transformation; E. Products of 4-chloroindole transformation; F. Products of 7-chloroindole transformation; G. Products of 5-methoxyindole transformation. 1H NMR was carried out at 298 K with Bruker Avance II 400 M and 400 MHz instrument and the samples were dissolved in d26 Me2SO. 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J Mol Evol 56: 435– 445. 37. Carredano E, Karlsson A, Kauppi B, Choudhury D, Parales RE, et al. (2000) Substrate binding site of naphthalene 1,2-dioxygenase: functional implications of indole binding. J Mol Biol 296: 701–712. 28. Powlowski J, Shingler V (1994) Genetics and biochemistry of phenol degradation by Pseudomonas sp. CF600. Biodegradation 5: 219–236. 38. Kim D, Wu ZL, Guengerich FP (2005) Analysis of coumarin 7-hydroxylation activity of cytochrome P450 2A6 using random mutagenesis. J Biol Chem 280: 40319–40327. 29. Sazinsky MH, Dunten PW, McCormick MS, DiDonato A, Lippard SJ (2006) X- ray structure of a hydroxylase-regulatory protein complex from a hydrocarbon- oxidizing multi component monooxygenase, Pseudomonas sp. OX1 phenol hydroxylase. Biochemistry 45: 15392–15404. 39. Wang P, Qu YY, Zhou JT (2009) Biodegradation of mixed phenolic compounds under high salt conditions and salinity fluctuations by Arthrobacter sp. W1. Appl Biochem Biotechnol 159: 623–633. 30. Powlowski J, Sealy J, Shingler V, Cadieux E (1997) On the role of DmpK, an auxiliary protein associated with multi component phenol hydroxylase from Pseudomonas sp. strain CF600. J Biol Chem 272: 945–951. 40. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG (2000) Current protocols in molecular biology. Greene Publishing Associates-Wiley Interscience, New York. 31. Murry LJ, Lippard SJ (2007) Substrate trafficking and dioxygen activation in bacterial multi component monooxygenases. Acc Chem Res 40: 466–474. 41. Tang H, Yao Y, Zhang D, Meng X, Wang L, et al. (2011) A novel NADH- dependent and FAD-containing hydroxylase is crucial for nicotine degradation by Pseudomonas putida. J Biol Chem 286: 39179–39187. cterial multi component monooxygenases. Acc Chem Res 40: 466–47 32. References Guengerich FP, Sorrells JL, Schmitt S, Krauser JA, Aryal P, et al. (2004) Generation of new protein kinase inhibitors utilizing cytochrome P450 mutant enzymes for indigoid synthesis. J Med Chem 47: 3236–3241. 20. Meyer A, Wu¨rsten M, Schmid A, Kohler HP, Witholt B (2002) Hydroxylation of indole by laboratory-evolved 2-hydroxybiphenyl 3-monooxygenase. J Biol Chem 277: 34161–34167. 9. Hoessel R, Leclerc S, Endicott JA, Nobel ME, Lawrie A, et al. (1999) Indirubin, the active constituent of a Chinese anti leukaemia medicine, inhibits cyclin- dependent kinases. Nat Cell Biol 1: 60–67. 21. Murdock D, Ensley BD, Serdar C, Thalen M (1993) Construction of metabolic operons catalyzing the de novo biosynthesis of indigo in Escherichia coli. Nat Biotechnol 11: 381–386. 10. Doukyu N, Nakano T, Okuyama Y, Aono R (2002) Isolation of an Acinetobacter sp. ST-550 which produces a high level of indigo in a water-organic solvent two- phase system containing high levels of indole. Appl Microbiol Biotechnol 58: 543–546. 22. O’Connor KE, Dobson AD, Hartmans S (1997) Indigo formation by microorganisms expressing styrene monooxygenase activity. Appl Environ Microbiol 63: 4287–4291. 11. 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W2891078453.txt	https://molecularbrain.biomedcentral.com/track/pdf/10.1186/s13041-018-0394-3	en		The effect of Neuroligin-2 absence on sleep architecture and electroencephalographic activity in mice	Molecular brain	2,018	cc-by	8,156	Seok et al. Molecular Brain (2018) 11:52 https://doi.org/10.1186/s13041-018-0394-3 RESEARCH Open Access The effect of Neuroligin-2 absence on sleep architecture and electroencephalographic activity in mice Bong Soo Seok1,2, Erika Bélanger-Nelson1, Chloé Provost1, Steve Gibbs1,2 and Valérie Mongrain1,2* Abstract Sleep disorders are comorbid with most psychiatric disorders, but the link between these is not well understood. Neuroligin-2 (NLGN2) is a cell adhesion molecule that plays roles in synapse formation and neurotransmission. Moreover, NLGN2 has been associated with psychiatric disorders, but its implication in sleep remains underexplored. In the present study, the effect of Nlgn2 knockout (Nlgn2−/−) on sleep architecture and electroencephalographic (EEG) activity in mice has been investigated. The EEG and electromyogram (EMG) were recorded in Nlgn2−/− mice and littermates for 24 h from which three vigilance states (i.e., wakefulness, rapid eye movement [REM] sleep, non-REM [NREM] sleep) were visually identified. Spectral analysis of the EEG was performed for the three states. Nlgn2−/− mice showed more wakefulness and less NREM and REM sleep compared to wild-type (Nlgn2+/+) mice, especially during the dark period. This was accompanied by changes in the number and duration of individual episodes of wakefulness and sleep, indexing changes in state consolidation, as well as widespread changes in EEG spectral activity in all states. Abnormal ‘hypersynchronized’ EEG events have also been observed predominantly in Nlgn2−/− mice. These events were mainly observed during wakefulness and REM sleep. In addition, Nlgn2−/− mice showed alterations in the daily time course of NREM sleep delta (1–4 Hz) activity, pointing to modifications in the dynamics of sleep homeostasis. These data suggest that NLGN2 participates in the regulation of sleep duration as well as EEG activity during wakefulness and sleep. Keywords: Neuroligin, Cell adhesion molecule, Knockout mice, Electroencephalography, Sleep regulation, Wakefulness, Delta activity Introduction Research from the last decades suggests that people suffering from a sleep disorder such as insomnia show higher risk of developing medical and/or psychiatric disorders [1, 2]. On the other hand, most psychiatric disorders are associated with sleep disturbances [3]. For instance, decreased sleep efficiency and consolidation is characterizing autism spectrum disorders (ASDs) and schizophrenia [3]. However, the molecular links between sleep alterations and comorbid disorders remain poorly understood. Neurodevelopmental psychiatric disorders in particular, such as ASDs and schizophrenia, have been proposed to originate from changes in the relative * Correspondence: valerie.mongrain@umontreal.ca 1 Research Center and Center for Advanced Research in Sleep Medicine, Hôpital du Sacré-Cœur de Montréal (CIUSSS-NIM), 5400 Gouin West blvd, Montréal, QC H4J 1C5, Canada 2 Department of Neuroscience, Université de Montréal, 2960 chemin de la Tour, Montreal, QC H3T 1J4, Canada balance of excitation to inhibition (E/I) in the central nervous system. For instance, indications of reduced γ-aminobutyric acid (GABA) ergic tone can result in an increased E/I ratio in ASDs [4], while N-methyl-D-aspartate (NMDA) receptor hypofunction likely also modifies the E/I ratio in schizophrenia [5]. Neuroligins (NLGNs) are postsynaptic adhesion proteins that have been shown to regulate synaptogenesis, synaptic function, and E/I balance [6, 7]. Importantly, mutations in Nlgn genes have been linked to the abovementioned neuropsychiatric disorders [6], such as missense mutations of Nlgn2 in schizophrenia [8]. Therefore, understanding the roles of NLGNs in the regulation of sleep quantity and quality could help to identify mechanisms underlying the relationship between neuropsychiatric disorders and sleep disturbances. Research in both rodents and flies has indeed provided support for an implication of specific NLGNs in sleep © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Seok et al. Molecular Brain (2018) 11:52 regulation [9]. In mice, the absence of NLGN1 (knockout, KO) results in a decreased duration of wakefulness and increased duration of NREM sleep accompanied by changes in wakefulness and NREM sleep quality as quantified using EEG spectral analysis and slow wave detection [10, 11]. In rats, the absence of NLGN3 (KO) was also recently shown to impact sleep duration and quality, and more precisely to result in decreased NREM sleep duration and increased REM sleep duration, and in multiple modifications in EEG activity in all states [12]. Nlgn3R451C knock-in mice, carrying an ASD-associated missence mutation, have been shown to exhibit normal sleep architecture but a decrease in low frequency (< 10 Hertz [Hz]) activity during NREM sleep [13]. To our knowledge, no data is available yet regarding the sleep phenotype in rodents with genetic manipulation of Nlgn2 or Nlgn4. NLGN2 is preferentially localized at inhibitory synapses [7, 14]. It regulates inhibitory synaptic transmission, which has been unveiled through manipulations of Nlgn2 expression level. Nlgn2 overexpression in mice increased the frequency of miniature inhibitory postsynaptic currents (mIPSCs) in pyramidal cells of the prefrontal cortex compared to control mice [15]. On the other hand, in the absence of NLGN2, both the amplitude and frequency of mIPSCs are decreased in the mouse medial prefrontal cortex [16] and the amplitude of mIPSCs is decreased in granule cells of the dentate gyrus of the hippocampus [17]. These modulations of NLGN2 level did not affect miniature excitatory postsynaptic currents [15, 16], thus modifying the E/I balance [17]. Of importance is that NLGN2 was shown to impact inhibition in a cell type-specific manner within the same circuit (i.e., neocortex) [18]. Inhibitory/GABAergic transmission has been implicated in sleep regulation, notably in the context of the flip-flop switch model of sleep regulation [19], and in that of the development of hypnotic drugs for insomnia treatment [20]. Indeed, the flip-flop switch model suggests that GABAergic sleep-promoting regions are inhibiting monoaminergic wake-promoting regions to control vigilance state transitions [19], whereas hypnotic drugs such as benzodiazepines are GABAA receptor agonists promoting light NREM sleep [21, 22]. In the absence of NLGN2, the decrease in mIPSC could result in weakening of sleep-promoting regions and to opposite effects than GABAA receptor agonists, therefore to more wakefulness and less sleep. Given the role of GABAA receptors in shaping EEG activity [20], and the results showing impaired mechanisms linked to the clustering of GABAA receptors at synaptic sites in Nlgn2−/− mice [23], alterations in EEG activity are also expected in absence of NLGN2. Page 2 of 11 The general objective of this study was to evaluate the role of NLGN2 in wakefulness and sleep regulation. EEG-EMG recordings have been used to assess changes in normal/undisturbed wake/sleep architecture as well as EEG activity in mice lacking NLGN2 (Nlgn2−/− mice) in comparison with wild-type (Nlgn2+/+) and heterozygous (Nlgn2+/−) littermates. Results are indicative of changes in both wake/sleep quantity and quality in Nlgn2−/− mice, and reveal a state-dependent occurrence of abnormal EEG events. Our findings thus suggest that NLGN2, in addition to NLGN1 and NLGN3, modulates both the architecture and quality of wakefulness and sleep, which will help to understand mechanisms underlying comorbidity between brain diseases and sleep disorders. Methods Animals Mixed genetic background (B6;129-Nlgn2tm1Bros/J) mice were purchased from Jackson Laboratories and bred on site by placing one male and one female Nlgn2+/− mice in a breeding cage to generate three genotypes: homozygous Nlgn2−/− mice, and Nlgn2+/− and Nlgn2+/+ littermates. Nlgn2−/− mice were previously generated by homologous recombination [24]. Briefly, a targeting vector containing a neomycin resistance cassette was electroporated into 129Sv-derived embryonic stem cells to disrupt the sequence of the first Nlgn2 exon covering the translational start site and 380 base-pair of the 5′ coding sequence, and recombinant stem cells were transfected into C57BL/6 blastocysts. Male mice only were used in our study to reduce variability due to sex because sex differences in sleep architecture and EEG activity have been reported [25, 26]. We studied 14 Nlgn2+/+ mice, 14 Nlgn2+/− mice, and 12 Nlgn2−/− mice. At the time of the surgery (see below), Nlgn2+/+ mice were 69.2 ± 2 days old (range 57 to 83 days) and weighing 27.5 ± 0.6 g; Nlgn2+/− mice were 71.8 ± 1.9 days old (range 57 to 83 days) and 27.3 ± 0.8 g; and Nlgn2−/− mice were 70.8 ± 2.0 days old (range 58 to 83 days) and 24.3 ± 0.8 g. Weight was significantly different between genotypes (F2,37 = 6.3, p = 0.004). Nlgn2−/− mice were significantly lighter than both Nlgn2+/+ and Nlgn2+/− mice (p = 0.003 and p = 0.005, respectively) while there was no significant difference between Nlgn2+/+ and Nlgn2+/− mice (p = 0.8). Electrode implantation surgery The surgery of electrode implantation for recording the EEG and EMG was performed as detailed previously [10, 27]. Briefly, when mice were between 9 and 10 weeks of age, EEG/EMG implantation surgery was performed under deep Ketamine/Xylazine anesthesia (120/10 mg/kg, intraperitoneal injection). Mice were placed in a stereotaxic frame and two gold-plated Seok et al. Molecular Brain (2018) 11:52 screws (diameter 1.1 mm), which served as EEG electrodes, were screwed through the skull over the right cerebral hemisphere (anterior: 1.7 mm lateral to midline, 1.5 mm anterior to bregma; posterior: 1.7 mm lateral to midline, 1.0 mm anterior to lambda). An additional screw, which served as a reference, was implanted on the right hemisphere (2.6 mm lateral to midline, 0.7 mm posterior to bregma). Three anchor screws were implanted on the left hemisphere. Two gold wires serving as EMG electrodes were inserted between neck muscles. EEG and EMG electrodes were soldered to a connector and secured on the skull with cement. After four days of recovery, mice were connected to a swivel contact and habituated to the cabling condition for a week before recording. Protocol and EEG recording Starting 2 weeks before surgery and throughout the experiment, mice were housed in individual cages and kept under a 12-h light/12-h dark cycle at a temperature between 23 and 25 °C, with free access to food and water. Electrophysiological signals were continuously recorded for 24 h starting at light onset (Zeitgeber time 0: ZT0). Signals were amplified (Lamont amplifiers), sampled at 256 Hz, and filtered using the software Stellate Harmonie (Natus, San Carlos, CA). A bipolar montage was subsequently employed for identification of vigilance states and spectral analysis (signal used representing the difference between the anterior and the posterior EEG electrodes). EEG analyses The sampled signals were segmented in 4-s epochs to visually identify vigilance states (wakefulness, NREM sleep, REM sleep) based on EEG and EMG features characterizing the different vigilance states as previously described [28]. During state identification, artifacts such as abnormal peaks and abnormal mixture of frequencies, and transitions were identified and excluded from subsequent EEG spectral analysis (see below). Parameters related to wakefulness and sleep duration/architecture as well as to wakefulness and sleep consolidation/fragmentation were directly calculated from vigilance state identification. More precisely, the time spent in vigilance states (in min) was calculated for the full 24-h, the first 12-h (Light period), the second 12-h (Dark period) and per hour. Mean duration of individual episodes of vigilance states (in sec) and total number of individual vigilance state episodes during the 12-h Light and the 12-h Dark have also been computed. Spectral analysis was performed to investigate EEG activity during the different vigilance states (as a measure of state quality). The bipolar EEG signal was decomposed into its constituent frequency components using fast Fourier transform. The EEG power density was Page 3 of 11 calculated between 1 and 50 Hz (1-Hz resolution) during wakefulness, NREM sleep and REM sleep for the full 24-h. In addition to absolute power spectra computed as performed previously [10], relative power density was also computed for which the activity of each Hz-bin was expressed relative to the mean power density of all bins of all states of the mouse as previously done [13, 27]. Absolute power spectra can quantify genotype differences in vigilance state quality while providing information regarding the cytoarchitecture of the cerebral cortex, whereas, relative power spectra removes the variability of signal due to, for instance, general alterations in cortical structure and/or differences in the depth of EEG electrodes, and can allow for more accurate observations of vigilance state-specific genotype differences. The time course of NREM sleep EEG delta (between 1 and 4 Hz) activity was computed to analyze the dynamics of the sleep homeostat [29, 30]. The change in absolute delta power was monitored throughout 18 intervals over the 24-h (12 intervals comprising the same number of epochs of NREM sleep during the light phase and 6 intervals with the same number of NREM sleep epochs in the dark phase) similar to previously performed [10]. Relative delta power dynamics was also computed with respect to the 24-h mean delta power of each animal, which allow to better highlight delta activity dynamics by removing intra-individual differences in absolute activity [27, 28]. The time course of theta (6–9 Hz) activity during wakefulness was also analyzed in absolute and relative values for 18 intervals comprising 6 equal intervals during the 12-h Light and 12 equal intervals during the 12-h Dark to take into account the 24-h distribution of wakefulness. For this last analysis, one Nlgn2+/+ mouse was not included because of an absence of wakefulness for one interval. Abnormal EEG event identification During vigilance state identification, occasional and distinct events of high amplitude EEG bursts were observed. Since alteration in E/I balance has been postulated as a mechanism underlying epileptogenesis and seizure generation [31, 32], this abnormal EEG activity might be indicative of hypersynchronisation and/or epileptiform activity. Thus, the abnormal EEG events were marked on EEG traces (and excluded from the spectral analysis of the vigilance states described above). More precisely, the number and duration of abnormal events were quantified by marking them according to the following two criteria: amplitude at least twice that of the background EEG signal, and duration of at least one second. Two events separated by less than 0.5 s were considered as a single one. The EEG power density was calculated between 1 and 50 Hz (1-Hz resolution) for abnormal events, from which the peak frequency was determined for each mouse. Seok et al. Molecular Brain (2018) 11:52 Statistical analyses Vigilance state variables and event features calculated for the 24-h, 12-h Light and 12-h Dark were compared between genotypes using one-way analyses of variance (ANOVAs). Vigilance state variables with significant genotype difference were decomposed using Tukey post hoc or planned comparisons. Vigilance state variables calculated per hour and time course of delta and theta activity per intervals were analyzed using two-way repeated-measure ANOVAs. Significance level for repeated-measure ANOVAs was adjusted by Huynh-Feldt correction and significant differences were decomposed by planned comparisons. Finally, comparison of the number of events between the 12-h Light and 12-h Dark as well as between states in Nlgn2−/− mice was performed using one-way repeated-measure ANOVAs. Data are reported as mean and standard error of the mean (SEM), and the threshold for statistical significance was set to 0.05. Results More time spent awake in Nlgn2−/− mice During the full 24-h recording, Nlgn2−/− mice spent more time in wakefulness and less time in REM sleep compared to Nlgn2+/+ littermates (Fig. 1a). A significant Page 4 of 11 difference in REM sleep amount was also observed between Nlgn2+/− mice and the other two genotypes, indicating a gene dosage-dependent effect. The increased time spent in wakefulness in Nlgn2−/− mice originated from a change specific to the 12-h Dark period during which significant differences were observed in comparison to both Nlgn2+/− and Nlgn2+/+ littermates (Fig. 1a). Also specifically for the 12-h Dark period, Nlgn2−/− mice showed significantly less NREM sleep than Nlgn2+/− and Nlgn2+/+ mice (Fig. 1a). Less REM sleep in both Nlgn2−/ − and Nlgn2+/− mice compared to Nlgn2+/+ mice was observed for the 12-h Light period, while less REM sleep in Nlgn2−/− mice compared to Nlgn2+/− and Nlgn2+/+ mice was found for the 12-h Dark period (Fig. 1a). These observations suggest impairment in mechanisms generating sleep in the absence of NLGN2. The 24-h distribution of wakefulness and sleep states in Nlgn2−/− mice was further analyzed using hourly values (Fig. 1b). The typical predominance of sleep during the light period and of wakefulness during the dark period was equally observed in all three genotypes. Moreover, the distribution was not significantly affected by genotype for all three vigilance states indicating a dispersed effect of the mutation that accumulates over Fig. 1 Time spent (min ± SEM) in wakefulness, NREM sleep and REM sleep in Nlgn2+/+, Nlgn2+/− and Nlgn2−/− mice measured using EEG and EMG recordings. a) Total time spent in wake, NREM sleep and REM sleep for the total 24 h recording, the 12 h Light and the 12 h Dark periods. Significant genotype effects were found for 24 h wake (F2,37 = 4.8, p = 0.014), 24 h REM sleep (F2,37 = 16.9, p < 0.0001), 12 h Light REM sleep (F2,37 = 8.5, p < 0.001), 12 h Dark wake (F2,37 = 6.8, p = 0.003), 12 h Dark NREM sleep (F2,37 = 6.4, p = 0.004) and 12 h Dark REM sleep (F2,37 = 6.4, p = 0.004). No significant genotype effect was found for 24 h NREM sleep (F2,37 = 2.7, p = 0.08), 12 h Light wake (F2,37 = 1.3, p = 0.3) and 12 h Light NREM sleep (F2,37 = 1.1, p = 0.3). Stars show significant post-hoc Tukey HSD comparisons between indicated genotypes (: p < 0.05; : p < 0.01). b) Hourly distribution of wake, NREM sleep and REM sleep. For wakefulness, significant genotype and time effects were found (respectively, F2,37 = 4.8, p = 0.014 and F23,851 = 51.3, p < 0.0001), but no significant interaction was observed (F46,851 = 1.2, p = 0.14). For NREM sleep, a significant time effect was found (F23,851 = 47.0, p < 0.0001), but no significant genotype effect or interaction was observed (respectively, F2,37 = 2.7, p = 0.08 and F46,851 = 1.3, p = 0.07). For REM sleep, significant genotype and time effects were found (respectively, F2,37 = 16.9, p < 0.0001 and F23,851 = 52.3, p < 0.0001), but no significant interaction was observed (F46,851 = 0.8, p = 0.7). Grey backgrounds indicate the 12 h dark period Seok et al. Molecular Brain (2018) 11:52 Page 5 of 11 many hours to result in the changes in wakefulness and sleep amount described above. Increased consolidation of vigilance states in Nlgn2 mice −/− The mean duration of individual episodes of vigilance states and the total number of individual episodes of each state were then calculated in Nlgn2−/− mice separately for the 12-h Light and Dark periods to better understand the origin of alterations in vigilance state duration. Moreover, these variables are indicative of wakefulness and sleep consolidation/fragmentation. The mean duration of individual episodes of NREM sleep was longer in Nlgn2−/− mice than in the two other genotypes during the 12-h Light period, and the same finding emerged regarding the mean duration of individual wakefulness episodes during the 12-h Dark period (Fig. 2a). Concerning the number of individual episodes, it was lower in Nlgn2−/− mice than in Nlgn2+/+mice only for all three vigilance states and for both the 12-h Light and Dark periods (Fig. 2b). In sum, longer and fewer episodes of the different vigilance states suggest increased wakefulness and sleep consolidation in the absence of NLGN2. Abnormal EEG events in Nlgn2−/− mice In the course of EEG visual inspection, abnormal bursts of high amplitude ‘hypersynchronized’ EEG activity were observed predominantly in Nlgn2−/− animals and in wakefulness and REM sleep (Fig. 3a). These abnormal events were observed in 3 out of 14 Nlgn2+/+ mice (21.4% of the group; between 1 and 26 events per mouse), in 5 out of 14 Nlgn2+/− mice (35.7%; between 1 and 16 events per mouse), and in all 12 Nlgn2−/− mice (between 1 and 2116 events per mouse). Spectral analysis of these events revealed a high peak in the theta range (i.e., 4–8 Hz; Fig. 3b), with an average peak frequency of 6.5 ± 1.0 Hz in Nlgn2+/+ mice, 7.7 ± 1.4 Hz in Nlgn2+/− mice, and 6.2 ± 0.1 Hz in Nlgn2−/− mice. The average peak frequency was not significantly affected by genotype (F2,17 = 1.4, p > 0.2), but Nlgn2−/− mice showed significantly more events than the other two genotypes (Fig. 3c). The duration of these abnormal events was also significantly affected by genotype with Nlgn2−/− mice showing longer events than Nlgn2+/− mice (Fig. 3c). In Nlgn2−/− mice, there was no difference in the number of observed events between the 12-h Light and the 12-h Dark periods (Fig. 3d), but events were significantly more frequent during wakefulness than during REM sleep, and very rare during NREM sleep (Fig. 3e). Therefore, the quality of the EEG during both wakefulness and REM sleep seems to be particularly affected by the absence of NLGN2. Widespread changes in EEG activity in Nlgn2−/− mice Fig. 2 Parameters of vigilance state consolidation/fragmentation in Nlgn2+/+, Nlgn2+/− and Nlgn2−/− mice quantified for the 12 h Light and the 12 h Dark periods using EEG and EMG recordings. a) Mean duration of individual episodes of vigilance states. Significant genotype effect was found for 12 h Light NREM sleep (F2,37 = 4.3, p = 0.02) and 12 h Dark wake (F2,37 = 4.9, p = 0.01). No significant genotype effect was found for 12 h Light wake (F2,37 = 3.2, p = 0.052), 12 h Light REM sleep (F2,37 = 0.2, p = 0.8), 12 h Dark NREM sleep (F2,37 = 1.1, p = 0.3), and 12 h Dark REM sleep (F2,37 = 0.1, p = 0.9). b) Number of individual episodes of vigilance states. Significant genotype effects were observed for all states for both the 12 h Light and 12 h Dark (12 h Light wake F2,37 = 3.6, p = 0.04; 12 h Light NREM sleep F2,37 = 3.5, p = 0.04; 12 h Light REM sleep F2,37 = 3.3, p = 0.048; 12 h Dark wake F2,37 = 4.3, p = 0.02; 12 h Dark NREM sleep F2,37 = 4.2, p = 0.02; 12 h Dark REM sleep F2,37 = 3.5, p = 0.04). Stars show significant post-hoc Tukey HSD comparisons between indicated genotypes (: p < 0.05; : p < 0.01). Grey backgrounds indicate the 12 h dark period Spectral analysis of the EEG revealed extensive alterations in spectral activity in all three vigilance states in Nlgn2−/− mice. First, absolute spectral power was higher in Nlgn2−/ − mice than in Nlgn2+/+ mice for most frequencies below 23 Hz during wakefulness, below 38 Hz during NREM sleep and below 28 Hz during REM sleep (Fig. 4a). In general, vigilance state power spectra in Nlgn2+/− mice were very similar to Nlgn2+/+ mice for the absolute computation. The global increase in absolute power in Nlgn2−/− mice compared to Nlgn2+/+ mice could be the result of differences in the general organization of the cerebral cortex that would impact EEG activity in all states. Second, in order to more adequately capture state-specific genotype differences, spectral power was expressed relative to the individual mean of all frequencies of all states. This revealed a different pattern of genotype differences where wakefulness spectral activity was lower in Nlgn2−/− mice than in Nlgn2+/+ mice for some Hz-bins below 8 Hz and all Hz-bins above 14 Hz; NREM sleep spectral activity was higher in Nlgn2−/− mice than in Nlgn2+/+ mice for some Hz-bins below Seok et al. Molecular Brain (2018) 11:52 Page 6 of 11 Fig. 3 Abnormal EEG events in Nlgn2+/+, Nlgn2+/− and Nlgn2−/− mice. a) Representative 12-s EEG traces (black), and corresponding EMG traces (blue), showing abnormal EEG events for mice of the three genotypes during wakefulness (upper traces) and REM sleep (lower traces). Scale bars are the same for the two states of all mice. b) Absolute spectral power of events calculated between 1 and 50 Hz and averaged for each genotype for mice showing events (n = 3 Nlgn2+/+, n = 5 Nlgn2+/−, n = 12 Nlgn2−/−). c) Total number of events (upper panel) observed for the 24 h recording in the three genotypes, and their mean duration for mice showing events (lower panel). The number of events was significantly affected by genotype (F2,37 = 13.7, p < 0.0001). : p < 0.01 between indicated points (planned comparisons). For mice showing events, the duration of events was significantly affected by genotype (F2,17 = 3.7, p < 0.05). : p < 0.05 between indicated points (planned comparisons). d) Number of events calculated separately for the 12 h Light and the 12 h Dark periods in Nlgn2−/− mice only. The number of events did not significantly differ between the 12 h Light and the 12 h Dark periods (F1,11 = 3.5, p = 0.09). e) Number of events calculated separately for the three vigilance states in Nlgn2−/− mice only. The number of events was significantly different between vigilance states (F2,22 = 9.9, p < 0.001). : p < 0.01 compared to the other two states; : p < 0.05 compared to wakefulness 6 Hz, but lower in Nlgn2−/− than in Nlgn2+/+ mice for most Hz-bins between 10 and 19 Hz and all bins above 27 Hz; REM sleep spectral activity was higher in Nlgn2−/ − than Nlgn2+/+ mice for frequencies 4–6 Hz and 11– 13 Hz, but lower in Nlgn2−/− than Nlgn2+/+ mice for frequencies 1–3 Hz, 6–9 Hz, 15–16 Hz, and most Hz-bins above 20 Hz (Fig. 4b). Also in the case of relative computation, vigilance state power spectra in Nlgn2+/− mice were very similar to Nlgn2+/+ mice. Finally, the peak of absolute theta activity during both wakefulness and REM sleep appeared to locate at slower frequencies in Nlgn2−/− mice compared to littermates (i.e., at around 5 Hz in Nlgn2−/− mice and around 6 Hz in Nlgn2+/+ mice for wakefulness; at around 5.5 Hz in Nlgn2−/− mice and around 7.5 Hz in Nlgn2+/+ mice for REM sleep; Fig. 4a). However, only a difference in theta peak activity seemed to remain for REM sleep when relative power spectra were considered (5 Hz in Nlgn2−/− mice and Nlgn2+/+ mice for wakefulness; 5.5 Hz in Nlgn2−/− mice and 7.5 Hz in Nlgn2+/+ mice for REM sleep; Fig. 4b). Slower dynamics of NREM sleep delta activity in Nlgn2−/− mice The time course of NREM sleep delta activity was also compared between genotypes using absolute and relative power calculations (Fig. 5a). Absolute delta activity was significantly and globally higher in Nlgn2−/− mice compared to Nlgn2+/+ mice (more than two times higher as also observed in Fig. 4a), but the 24-h dynamics only showed a tendency to be affected by genotype. However, a between-genotype difference in the 24-h dynamics was revealed by analyzing the relative delta activity time course, which showed higher delta in Nlgn2−/− mice compared to Nlgn2+/+ mice for many intervals during the light period and lower delta in Nlgn2−/− than in Nlgn2+/+ mice for the first two intervals of the dark period. Given that Nlgn2−/− mice showed preserved NREM sleep duration during their main rest period (i.e., 12-h Light; Fig. 1b) and unaltered delta activity level at the beginning and end of the rest period, this observation suggests a slower dissipation of delta Seok et al. Molecular Brain (2018) 11:52 Page 7 of 11 Fig. 4 Power spectra of the 24 h EEG recording computed between 1 and 50 Hz with a 1-Hz resolution separately for the three vigilance states in Nlgn2+/+, Nlgn2+/− and Nlgn2−/− mice. a) Absolute spectral power for wakefulness, NREM sleep and REM sleep. For wakefulness, significant genotype effects were found for frequencies between 1 and 6 Hz and between 9 and 22 Hz (F2,37 > 3.9, p < 0.03). For NREM sleep, significant genotype effects were found for frequencies between 1 and 37 Hz (F2,37 > 3.4, p < 0.05). For REM sleep, significant genotype effects were found for frequencies between 1 and 6 Hz and between 9 and 27 Hz (F2,37 > 3.2, p < 0.05). b) Relative spectral power for wakefulness, NREM sleep and REM sleep. For wakefulness, significant genotype effects were found for frequencies 2 to 3 Hz, 6 to 8 Hz, and 14 to 50 Hz (F2,37 > 3.6, p < 0.04). For NREM sleep, significant genotype effects were found for frequencies 2 to 3 Hz, 4 to 6 Hz, 9 to 19 Hz, and 27 to 50 Hz (F2,37 > 3.3, p < 0.05). For REM sleep, significant genotype effects were found for frequencies 1 to 3 Hz, 4 to 9 Hz, 11 to 13 Hz, 15 to 16 Hz, 20 to 21 Hz, and 22 to 50 Hz (F2,37 > 3.3, p < 0.05). Red symbols indicate Hz-bins for which Nlgn2−/− mice are significantly different from Nlgn2+/+ mice (simple effect analysis; p < 0.05). For clarity, significant differences between Nlgn2+/− and Nlgn2+/+ mice have not been represented activity during the light (rest) period as well as a slower build-up of delta activity during the dark (active) period in Nlgn2−/− mice. A recent study proposed that it is specifically wakefulness dominated by theta (6–9.5 Hz) activity that drives homeostatic build-up of NREM sleep delta activity [33]. Accordingly, we investigated the 24-h time course of both absolute and relative theta activity during wakefulness in Nlgn2−/− mice and littermates (Fig. 5b). Although significant daily variations in both absolute and relative theta activity were found, both the dynamics and the global theta activity level were not significantly affected by genotype. Discussion We here report various alterations in wakefulness and sleep quantity and quality in mice lacking NLGN2. More precisely, Nlgn2−/− mice showed an increased duration of wakefulness and decreased durations of sleep states, with changes in wakefulness and NREM sleep attributable to the active (dark) period, and those in REM sleep found for the full nychthemeron. In addition, Nlgn2−/− mice exhibited a general increase in the consolidation of wakefulness and NREM sleep as well as abnormal EEG bursts of high amplitude predominately during wakefulness and REM sleep. Vigilance state quality was further affected in mice lacking NLGN2 as indexed with widespread changes in EEG activity in all states, and indications of dampened dynamics of sleep homeostasis (i.e., time course of NREM sleep delta activity). These observations point to different roles of NLGN2 in sleep regulation, and suggest that multiple sleep regulatory systems are impacted by the absence of NLGN2. The first hypothesis of the current study was that the absence of NLGN2 would result in more wakefulness and less sleep, which is supported by the results. This assumption was based on the flip-flop switch model of sleep regulation [19, 34], supposing that, in the absence of NLGN2, GABAergic outputs from sleep-inducing regions such as the hypothalamic ventrolateral preoptic nucleus (VLPO) and extended VLPO are reduced, thus favoring wakefulness. In this model, inhibitory outputs of sleep-inducing regions are notably targeting orexin/ hypocretin neurons of the lateral hypothalamus, a cell population on which NLGN2 was specifically shown to be increased under high sleep need (i.e., after sleep deprivation) [35]. Given the role of NLGN2 in promoting inhibitory transmission [16] and the role of orexin/ Seok et al. Molecular Brain (2018) 11:52 Page 8 of 11 Fig. 5 Twenty-four hour time course of delta activity (1–4 Hz) during NREM sleep and theta activity (6–9 Hz) during wakefulness in Nlgn2+/+, Nlgn2+/− and Nlgn2−/− mice measured using EEG. a) 24 h dynamics of NREM sleep absolute delta activity (upper panel) and relative delta activity (lower panel). For absolute activity, significant genotype and interval effects have been found (respectively, F2,37 = 11.7, p < 0.001 and F17,629 = 38.0, p < 0.0001), as well as a tendency for significant interaction (F34,629 = 2.3, p = 0.05). For relative activity, a significant interaction has been observed (F34,629 = 3.4, p < 0.0001). Red symbols indicate intervals for which Nlgn2−/− mice are significantly different from Nlgn2+/+ mice, and dark red symbols indicate intervals for which Nlgn2+/− mice are significantly different from Nlgn2+/+ mice (simple effect analysis; p < 0.05). b) 24 h dynamics of waking absolute theta activity (upper panel) and relative theta activity (lower panel). For both absolute and relative activity, a significant interval effect was found (respectively, F17,612 = 3.6, p < 0.01 and F17,612 = 5.3, p < 0.0001), but no significant genotype effect (respectively, F2,36 = 2.2, p = 0.13 and F2,36 = 0.7, p = 0.5) or interaction (respectively, F34,612 = 1.2, p = 0.2 and F34,612 = 1.3, p = 0.13) was observed. Grey backgrounds indicate the 12 h dark period hypocretin neurons in promoting transitions to wake [36], upregulation of NLGN2 could favor inhibition of orexin/hypocretin neurons and sleep. In Nlgn2−/− mice, a global reduction of the inhibition on orexin/hypocretin neurons would favor wake. Furthermore, a role for NLGN2 in orexin/hypocretin neurons could represent a mechanism by which alterations in wakefulness and NREM sleep consolidation occur in Nlgn2−/− mice, because pharmacological and genetic manipulations of the orexin/hypocretin system have been shown to impact variables linked to wake/sleep consolidation/fragmentation in rodents [33, 37]. In parallel to the orexin/hypocretin system, other GABAergic innervated regions could also be implicated in the wakefulness/sleep phenotype observed in the absence of NLGN2. For instance, GABAergic neurons from the medullary parafacial zone were shown to promote NREM sleep in mice likely via projections to the parabrachial nucleus [38]. An absence of NLGN2 in parabrachial neurons receiving parafacial GABA projections may thus alleviate inhibitory transmission and decrease NREM sleep. In addition, a subtype of GABAergic neurons (i.e., expressing Lhx6) of the zona incerta was recently shown to promote both NREM and REM sleep [39]. Nlgn2−/− mice could thus express less sleep via impairment in GABAergic transmission in areas targeted by this specific cell population. Additional studies aiming at quantifying NLGN2 presence in these defined brain regions and eventually assessing sleep variables under downregulation of NLGN2 in some of these regions will further the understanding of mechanisms behind the role of NLGN2 in regulating wakefulness and sleep duration. The second hypothesis of this study stated that the absence of NLGN2 would modify EEG activity because NLGN2 regulates GABAA receptors [17, 23], which are shaping EEG activity [20]. Our observations of hypersynchronized EEG events during wakefulness and REM sleep as well as of multiple alterations in EEG activity in all vigilance states support this hypothesis. The report of increased excitability under NLGN2 downregulation [17] is consistent with the implication of NLGN2 in inhibitory synaptic transmission [15, 16], and may suggest a role for NLGN2 in epileptogenesis. Such a role could also be supported by our observation of abnormal ‘hypersynchronized’ EEG events in Nlgn2−/− mice. Interestingly, very similar ‘hypersynchronized’ EEG events, which were considered as seizure spiking activity, have also been observed under overexpression of Nlgn2 in the forebrain [15]. Indeed, this study found spiking events of, on average, 7.4 Hz and 1.7 s that predominated during wakefulness and REM sleep [15]. Together with this study, our findings suggest that a delicate balance of NLGN2 is required to prevent ‘hypersynchronized’, potentially epileptiform, EEG activity. Of note is that a predominance of seizures during wakefulness and REM sleep has been reported for another mouse model showing increased wakefulness amount (i.e., Kcna2 KO mice) [40]. The predominance of abnormal EEG events during wakefulness and REM sleep could point to a role for NLGN2 in cholinergic transmission, given that Seok et al. Molecular Brain (2018) 11:52 cholinergic tone is high specifically during these two vigilance states [19]. In fact, NLGN2 has been reported to localize at synapses from cholinergic cells in multiple areas of the mouse brain (e.g., hippocampus, somatosensory and medial prefrontal cortex) [41]. Cholinergic activity is well-known to shape activity and responsiveness of neurons of the cerebral cortex [42], and a role for NLGN2 in the regulation of cholinergic outputs could thus also explain the widespread changes in EEG activity observed in Nlgn2−/− mice in the present study. Accordingly, future investigations should assess the role of NLGN2 in cholinergic neurotransmission and the contribution of this specific role to EEG activity. Alterations of GABAergic neurotransmission in thalamocortical circuits are also likely to contribute to modifications of EEG activity during wakefulness, NREM and REM sleep in Nlgn2−/− mice. The general increase in absolute spectral activity, especially for frequencies overlapping alpha, sigma and beta activity bands (i.e., 9 to 22 Hz) could originate from a global deficit in inhibition as pointed out by decreased mIPSCs and increased excitability reported under Nlgn2 downregulation [16, 17], and by the role of NLGN2 in the development of GABAergic synapses [43]. In parallel, our observation of high delta activity in Nlgn2−/− mice is consistent with findings of decreased spectral power in the delta range under administration of GABAA receptor agonists [21, 22, 44], given the regulation of the clustering of GABAA receptors by NLGN2 [23]. Of interest is that even if Nlgn2−/− mice express higher absolute delta power during NREM sleep compared to littermates, they show a slower daytime dynamics of delta activity as observed using relative quantification. Indeed, the decay of relative delta activity during the light phase is more gradual, apparently following a linear instead of an exponential trend, and the build-up of delta activity during the dark phase is also less abrupt. According to the two-process model of sleep regulation [29], such a time course of delta activity may suggest a slower dynamics of homeostatic sleep pressure. Furthermore, a slower build-up of homeostatic sleep pressure (or need) may represent another explanation for an increase capacity to stay awake and a reduced NREM sleep duration observed in absence of NLGN2. Here, we have assessed the dynamics of the main sleep homeostasis marker (i.e., delta activity) in absence of NLGN2 under normal/undisturbed conditions, and found indications of slower dynamics of both decay and build-up as detailed above. Although a sleep homeostasis phenotype could be more evident under baseline/undisturbed conditions for some genetic models, as shown for Orexin/Hypocretin KO mice [33], an altered dynamics of sleep need could rather be more apparent under challenged conditions such as enforced wakefulness. For Page 9 of 11 instance, in Nlgn1 KO mice, sleep deprivation specifically revealed a major exacerbation of the delta activity rebound indicative of a faster build-up of sleep need in absence of NLGN1 [10]. A next step in understanding the role of NLGN2 in sleep regulation could thus be to monitor recovery sleep after sleep deprivation in mice with modifications of Nlgn2 expression. The full KO model studied in the current study has allowed to verify the implication of NLGN2 in the regulation of sleep quantity and quality, but sleep duration and EEG alterations observed after total gene ablation could be attributable to developmental defects and/or compensations [45]. To avoid such confounding effects, in addition to investigating the precise brain and cellular locations mediating the roles of NLGN2, future research will need to downregulate Nlgn2 expression in specific brain areas and cell types at given developmental ages using, for instance, viral strategies or conditional gene KO techniques. Nonetheless, the present findings have the value of justifying such additional, more targeted, investigations. Sleep disorders have been considered as a serious economic burden because people suffering from sleep disorders utilize more medical resources and have higher risks of developing comorbid medical or psychiatric disorders [46, 47]. Understanding mechanisms underlying the role of NLGN2 in sleep regulation will uncover, at least to some extent and mainly for neuropsychiatric conditions, the etiology of comorbidity as well as provide potential therapeutic targets for comorbid disorders. Our findings support roles of NLGN2 in the regulation of multiple sleep dimensions and encourage future investigations aimed at unravelling the cellular and molecular mechanisms behind. Abbreviations ANOVA: Analysis of variance; ASDs: Autism spectrum disorders; E/I: Excitation to inhibition; EEG: Electroencephalographic; EMG: Electromyographic; GABA: γ-aminobutyric acid; Hz: Hertz; KO: Knockout; mIPSCs: Miniature inhibitory postsynaptic currents; NLGN: Neuroligin; NMDA: N-methyl-Daspartate; NREM: Non-rapid eye movement; REM: Rapid eye movement; SEM: Standard error of the mean; VLPO: Ventrolateral preoptic nucleus of the hypothalamus; ZT: Zeitgeber time Acknowledgements The authors thank Cassandra Areal who helped with EEG scoring and analysis, colleagues who helped with SD (Julien Dufort-Gervais, Lydia Hannou, Marlène Freyburger), Gaétan Poirier for technical help, and Dr. Zhengping Jia from University of Toronto/Sick Kids Hospital for advice on abnormal EEG event identification. Availability of data and material Not applicable. Funding This study was funded by a Bourse de recrutement maitrise of the Faculty of Graduate and Postdoctoral studies and the Department of Neuroscience of the Université de Montréal to BSS, salary awards from the Canadian Institutes of Health Research and the Fonds de Recherche du Québec-Santé to VM, and the Canada Research Chair in Sleep Molecular Physiology. Seok et al. Molecular Brain (2018) 11:52 Authors’ contributions BSS analyzed and interpreted the data, and wrote the paper. EBN and CP performed the experiments. SG participated in data interpretation. VM designed the experiment, analyzed and interpreted the data, and wrote the paper. All authors read and approved the final manuscript. Page 10 of 11 16. 17. Ethics approval All of the protocols and experimental procedures were performed according to the guidelines of the Canadian Council on Animal Care and approved by the Ethical Committee for Animal Experimentation of the Hôpital du SacréCoeur de Montréal. 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https://openalex.org/W2765843961	https://jyx.jyu.fi/bitstream/123456789/55675/1/sports0500081.pdf	English	null	Organized Sport Participation and Physical Activity Levels among Adolescents with Functional Limitations	Sports	2,017	cc-by	6,622	Received: 21 August 2017; Accepted: 17 October 2017; Published: 19 October 2017 Abstract: Sufficient and regular physical activity is considered a protective factor, reducing the onset of secondary disability conditions in adolescents with chronic diseases and functional limitations. The aim of this study was to explore whether participation in organized sport may be associated to higher levels of physical activity in adolescents with functional limitations, based on a national representative sample. Data from the Health Behaviour in School-aged Children (HBSC) study collected in Finland from two data collection rounds (2002 and 2010) were conducted and pooled from adolescents aged between 13 and 15 years old with functional limitations (n = 1041). Differences in self-reported physical activity over the past week and participation in organized sport activity were analysed for each function. Overall, four in ten (n = 413) participated in organized sport and were significantly (p < 0.001) more physically active (mean = 4.92days, SD = 1.81) than their non-participating (mean = 3.29, SD = 1.86) peers with functional limitations. Despite low population prevalence, adolescents with epilepsy or visual impairments were the least active if they were not participating in organized sport, yet were the most active if they were involved in organized sport. Participating in organized sport appears to be an important factor promoting resources for maintaining recommended levels of physical activity in Finnish adolescents with functional limitations. Keywords: salutogenesis; chronic disease; epilepsy; visual impairment; International Classification of Functioning, Disability and Health ICF; generalized resistance resources This is an electronic reprint of the original article. This reprint may differ from the original in pagination and typographic detail. Author(s): Title: Year: Version: Please cite the original version: Organized Sport Participation and Physical Activity Levels among Adolescents with Functional Limitations Ng, Kwok; Rintala, Pauli; Hutzler, Yeshayahu; Kokko, Sami; Tynjälä, Jorma Ng, K., Rintala, P., Hutzler, Y., Kokko, S., & Tynjälä, J. (2017). Organized Sport Participation and Physical Activity Levels among Adolescents with Functional Limitations. Sports, 5(4), Article 81. https://doi.org/10.3390/sports5040081 2017 This is an electronic reprint of the original article. This reprint may differ from the original in pagination and typographic detail. the original version: Ng, K., Rintala, P., Hutzler, Y., Kokko, S., & Tynjälä, J. (2017). Organized Sport Participation and Physical Activity Levels among Adolescents with Functional Limitations. Sports, 5(4), Article 81. https://doi.org/10.3390/sports5040081 All material supplied via JYX is protected by copyright and other intellectual property rights, and duplication or sale of all or part of any of the repository collections is not permitted, except that material may be duplicated by you for your research use or educational purposes in electronic or print form. You must obtain permission for any other use. Electronic or print copies may not be offered, whether for sale or otherwise to anyone who is not an authorised user. Organized Sport Participation and Physical Activi Levels among Adolescents with Functional Limitations Kwok Ng 1,, Pauli Rintala 1, Yeshayahu Hutzler 2, Sami Kokko 1 and Jorma Tynjälä 1 1 Faculty of Sport and Health Sciences, University of Jyvaskyla, 40014, Jyvaskyla, Finland; Pauli.rintala@jyu.fi (P.R.); sami.p.kokko@jyu.fi (S.K.); jorma.a.tynjala@jyu.fi (J.T.) 2 Wingate Academic College, Wingate Institute, Netanya 42902, Israel; shayke@wincol.ac.il Correspondence: kwok.ng@jyu.fi; Tel.: +358-4514-99919 Kwok Ng 1,, Pauli Rintala 1, Yeshayahu Hutzler 2, Sami Kokko 1 and Jorma Tynjälä 1 1 Faculty of Sport and Health Sciences, University of Jyvaskyla, 40014, Jyvaskyla, Finland; Pauli.rintala@jyu.fi (P.R.); sami.p.kokko@jyu.fi (S.K.); jorma.a.tynjala@jyu.fi (J.T.) 2 Wingate Academic College, Wingate Institute, Netanya 42902, Israel; shayke@wincol.ac.il Correspondence: kwok.ng@jyu.fi; Tel.: +358-4514-99919 Kwok Ng 1,, Pauli Rintala 1, Yeshayahu Hutzler 2, Sami Kokko 1 and Jorma Tynjälä 1 1 Faculty of Sport and Health Sciences, University of Jyvaskyla, 40014, Jyvaskyla, Finland; Pauli.rintala@jyu.fi (P.R.); sami.p.kokko@jyu.fi (S.K.); jorma.a.tynjala@jyu.fi (J.T.) 2 Wingate Academic College, Wingate Institute, Netanya 42902, Israel; shayke@wincol.ac.il Correspondence: kwok.ng@jyu.fi; Tel.: +358-4514-99919 Received: 21 August 2017; Accepted: 17 October 2017; Published: 19 October 2017 www.mdpi.com/journal/sports Sports 2017, 5, 81; doi:10.3390/sports5040081 1. Introduction Given the important role of physical activity in the prevention of non-communicable diseases [1], studies of adolescents with long-term illnesses or disabilities (LTID) have been gaining much attention. Adolescents with LTID can comprise of non-specified chronic diseases. During adolescence, children experience many biopsychosocial changes that influence the transition into adulthood, making it a critical period for study [2]. The physiological benefits from regular participation in physical activity have been well-documented [3], and so have the psychosocial aspects of health, to a lesser extent [4]. Despite these known benefits, few adolescents with LTID take part in regular physical activities, and the extent of participation varies by different functional limitations. For example, from the Finnish School-aged Physical Activity (SPA) study, adolescent boys with motor impairments reported a mean average of 3.5 days of at least 60 min of moderate to vigorous physical activity per day (MVPA), whereas adolescent boys with visual impairments reported a mean average of 4.8 days of MVPA [5]. According to the World Health Organization, moderate physical activity is considered to be an activity requiring three to six times more energy www.mdpi.com/journal/sports Sports 2017, 5, 81; doi:10.3390/sports5040081 Sports 2017, 5, 81 2 of 11 than sitting quietly (that is, at resting metabolic rate (MET), and vigorous physical activity requires more than six times relative to this resting MET. There are known issues that adolescents with LTID face when it comes to the promotion of physical activity, and are noted as barriers and facilitators to physical activity. The barriers to physical activity in adolescents with LTID may include issues such as low self-confidence to perform exercises [6–8]; fear of the individual’s health condition impeding on exercising [6,7,9,10], lack of professional knowledge and attitudes of others [7,11,12], as well as physical environmental factors [7,11]. Yet, known facilitators for increasing participation in physical activity have primarily been related to personal and inter-personal factors such as; having fun, involvement with peers; perceived health benefits and own motivation to be active [7,13,14]. When exploring the physical activity levels among adolescent groups with different functional limitations, a salutogenic approach [15] was used as a theoretical framework in this study. According to Salutogenesis, a person copes with external or internal stressors by a sense of coherence (SOC) and the utilization of generalized resistance resources (GRRs). The SOC can predict the way a person lives their life despite some health condition. 1. Introduction Individuals can deal with their existing conditions by structuring their life into manageable ways and finding meaningfulness in their activities. This is a state of mind that people have, with studies among adolescents demonstrating stronger SOC are related to increased quality of life, reduced substance use, more coping with difficulties [16]. Physical activity was the strongest predictor for SOC among Finnish adolescents [17]. Through physical activities, adolescents constantly face stressors that reinforce the salutogenic approach to health. Physical activities can take place from an array of settings, where organized sports are the most common type among adolescents in the western culture [18]. Participation in organized sports has been reported to have many benefits for society through the promotion of inclusion, health promoting behaviours [14], as well as the individual’s social and mental resources [4]. However, there is a lack of evidence of organized sport as GRRs for adolescents with LTID. The GRRs are either available at the personal or environmental level, and are utilized for enhancing health [15]. A standardised framework for describing health and health-related states is the WHO International Classification of Functioning, Disability and Health (ICF) [19]. The main ways of operationalising the ICF are to use the alphanumeric coding system organised in lists. One list is for body structures and body functions. The other list is for indicating actions in life areas as activities and participation. One such code is the engagement in sports (d9201), and several studies point out the barriers to [20] and benefits of full participation in organized sport [21]. Due to favourable social attitudes, accessibility to venues and programs adolescents from the general population context who take part in organized sport activities have opportunities to increase their positive perceptions of physical competence. This combination has been demonstrated to continue into physical activity habits during adulthood [22,23] thus, suggesting organized sport may be an example of effective GRRs. Moreover, organized sport participants with LTID are more likely to meet the international physical activity recommendations for health than non-participants [24]. Less is known about the associations with participation in organized sport and physical activity outcomes among adolescents with LTID [25]. This is even more evident in studies that make comparisons among adolescents with functional impairment. The resources needed to facilitate regular physical activity for one type of functional limitation, such as breathing difficulties are likely to differ from, for example, visual impairments. 1. Introduction Therefore, the purpose of this study was to explore whether participation in organized sport may be linked to increased physical activity and therefore considered as a GRR in adolescents with functional limitations, based on a national representative sample. 2.1. Participants Data extracted from the Finnish version of the WHO Collaborative Health Behaviour in School-aged Children (HBSC) study were used. The HBSC study is a quadrennial cross-sectional study that involves adolescents aged between 11 and 15 years old. Minority populations such as 3 of 11 Sports 2017, 5, 81 adolescents with long-term illnesses or disabilities (LTID) in one data collection cycle in 2002 were pooled with another cycle in 2010 to improve the statistical power. On both occasions, the data was collected in the Spring months of the year. adolescents with long-term illnesses or disabilities (LTID) in one data collection cycle in 2002 were pooled with another cycle in 2010 to improve the statistical power. On both occasions, the data was collected in the Spring months of the year. The sampling procedures were identical in both data collection years. This involved producing a sample frame based on the total number of pupils supplied through the national school register. From this information, a cluster sampling method that took into account the number of pupils in the school, and from inside the selected school, the class was chosen at random. National representativeness was based at two levels, one at the regional level, and the second, based on municipality types; urban, semi-urban, and rural. Adolescents in Swedish speaking schools, special educational schools, or home schooled were not included. The sampling procedures were identical in both data collection years. This involved producing mple frame based on the total number of pupils supplied through the national school register For this study, respondents from the adolescents in 13 (Mage = 13.7 y, SD = 0.30) and 15 (Mage = 15.7 y, SD = 0.32) year old age groups were used because they were given the essential questions used in this analysis. The survey was administered by teachers in school classrooms during a lesson. Teachers were given instructions on how to provide assistance if necessary, thus ensuring standardise protocols throughout the data collection. The starting sample size from the HBSC study in 2002 (n = 3932) and 2010 (n = 4874) were pooled and cleaned to filter out unreadable data, non-respondents, outliers in age range, removal of missing values to the final sample, and pupils who did not have LTID (n = 1041). Details of these procedures can be found elsewhere [26]. 2.1. Participants This study adds to previous analyses [27] from the same dataset by focusing on participants and non-participants of organized sport after taking into account different functional limitation categories. The survey was carried out voluntarily, through passive consent, and the respondent had the right to withdraw. No personal identifiers were used, and data was anonymous. The Finnish HBSC study was approved by the Finnish Teachers Trade Union and the Finnish National Agency for Education when the survey was collected the first time. Approval has not been requested in each survey year, and no known objections have been raised. There were no incentives or rewards for participation in the survey. Requests to access the data can be made through the international HBSC data bank in Bergen, Norway—http://www.hbsc.org. 2.2. Materials Adolescents were asked to respond to the following question, “Do you have a long-term illness, disability, or medical condition (like diabetes, arthritis, allergy, or cerebral palsy) that has been diagnosed by a doctor? Please do not include learning disabilities”. Response options were “yes” and “no”. A further question was available to categorise functional limitations to adolescents who selected “yes”. This question was only available in the 2002 and 2010 questionnaires, and was worded as; “If you answered yes, does this disability, illness, or medical condition cause you (1) difficulty seeing things (does not include prescription eye glasses), (2) difficulty in hearing what others say, (3) difficulty in speaking to others, (4) difficulty in moving around, (5) difficulty in handling objects, (6) difficulty in breathing, or (7) epileptic seizures (fits)?” The respondents proceeded to check boxes under columns of “yes” or “no” for each functional limitation. This question was missing from 2006 and 2014 survey, and pooled data was only from the 2002 and 2010 surveys. For the study analyses, respondents who checked “difficulty in speaking to others” (n = 7), “difficulty in hearing what others say” (n = 21), or both speaking and hearing difficulties (n = 1) were combined into a communication impairment category. In addition, a motor impairment category was composed of the combination of “difficulty in moving around” (n = 50), “difficulty in handling objects” (n = 11), and both moving and handling object difficulties (n = 6). Respondents who reported “yes” to the screener question about LTID, but did not check any functional limitation boxes were grouped into a “not specified” category. The “not specified” category is an important group to indicate the variety of other existing health conditions such as chronic diseases and rare conditions. The International Classification of Diseases 10th revision has over 14,000 codes for various conditions, and there are too many to list in a child-friendly format in national representative survey. Co-existing functional limitations of more than two categories did exist (n = 11) and were removed from the analysis. 4 of 11 Sports 2017, 5, 81 Physical activity was measured through a single self-reported item in both surveys. A preamble to contextualise moderate intensity physical activity was presented before the following question, Physical activity was measured through a single self-reported item in both surveys. 2.3. Analyses Potential confounders that could influence the levels of physical activity were explored, including gender, age [31], and year of data collection [24]. The confounders were used in adjusting the models to describe the differences of physical activity levels between non-participant and participants of organized sport. To check for confounding on the dependent variable, independent T-tests were used for gender, age, and year of data collection. To report the adjusted mean levels of physical activity by gender, age, and year of data collection, a univariate analysis of covariance test [32] were performed with no independent variables, and repeated for each functional limitation category. Statistical analyses were conducted through non-parametric t-test (Mann-Whitney) using SPSS 24.0 between organized sport participants and non-participants among the various functional impairment categories. Statistical significance was tested at p < 0.05. Cohen’s d effect sizes were computed and reported. 2.2. Materials A preamble to contextualise moderate intensity physical activity was presented before the following question, “Over the past 7 days, on how many days were you physically active for a total of at least 60 min per day? Please add up all the time you spent in physical activity each day.” (emphasis is the same as in the questionnaire). The response options ranged from 0 days through to 7 days, with a checkbox on each whole number increment between and including this range. This single item has been used in the HBSC study since 2001 and has featured as a key indicator of physical activity to meet the WHO physical activity recommendations [28] within international surveys [25]. Moderate correlation coefficients (r = 0.46; p < 0.001) with objective measures have been reported [29] and the item test-retest scores among a representative sample of Finnish adolescents in general schools have indicated acceptable reliability with ICC values ranged between 0.7 and 0.8, depending upon age and gender [30]. Groups of non-participants and participants of organized sport were based on a single item sports club membership question. The question was, “Are you a member of a sports club?” There were three response options, “No”, “Yes, and I am training in a sports club”, and “Yes, but I do not participate in training”. The main purpose of the study was aligned with the framework of the ICF, and members of a sports club, but did not participate in training (n = 40), were grouped with the group of non-participants. Confirmed through Kruskal-Wallis pairwise comparisons between the three groups, the number of physically active days between the “no” and “yes, but I do not participate in training” groups did not differ (p = 0.172). Whereas, there were significant differences between the group of “yes, and I am training in a sports club” with the other two groups (p < 0.001). 3. Results The effect size calculations, according to Cohen [33] were large for the categories with except the combination of motor & breathing with a medium sized effect. Table 2. Differences in means of moderate to vigorous physical activity per day (MVPA), after adjusting for gender, age, and year of data collection, between participant and non-participant groups. Non-Participant (n = 628) Participant (n = 413) MWtest a Cohen’s Mean LCI UCI Mean LCI UCI p d Not Specified 3.31 3.11 3.51 4.84 4.60 5.07 <0.001 0.90 Breathing 3.80 3.23 3.72 4.90 4.59 5.20 <0.001 0.88 Motor 3.27 2.70 3.84 4.56 3.75 5.37 0.010 0.65 Communication 3.31 2.44 4.17 5.16 3.41 6.91 0.135 0.74 Visual 2.99 2.16 3.82 5.40 4.09 6.70 0.002 1.58 Epilepsy 2.87 1.85 3.88 5.58 4.33 6.82 0.002 1.84 Motor & Breathing 3.66 2.64 4.69 3.61 2.32 4.91 0.351 0.33 Total 3.34 3.20 3.48 4.85 4.67 5.02 <0.001 0.89 a Mann-Whitney U Exact Significant Test (2-tailed). LCI—Lower confidence interval, UCI—Upper confidence interval. Participating boys in organized sport reported significantly more (p = 0.001) MVPA (mean = 5.44 days, SD = 1.49) than girls (mean = 4.59 days, SD = 1.88), whereas there was no significant gender difference among non-participants. There were no significant age differences among organized sport participants, although younger non-participants (13 year old; mean = 4.26 days, SD = 2.02) reported significantly more (p = 0.045) MVPA than older (15 year old; mean = 3.62 days, SD = 1.95). For each LTID category, participants in organized sport reported more days of 60 min MVPA than what non-participants in their respective category did, in particular adolescents with not specified, breathing, visual and epilepsy groups (Figure 1). Of non-participants, adolescents with breathing difficulties (mean = 3.42 days, SD = 1.81), communicating difficulties (mean = 3.39 days, SD = 1.90), and motor and breathing difficulties (mean = 3.35 days, SD = 2.29) reported, on average, the highest amounts of non-adjusted MVPA. At the other end of the spectrum, non-participant adolescents with visual impairments (mean = 2.94 days, SD = 1.56) and epilepsy (mean = 2.83 days, SD = 1.59) reported, on average, the lowest amounts of non-adjusted MVPA. Participants in organized sport with epilepsy were, on average, 2.8 days more active than non-participants with epilepsy. 3. Results Approximately four in 10 (n = 413/1041) of the sample participated in organized sport. The sample consisted of slightly more girls than boys (n = 591/1041) although there were no differences in organized sports participation (p = 0.533). Age groups were evenly distributed, although significantly (χ2 = 23.77, p < 0.001) more 13 year olds (58.6%) were active participants of organized sports than 15 year olds (41.4%). There were more adolescents from the 2002 data collection (n = 561/1041) than in 2010. There was a higher (χ2 = 4.02, p = 0.045) proportion of participation in organized sports in 2010 (50.1%) than 2002 (43.8%). These distributions varied by LTID category and are reported in Table 1. The most commonly identified LTID category in general school was breathing difficulties (31.1%), and the least was epilepsy (1.9%). Differences in proportion of each functional limitations group, were similar in both 2002 and 2010, except adolescents with motor difficulties in 2002 (n = 48) and in 2010 (n = 19). The proportion of adolescents with breathing difficulties who were participants of organized sports in 2002 (45.3%) and 2010 (54.7%) were significantly different (χ2 = 4.18 p = 0.042), whereas other functional difficulty groups involved in organized sports did not differ between the two data collection cycles. 26) (n = 20) (n = 28) % 60.0% 85.7% % 40.0% 14.3% % 75.0% 57.1% % 25.0% 42.9% % 40.0% 35.7% % 60.0% 64.3% % 60.0% 60.7% % 40.0% 39.3% 6 of 11 Sports 2017, 5, x FOR PEER REVIEW Mean physical activity levels differed by gender (Girls m = 3.67 SD = 1.94, Boys m = 4.17 SD = 2.07, t-test p = 0.001), age (13 y m = 4.26 SD = 2.02, 15 y m = 3.63 SD = 1.95, t-test p < 0.001), and year of data collection (2002 m = 3.54 SD = 1.96, 2010 m = 4.40 SD = 1.97, t-test p < 0.001). The overall mean PA levels of adolescents with LTID was 3.94 days with a pooled (by year) standard deviation of 1.96. The levels of physical activity between participants of organized sport was significantly higher than of non-participant in the following functional limitation categories; Not specified, breathing, motor, visual, and epilepsy categories (Table 2). 3. Results Similar results were noticed with adolescents with reported visual impairments, because participants in organized sport were, on average, 2.6 days more active than their matched non-participant peers. The mean number of days were higher than other functional difficulty groups including difficulties in breathing (mean = 4.99 days, SD = 1.77) and communication (mean = 4.83 days, SD = 2.14). 7 of 11 Sports 2017, 5, 81 Figure 1. Non-adjusted means and error bars of MVPA by sport club participation. Figure 1. Non-adjusted means and error bars of MVPA by sport club participation. 4. Discussion According to the results of this study, adolescents with LTID reported, on average, more days of physical activity when they were participants of organized sport than their matched non-participant peers. More specifically, the difference between participants and non-participants of organized sport was highest among adolescents with visual impairments or epilepsy. A large effect was observed in the difference of physical activity levels between participants and non-participants of organized sport. The group with the most adolescents with LTID and specified functional limitation was the group of breathing difficulties. Consistent with the principle of participating adolescents in organized sport are more physically active than their non-participating counterparts [34], in this study, the levels of physical activity were greater from participants of organized sport than non-participants. These findings compliment what Geidner and Jerlinder [14] recently reported, whereby one of the main reasons for including adolescents with LTID into organized sports is to promote physical activity. The difference in physical activity levels were exemplified among adolescents with visual impairments or epilepsy. On average, more than two days difference in moderate to vigorous physical activity was observed between organized sport participants and non-participants. Adolescents with visual impairments or epilepsy who were non-participants in organized sports had the lowest levels of PA. Considering 30% of adolescents with visual impairments were organized sport participants, these findings concur with previous studies focused on adolescents with visual impairments, where low levels of physical activity were reported [35]. The enhancing benefits of organized sport may explain how Aslan and colleagues [36] reported low amounts of low intensity physical activity during the week, whereas during the weekend, there were increased moderate intensity levels among adolescents with visual impairments. This could be because the majority of adolescent organized sports may have competitions held on weekends in addition to their regular training schedules during the week. Based on this interaction, it may be suggested that participation in organized Sports 2017, 5, 81 8 of 11 sports could be seen as a GRR for maintaining physical activity participation and ultimately health benefits. Adolescents with LTID may find their health conditions impede their participation in organized sports [10], whereas similar adolescents with desire to be active and find enjoyment from exercise may find they can thrive is such situations [7]. 4. Discussion Enrichment through participation of adolescents with LTID in organized sport may also depend on the activities available, accessibility to the sports, the purpose of the club activities, the personnel that run and operate the activities, and the level of inclusion [14]. These determinants act as GRRs in a salutogenic approach to health. With regard to participants with visual impairments, Lieberman and colleagues [37] suggest practitioners should implement successful modifications through awareness of individual needs. As the majority of organized sports are run by volunteers, these dedicated resources are essential in the promotion of physical activities. Adolescents with epilepsy need to learn to deal with self-management skills, including participation in physical activity and sport [38]. It is especially important, because the risk of injuries during sport is higher than peers without epilepsy [39]. Managing this risk can be mitigated through education and support. The organized sport environment may be perceived to be safer for adolescents with epilepsy and could explain the large differences in physical activity reported in our study. Other determinants that influence participation in leisure activities such as physical activity include age, gender, degree of activity limitations, family preferences and coping, motivation, and environmental resources and supports [6,40]. Additional research is needed to better establish physical activity determinants related to organized sport participation in children with functional limitations. Examples for potential determinants for increased participation that have been indicated in other populations are self-efficacy [8], group cohesion [41], and athletic self-identity [42]. The resources available to adolescents with LTID need to be carefully designed. It is often assumed that adolescents with LTID have to cope with stressors in their lives such as maintenance of the condition, sometimes through medication, regular health care visits, poorer mental health, and difficulties to make transitions in life [43]. Therefore, the need to focus on the resources available to the individual and not only the aetiology and treatment are essential ways to improve health. Many studies in the phenomenon of drop-out of physical activity have been attributed to low motivation, bad experiences, a lack of time, and unsuitable activities and there could be further barriers for individuals with disabilities [10]. Instructors with specialist knowledge of inclusion may encourage more engagement in organized sport, as activities may foster more social interaction among participants, and this can lead to increased levels of support between adolescents with and without limitations [44]. Reference 1. Lee, I.; Shiroma, E.J.; Lobelo, F.; Puska, P.; Blair, S.N.; Katzmarzyk, P.T. Effect of physical inactivity on major non-communicable diseases worldwide: An analysis of burden of disease and life expectancy. Lancet 2012, 380, 219–229, doi:10.1016/S0140-6736(12)61031-9. 1. Lee, I.; Shiroma, E.J.; Lobelo, F.; Puska, P.; Blair, S.N.; Katzmarzyk, P.T. Effect of physical inactivity on major non-communicable diseases worldwide: An analysis of burden of disease and life expectancy. Lancet 2012, 380, 219–229, doi:10.1016/S0140-6736(12)61031-9. 2. Sheehan, P.; Sweeny, K.; Rasmussen, B.; Wils, A.; Friedman, H.S.; Mahon, J.; Patton, G.C.; Sawyer, S.M.; Howard, E.; Symons, J.; et al. Building the foundations for sustainable development: A case for global investment in the capabilities of adolescents. Lancet 2017, doi:10.1016/S0140-6736(17)30872-3. 2. Sheehan, P.; Sweeny, K.; Rasmussen, B.; Wils, A.; Friedman, H.S.; Mahon, J.; Patton, G.C.; Sawyer, S.M.; Howard, E.; Symons, J.; et al. Building the foundations for sustainable development: A case for global investment in the capabilities of adolescents. Lancet 2017, doi:10.1016/S0140-6736(17)30872-3. Durstine, J.L.; Painter, P.; Franklin, B.A.; Morgan, D.; Pitetti, K.H.; Roberts, S.O. Physical activity for the chronically ill and disabled. Sports Med. 2000, 30, 207–219, doi:0112-1642/00/0009-0207. 4. Eime, R.M.; Young, J.A.; Harvey, J.T.; Charity, M.J.; Payne, W.R. A systematic review of the psychological and social benefits of participation in sport for children and adolescents: Informing development of a conceptual model of health through sport. Int. J. Behav. Nutr. Phys. Act. 2013, 10, 98, doi:10.1186/1479-5868-10-98. 4. Eime, R.M.; Young, J.A.; Harvey, J.T.; Charity, M.J.; Payne, W.R. A systematic review of the psychological and social benefits of participation in sport for children and adolescents: Informing development of a conceptual model of health through sport. Int. J. Behav. Nutr. Phys. Act. 2013, 10, 98, doi:10.1186/1479-5868-10-98. 5. Ng, K.W.; Rintala, P.; Saari, A. Functional Difficulties and Physical Activity and Sedentariness [Toimintakyvyn ja-Rajoitteiden Yhteydet Liikunta-Aktiivisuuteen ja Paikallaanoloon]; Kokko, S., Mehtälä, A., Eds.; The Finnish school-aged physical activity monitoring study: Results from 2016. [Lasten ja nuorten liikuntakäyttäytyminen Suomessa (LIITU)—Tutkimuksen aineistonkeräys ja menetelmät 2016]; Valtion Liikuntaneuvoston Julkaisuja: Helsinki, Finland, 2016; pp. 73–78. 5. Ng, K.W.; Rintala, P.; Saari, A. Functional Difficulties and Physical Activity and Sedentariness [Toimintakyvyn ja-Rajoitteiden Yhteydet Liikunta-Aktiivisuuteen ja Paikallaanoloon]; Kokko, S., Mehtälä, A., Eds.; The Finnish school-aged physical activity monitoring study: Results from 2016. [Lasten ja nuorten liikuntakäyttäytyminen Suomessa (LIITU)—Tutkimuksen aineistonkeräys ja menetelmät 2016]; Valtion Liikuntaneuvoston Julkaisuja: Helsinki, Finland, 2016; pp. 73–78. 6. Mulligan, H.F.; Hale, L.A.; Whitehead, L.; Baxter, D.G. 4. Discussion Moreover, positive and negative health behaviours are known within organized sport participation, such as reducing substance use, bullying, fighting, and improving sleep [45,46] and require further educating to create a more effective resource for health. The study has come from a national representative sample of school-aged children, and we provided an overall picture of the prevalence of functional limitations, physical activity levels, and participation in organized sport among Finnish adolescents. Comparisons among groups were possible because measures were the same allowing the possibility to be combined with larger national and international data sets. However, with large size and self-reporting surveys, it is worthy to note some study limitations. The data was cross-sectional, and no causal inference can be made between participation in organized sport and physical activity levels. Adolescents self-reported their functional limitations and there may be other reporting biases in the exposure and outcome variables. The level of representative data is of the national population, not necessarily of particular impairments, and this should be taken into consideration when interpreting the results. Finally, interpretation of the results are restricted to the general school population and not from participants outside of the HBSC study, such as from special schools and non-Finnish language schools. Sports 2017, 5, 81 9 of 11 Acknowledgments: Jari Villberg is acknowledged for data collection of the HBSC study in Finland. Author Contributions: K.N. conceived and designed the study, K.N. analyzed the data; J.T. contributed materials, K.N., P.R., S.K. and Y.H. wrote the paper. All authors have read and approved the content of the submitted manuscript. Conflicts of Interest: The authors declare no conflict of interest. 5. Conclusions Participation in organized sports is clearly associated with increased levels of physical activity among adolescents with LTID, although the level of association varied by functional limitation. The prevalence of organized sport participation also varied by functional limitation and needs to be addressed in future programs that include adolescents with LTID. Participation in organized sports has the potential to act as GRRs, with a large effect for adolescents with breathing difficulties, visual impairments, and epilepsy. Studies that can help underpin the reasons for the low frequency of participation in organized sports among adolescents with visual or communication impairments are needed. In addition, future studies may need to examine the relationships with other physical activity related behaviours, such as sleep, sedentariness, nutrition, peer and family support, and quality of life. One of the main findings of the current study were in relation to the largest difference in physical activity levels among adolescents with visual impairments, yet only 30% reported participating. More programs that can increase participation in organized sport may enhance the overall physical activity levels in these functional limitation categories. Acknowledgments: Jari Villberg is acknowledged for data collection of the HBSC study in Finland Reference Barriers to physical activity for people with long-term neurological conditions: A review study. Adapt. Phys. Act. 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https://openalex.org/W3028830068	https://europepmc.org/articles/pmc7266966?pdf=render	English	null	Interleukin-31 Receptor α Is Required for Basal-Like Breast Cancer Progression	Frontiers in oncology	2,020	cc-by	6,302	Edited by: Binhua Peter Zhou, University of Kentucky, United States Reviewed by: Yifan Wang, Tongde Hospital of Zhejiang Province, China Yadi Wu, University of Kentucky, United States Correspondence: Jian Shi jianshismu@126.com Edited by: Binhua Peter Zhou, University of Kentucky, United States Results: We found that silencing of IL31RA suppresses the cancer stem cell-like properties, migration and invasion of BLBC cells in vitro as well as tumor growth and metastasis in vivo. Knockdown of IL31RA ameliorates IL-31-mediated pro-oncogenic functions. Overexpression of IL31RA in luminal breast cancer cells enhances the cancer stem cell-like properties and cell motility. Our data further identiﬁed IL31RA as a target gene of Twist/BRD4 transcription complex. Reviewed by: Yifan Wang, Tongde Hospital of Zhejiang Province, China Yadi Wu, University of Kentucky, United States University of Kentucky, United States Conclusion: Overall, these data indicate that IL31RA promotes basal-like breast cancer progression and metastasis, suggesting that targeting of IL-31/IL31RA axis might be beneﬁcial to treatment of BLBC. †These authors share ﬁrst authorship Keywords: IL31RA, IL31, Twist, BRD4, BLBC Keywords: IL31RA, IL31, Twist, BRD4, BLBC Specialty section: This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Oncology Interleukin-31 Receptor α Is Required for Basal-Like Breast Cancer Progression Yanling He 1†, Xinyuan Zhang 1†, Weijun Pan 1†, Fang Tai 1, Li Liang 1 and Jian Shi 1,2 1 Department of Pathology, Nanfang Hospital & School of Basic Medical Science, Southern Medical University, Guangzhou, China, 2 School of Basic Medical Science, Guangzhou Medical University, Guangzhou, China Yanling He 1†, Xinyuan Zhang 1†, Weijun Pan 1†, Fang Tai 1, Li Liang 1 and Jian Shi 1,2* 1 Department of Pathology, Nanfang Hospital & School of Basic Medical Science, Southern Medical University, Guangzhou, China, 2 School of Basic Medical Science, Guangzhou Medical University, Guangzhou, China 1 Department of Pathology, Nanfang Hospital & School of Basic Medical Science, Southern Medical University, Guangzhou, China, 2 School of Basic Medical Science, Guangzhou Medical University, Guangzhou, China Purpose: Interleukin-31 receptor α (IL31RA) usually mediates IL-31 induced inﬂammation and allergic diseases. However, the functional roles of IL-31/IL31RA signaling in basal-like breast cancer (BLBC) progression remain totally unclear. Methods: Tumorsphere formation, transwell, and wound healing assays were used to measure the BLBC progression. We implanted tumor cells in mammary fat pad and tail vein of nude mice to detect the growth and metastasis of BLBC cells. Luciferase and ChIP assays were employed to measure the transcriptional regulation. Western blot and real-time PCR assays as well as bio-informatics analyses were conducted to observe the expression of IL31RA. INTRODUCTION Basal-like breast cancer (BLBC) is a cluster of breast tumor cells that characterized with low expression of estrogen receptor, progesterone receptor, and epidermal growth factor receptor 2; it frequently occurs in young women and has higher incidence rates of distant organ metastasis and recurrence than hormone receptor positive breast cancer. BLBC is insensitive to endocrine or HER2-targeted therapies with low 5-year survival rates. Few systemic treatment options exist besides chemotherapy (1–3). Received: 16 February 2020 Accepted: 27 April 2020 Published: 27 May 2020 ORIGINAL RESEARCH published: 27 May 2020 doi: 10.3389/fonc.2020.00816 Tumorsphere 1 × 104 cells were plated as single-cell suspensions on ultra-low attachment plates (Corning) in DMEM/F12 medium supplemented with 20 ng/ml EGF, 10 µg/ml insulin, 0.5 µg/ml hydrocortisone and B27. Tumorspheres were counted and taken photos after 5–7 days. In this study, we found that IL31RA is required for cancer stem cell (CSC)-like properties, invasion and metastasis of BLBC cells, implicating that speciﬁc targeting of this protein represents a potential therapy for BLBC treatment. RT-PCR Analysis Total RNA was extracted from cells by RNeasy Mini Kit (#74104, Qiagen, Valencia, CA), and 1 µg of RNA was reverse transcribed by SuperScriptR III Reverse Transcriptase (#18080044) from Thermo Fisher Scientiﬁc (Waltham, MA). Real-time PCR was analyzed using Power SYBR Green Master Mix (Applied Biosystems, USA). Expression values relative to control were calculated using the 11CT method. GAPDH was used as a housekeeping gene for normalization. Results were represented as relative fold change. The primers used for IL31RA gene were: 5′-ctggagtgactggagccaag-3′ and 5′- ctaggactggggctcctctt-3′. Reagents g For wound healing assay, cells were seeded at 80% conﬂuency and cultured overnight. The culture was scratched with a 200 µl pipette tip and the wound was allowed to heal for 48 h. The reduction of area between two wound edges was calculated and quantitated. For transwell assay, each insert (8-µm pore size, Falcon) was coated with matrigel overnight. 1 × 105 control or clone cells were re-suspended in 150 µl non-serum medium and seeded in the upper Boyden chamber coated with Matrigel (BD biosciences, San Jose, CA) while the bottom chambers were ﬁlled with 600 µl non-serum medium plus 100 nM LPA. After 24– 48 h, un-invasive cells on the membrane apical side were removed using wet cotton swabs and the invasive cells were stained with crystal violent and counted under microscope. Typical pictures of invaded cells are shown, Scale bar, 100 µM. Statistical data (mean ± SD) for numbers of invading cells are shown. Reagents Antibodies for p-STAT3 and BRD4 were purchased from Cell Signaling (Danvers, MA). Antibodies for Twist were from Abcam (Cambridge, MA). IL31RA antibody was from GeneTex (Irvine, CA). ShRNA against IL31RA and antibody against Actin were purchased from Sigma-Aldrich (St. Louis, MO). Puriﬁed human recombinant IL-31 protein was obtained from R&D systems (Minneapolis, USA). IL31RA lentivirus plasmid was purchased from GeneCopoeia (Guangzhou, China). Antibodies for p-STAT3 and BRD4 were purchased from Cell Signaling (Danvers, MA). Antibodies for Twist were from Abcam (Cambridge, MA). IL31RA antibody was from GeneTex (Irvine, CA). ShRNA against IL31RA and antibody against Actin were purchased from Sigma-Aldrich (St. Louis, MO). Puriﬁed human recombinant IL-31 protein was obtained from R&D systems (Minneapolis, USA). IL31RA lentivirus plasmid was purchased from GeneCopoeia (Guangzhou, China). p g SiRNAs for Twist and BRD4 were obtained from Dharmacon (USA). SiRNAs for Twist and BRD4 were obtained from Dharmacon (USA). Citation: He Y, Zhang X, Pan W, Tai F, Liang L and Shi J (2020) Interleukin-31 Receptor α Is Required for Basal-Like Breast Cancer Progression. Front. Oncol. 10:816. doi: 10.3389/fonc.2020.00816 Interestingly, BLBC is an inﬂammation-associated disease, its rapid growth and metastasis heavily relies on aberrant up-regulation of pro-oncogenic inﬂammatory pathways (4). For example, plenty of studies have observed that two critical cytokines, IL-6 and IL-8, are up-regulated in BLBC which are required for maintenance of breast cancer stem cell-like properties (5). Usually, BLBC has high level of inﬁltration of tumor associated macrophage and lymphocytes compared May 2020 \| Volume 10 \| Article 816 Frontiers in Oncology \| www.frontiersin.org IL31RA Promotes BLBC Progression He et al. with other subtypes, which is correlated with the high risk for progression and distant metastasis. It is well-established that Th2-type and CD4-positive T cells are basic constitutes of the tumor microenvironment (6). Interestingly, activated Th2- type or CD4-positive T cells are able to produce Interleukin- 31 (IL-31), a member of IL-6 cytokine family. IL-31 frequently activates JAK2-STAT3 signaling, promoting inﬂammation and allergic diseases, such as asthma and dermatitis (7). Normally, IL-31 recognizes Interleukin-31 receptor α (IL31RA), a common receptor subunit for IL-6-type cytokines on the cell membrane. IL31RA, frequently interacting with oncostatin M receptor, mediates biological or pathological functions of IL-31 (8), as evidenced by the fact that Il31ra-deﬁcient mice did not develop alopecia or pruritus in response to IL-31 treatment (9). Although it is realized that expression of IL31RA mRNA is induced after stimulation with IFN-γ (10), the transcriptional regulation manner of this gene is barely known. More importantly, the pathological role of IL-31/IL31RA axis in tumor progression is largely unclear. In this study, we tried to discern the functional role of IL31RA in BLBC progression and make clear its transcription regulation manner. cancer cell lines MDA-MB-231, MDA-MB-157, MDA-MB-453, MCF7 and BT474 were cultured using Dulbecco’s Modiﬁed Eagle Medium containing 10% FBS. Penicillin/streptomycin were added to cell culture medium. All cell lines were obtained from ATCC. Stable cell clones were constructed by selection using puromycin (1 µg/mL). For siRNA transfection, cells were seeded about 1 × 105 cells per well in medium with 10% FBS and transfected with siRNA using siRNA-mate reagent (GenePharma) after reaching 70% conﬂuency. Western Blot Cells were harvested and lysed, protein concentration was determined using BCA protein assay kit. Equal amounts of puriﬁed proteins were separated by SDS-PAGE electrophoresis and transferred onto PVDF membranes. Protein signals were detected using Enhanced chemiluminescence kit (FDbio science). Primary antibodies concentrations were used as following: IL31RA (1:1,000, GeneTex), p-STAT3 (1:1,000, Cell signaling), β-Actin (1:1,000, Transkgen). Anti–rabbit or anti-mouse secondary antibody (Earthox) was used at 1:5,000 dilution. p g Twist is a powerful epithelial-mesenchymal transition (EMT)- inducing transcription factor that activates the expression of mesenchymal-type genes and represses the epithelial phenotype (11). Twist mediated EMT process is critical for cancer cell invasion and metastasis (12, 13), and the acquisition of cancer stem-like property (14, 15). Our previous data indicated that activated Twist/BRD4 complex occupies the super- enhancer regions of oncogenes and maintains their expression. Pharmacologic inhibition of the Twist-BRD4 association by BET-speciﬁc BD inhibitors suppressed invasion, cancer stem cell (CSC)-like properties, and tumorigenicity of BLBC cells (16). However, whether Twist/BRD4 transcription complex modulates the cytokine signaling and inﬂammation process remains obscure. Chromatin Immunoprecipitation 6 PCR. The speciﬁc primers for the IL31RA promoter were: 5′- GAGACAGGAAGGCAGAGTGT-3′ and 5′-TTGCGGACATTC ACAGACAC-3′. Approximately 1 × 106 control and Twist-knockdown MDA- MB-231 cells as well as JQ1-treated cells were ﬁxed with cross- link solution and collected, ChIP assays were performed using Imprint Chromatin Immunoprecipitation Kit (Sigma, #CHP1) according to the manufacturer’s instructions. Twist or BRD4 antibody-immunoprecipitated DNA was analyzed by real-time Cell Culture Breast cancer cell lines BT549 and T47D were maintained in RPMI-1640 medium with 10% fetal bovine serum. Breast May 2020 \| Volume 10 \| Article 816 Frontiers in Oncology \| www.frontiersin.org 2 He et al. He et al. IL31RA Promotes BLBC Progression GURE 1 \| Silencing of IL31RA suppresses BLBC progression. (A) IL31RA gene was knocked down in MDA-MB-231 and MDA-MB-157 cells by shRNA and stable nes were constructed. Expression of IL31RA and STAT3 phosphorylation was detected by western blot. (B) Cancer stem cell-like properties were determined by morsphere assay in vector control and IL31RA-knockdown MDA-MB-231 and MDA-MB-157 cells. Scale bar, 100 µM. Statistical analysis (mean ± SD) for numbers tumorsphere is shown. (C) Cell migration was detected by wound healing assay in vector control and IL31RA-knockdown MDA-MB-231 cells for 48 h. Statistical ta (mean ± SD) for wound closure are shown. indicates P < 0.05, indicates P < 0.01. (D) Cell invasion was observed by transwell assay in vector control and 1RA-knockdown MDA-MB-231 cells. Scale bar, 100 µM. Statistical data (mean ± SD) for invaded cells are shown. *indicates P < 0.001. Scale bar, 100 µM. FIGURE 1 \| Silencing of IL31RA suppresses BLBC progression. (A) IL31RA gene was knocked down in MDA-MB-231 and MDA-MB-157 cells by shRNA and stable clones were constructed. Expression of IL31RA and STAT3 phosphorylation was detected by western blot. (B) Cancer stem cell-like properties were determined by tumorsphere assay in vector control and IL31RA-knockdown MDA-MB-231 and MDA-MB-157 cells. Scale bar, 100 µM. Statistical analysis (mean ± SD) for numbers of tumorsphere is shown. (C) Cell migration was detected by wound healing assay in vector control and IL31RA-knockdown MDA-MB-231 cells for 48 h. Statistical data (mean ± SD) for wound closure are shown. indicates P < 0.05, indicates P < 0.01. (D) Cell invasion was observed by transwell assay in vector control and IL31RA-knockdown MDA-MB-231 cells. Scale bar, 100 µM. Statistical data (mean ± SD) for invaded cells are shown. indicates P < 0.001. Scale bar, 100 µM. Chromatin Immunoprecipitation 6 Luciferase Reporter Assay The human IL31RA gene promoter region (988 bp) was cloned and its promoter-luciferase construct was generated. May 2020 \| Volume 10 \| Article 816 Frontiers in Oncology \| www.frontiersin.org 3 He et al. He et al. IL31RA Promotes BLBC Progression FIGURE 2 \| Silencing of IL31RA suppresses tumor growth and metastasis. (A–C) Vector control and two IL31RA-knockdown MDA-MB-231 clones were injected into mammary fat pad of BALB/c nude mice (n = 7), respectively. Xenograft tumor growth was determined by measuring tumor volume and weight. Statistical data are shown ( indicates P < 0.001). (D,E) MDA-MB-231 vector control and IL31RA-knockdown cells were, respectively, injected into tail vein of BALB/c nude mice. The images of mice lung with metastatic nodules (Left panel) and statistical data (Right panel) are shown (indicates P < 0.01, *indicates P < 0.001). White arrows indicate the metastatic nodules. FIGURE 2 \| Silencing of IL31RA suppresses tumor growth and metastasis. (A–C) Vector control and two IL31RA-knockdown MDA-MB-231 clones were injected into mammary fat pad of BALB/c nude mice (n = 7), respectively. Xenograft tumor growth was determined by measuring tumor volume and weight. Statistical data are shown (* indicates P < 0.001). (D,E) MDA-MB-231 vector control and IL31RA-knockdown cells were, respectively, injected into tail vein of BALB/c nude mice. The images of mice lung with metastatic nodules (Left panel) and statistical data (Right panel) are shown (indicates P < 0.01, indicates P < 0.001). White arrows indicate the metastatic nodules. HEK293T cells were seeded in 60 mm dishes and transfected with mentioned plasmids by FuGene 6 transfection reagent (Roche) for 24 h, cell lysates were extracted with passive lysis buﬀer (Promega, Madison, WI) and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). Relative luciferase activities were calculated as folds of induction compared with vector control. caliper measurements every 5 days, tumor volume was calculated according to the formula: length × width2/2. After 30 days, mice were euthanized, tumors were weighed and taken photos. For lung metastasis model, 2 × 105 MDA-MB-231 cells were re-suspended in 0.1 ml PBS and injected into the tail vein. After 6 weeks, the mice were sacriﬁced, tumor nodules on the surface of lung were counted and quantiﬁed. Statistical Analyses (Figure 3B). Similarly, tumor cell migration (Figure 3C) and invasive ability (Figure 3D) were also robustly stimulated upon IL31RA over-expression. These data further indicate that IL31RA is essential for cancer stem cell-like properties, cell migration, and invasion. Data are presented as mean ± SD. Student’s t test (two-tailed) was used to compare two-group data which satisfy normal distribution with homogeneous variance. Multiple comparisons were analyzed by one-way ANOVA and Welch’s test was used for data with unequal variance. P < 0.05 was considered signiﬁcant. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Data are presented as mean ± SD. Student’s t test (two-tailed) was used to compare two-group data which satisfy normal distribution with homogeneous variance. Multiple comparisons were analyzed by one-way ANOVA and Welch’s test was used for data with unequal variance. P < 0.05 was considered signiﬁcant. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Silencing of IL31RA Suppresses Tumor Growth and Metastasis in vivo To further study the functional roles of IL31RA in vivo, vector control and IL31RA-knockdown MDA-MB-231 clones were injected into mammary fat pad of Balb/c nude mice, respectively. After 30 days, vector control MDA-MB-231 developed into xenograft tumors with volume about 800 mm3, IL31RA-knockdown clones had signiﬁcantly reduced tumor size compared with vector control cells (Figures 2A–C). Next, we performed tail vein injection to construct in vivo lung metastasis model in order to measure the metastatic capacity of vector control and IL31RA-silencing MDA-MB-231 cells. As shown, robust reduction of pulmonary nodules from IL31RA- knockdown clones was observed compared with vector control group (Figures 2D,E). All these data indicate that IL31RA is required for breast cancer cell invasion and metastasis. Silencing of IL31RA Suppresses BLBC Progression We proceeded to investigate the functional dependence of IL- 31 on IL31RA in BLBC progression. Tumorsphere formation ability of shRNA-control and IL31RA-knockdown clones of MDA-MB-231 was measured in the absence or presence of IL- 31 (10 ng/ml) treatment. IL-31 treatment signiﬁcantly enhanced the tumorsphere formation in shRNA-control MDA-MB-231 cells, but not in IL31RA-knockdown clones (Figure 4A). We also detected the migratory and invasive capacity of these clones with or without IL-31 treatment; similar results were observed that IL- 31 induced the migration (Figure 4B) and invasion (Figure 4C) of control MDA-MB-231 cells, however, it completely lost the ability in IL31RA-silencing cells, indicating that IL-31 is able to promote BLBC tumor progression and its pro-oncogenic functions are strongly dependent on IL31RA. g To reveal the pathological role of IL31RA in BLBC, we knocked down IL31RA gene in MDA-MB-231 and MDA- MB-157 BLBC cell lines and constructed their stable clones. Silencing of IL31RA markedly repressed STAT3 tyrosine 705 phosphorylation, indicating the blockade of IL-31/IL31RA signaling (Figure 1A). Intriguingly, both IL31RA-knockdown clones showed the reduced cancer stem cell-like properties that determined by tumorsphere formation assay (Figure 1B). IL31RA-silencing also signiﬁcantly inhibited the cell migratory capacity of MDA-MB-231 cells, in which about 10–20% wound closure was observed in IL31RA-silencing cells compared with almost 40% closure in vector control cells (Figure 1C). Consistently, remarkably weaker invasive ability was revealed in IL31RA-knockdown MDA-MB-231 clones than vector control cells that detected by the transwell invasion assay (Figure 1D). These results indicate that IL31RA is critical for cancer stem cell-like properties and motility of BLBC cells. Gene Expression Correlation Analysis Gene Expression Correlation Analysis Gene expression data for breast carcinoma patients from 3 studies including GSE48390, GSE76275, and GSE93601 were downloaded from the GEO. The Pearson’s and Spearman’s correlation coeﬃcients were used to quantify the correlation in expression between Twist and IL31RA. P-values were calculated based on testing the hypothesis of correlation coeﬃcient equal to zero, i.e., the expressions of genes are independent. Balb/c female nude mice (4–6 weeks) were purchased from Animal Center of Guangdong Province. All animal experiments were approved by the Animal Care and Use Committee of Southern Medical University. Mice were housed in autoclaved, ventilated cages and provided with autoclaved water. 1 × 106 MDA-MB-231 vector control cells and IL31RA-knockdown cells were, respectively, injected into mammary fat pad of mice (n = 7). Tumor growth was monitored with May 2020 \| Volume 10 \| Article 816 Frontiers in Oncology \| www.frontiersin.org 4 IL31RA Promotes BLBC Progression He et al. Twist/BRD4 Complex Induces IL31RA Expression p Next, we sought to investigate the expression status of IL31RA in breast cancer. Three datasets from Gene expression omnibus (GEO) that contain gene expression information of breast cancer patients were analyzed; the data indicated that the mRNA level of IL31RA is positively correlated to that of Twist in breast cancer (Figure 5A). Interestingly, IL31RA was observed to have highest expression level in BLBC compared with other subtypes of breast cancer (Figure 5B). Subsequently, real-time PCR results in a cell line array also revealed that both Twist and IL31RA are highly expressed in BLBC cells (Figure 5C). Previously we generated stable Twist-overexpression clone in luminal breast cancer cell line T47D, and performed cDNA microarray analysis of vector-T47D and Twist-T47D cells (GSE53222) (16). This analysis revealed that the mRNA expression of IL31RA was greatly induced by Twist, while down-regulated by treatment of BET inhibitor JQ1. Real-time PCR assay showed that Twist over-expression robustly stimulated the mRNA level of IL31RA; pre-treatment of JQ1 markedly ameliorated this induction. Western blot results further veriﬁed this phenomenon (Figure 5D). Furthermore, knockdown of Twist and/or BRD4 in two BLBC cell lines (MDA-MB-231 and MDA-MB-157) signiﬁcantly decreased the expression of IL31RA (Figure 5E). Similarly, JQ1 treatment strongly down-regulated the protein levels of IL31RA in BLBC cells; meanwhile, JQ1 also inhibited the tyrosine 705 phosphorylation of STAT3 (Figure 5F). These results indicate that Twist/BRD4 complex induces the expression of IL31RA. Overexpression of IL31RA Promotes Cancer Stem Cell-Like Properties and Cell Motility To verify the biological roles of IL31RA in breast cancer, we constructed IL31RA stable over-expression clone in MCF- 7 luminal breast cancer cells by transfection of lentivirus- based expression plasmid (Figure 3A). As expected, this gene over-expression strongly enhanced the tumorsphere formation ability compared with vector control and parental MCF-7 cells May 2020 \| Volume 10 \| Article 816 Frontiers in Oncology \| www.frontiersin.org 5 He et al. He et al. IL31RA Promotes BLBC Progression FIGURE 3 \| Overexpression of IL31RA promotes cancer stem cell-like properties and cell motility. (A) Construction of stable IL31RA over-expression clone in MCF-7 cells was determined by western blot. (B) Cancer stem cell-like properties were determined by tumorsphere assay in parental MCF-7, vector control and IL31RA-overexpression clone. Typical images of tumorsphere were taken at 40 and 200 magnitudes. Statistical data (mean ± SD) for numbers of tumorsphere are shown. indicates P < 0.05 (OE vs. Vector). Scale bar, 100 µM. (C) Cell migration was detected by wound healing assay in parental MCF-7, vector control and IL31RA-overexpression clone for 48 h. Statistical data (mean ± SD) for wound closure are shown. indicates P < 0.01 (OE vs. Vector). (D) Cell invasion was observed by transwell assay in parental MCF-7, vector control and IL31RA-overexpression clone. Scale bar, 100 µM. Statistical data (mean ± SD) for invaded cells are shown. indicates P < 0.001 (OE vs. Vector). Scale bar, 100 µM. FIGURE 3 \| Overexpression of IL31RA promotes cancer stem cell-like properties and cell motility. (A) Construction of stable IL31RA over-expression clone in MCF-7 cells was determined by western blot. (B) Cancer stem cell-like properties were determined by tumorsphere assay in parental MCF-7, vector control and IL31RA-overexpression clone. Typical images of tumorsphere were taken at 40 and 200 magnitudes. Statistical data (mean ± SD) for numbers of tumorsphere are shown. indicates P < 0.05 (OE vs. Vector). Scale bar, 100 µM. (C) Cell migration was detected by wound healing assay in parental MCF-7, vector control and IL31RA-overexpression clone for 48 h. Statistical data (mean ± SD) for wound closure are shown. indicates P < 0.01 (OE vs. Vector). (D) Cell invasion was observed by transwell assay in parental MCF-7, vector control and IL31RA-overexpression clone. Scale bar, 100 µM. Statistical data (mean ± SD) for invaded cells are shown. indicates P < 0.001 (OE vs. Vector). Scale bar, 100 µM. Frontiers in Oncology \| www.frontiersin.org Overexpression of IL31RA Promotes Cancer Stem Cell-Like Properties and Cell Motility May 2020 \| Volume 10 \| Article 816 Frontiers in Oncology \| www.frontiersin.org e et al. IL31RA Promotes BLBC Progression FIGURE 4 \| Loss of IL31RA abrogates the oncogenic roles of IL-31. (A) Tumorsphere assay was performed in control and IL31RA-shRNA clones of MDA-MB-231 in the absence or presence of IL-31 (10 ng/ml) treatment for 3 days. Quantitative analysis of tumorsphere numbers is shown. indicates P < 0.05, #indicates no signiﬁcance. Scale bar, 100 µM. (B) Wound-healing assay was done in the absence or presence of IL-31 (10 ng/ml) treatment for 48 h. Statistical data (mean ± SD) for wound closure are shown. indicates P < 0.05, # indicates no signiﬁcance. (C) Transwell assay was conducted in IL31RA-knockdown and control MDA-MB-231 cells with or without IL-31 (50 ng/ml) treatment. Scale bar, 100 µM. Statistical data (mean ± SD) for invaded cells are shown. indicates P < 0.01, #indicates no signiﬁcance. L31RA I T t G f T i t/BRD4 IL31RA Promotes BLBC Progression He et al. FIGURE 4 \| Loss of IL31RA abrogates the oncogenic roles of IL-31. (A) Tumorsphere assay was performed in control and IL31RA-shRNA clones of MDA-MB-231 in the absence or presence of IL-31 (10 ng/ml) treatment for 3 days. Quantitative analysis of tumorsphere numbers is shown. indicates P < 0.05, #indicates no signiﬁcance. Scale bar, 100 µM. (B) Wound-healing assay was done in the absence or presence of IL-31 (10 ng/ml) treatment for 48 h. Statistical data (mean ± SD) for wound closure are shown. indicates P < 0.05, # indicates no signiﬁcance. (C) Transwell assay was conducted in IL31RA-knockdown and control MDA-MB-231 cells with or without IL-31 (50 ng/ml) treatment. Scale bar, 100 µM. Statistical data (mean ± SD) for invaded cells are shown. *indicates P < 0.01, #indicates no signiﬁcance IL31RA Is a Target Gene of Twist/BRD4 Complex IL31RA gene promoter region and generated its promoter- luciferase construct. When this construct was co-expressed with Twist and BRD4 expression plasmids in HEK293T cells, we found that ectopic expression of Twist and/or BRD4 To determine whether IL31RA is a direct transcriptional target gene of the Twist/BRD4 complex, we cloned the human May 2020 \| Volume 10 \| Article 816 Frontiers in Oncology \| www.frontiersin.org He et al. IL31RA Promotes BLBC Progression FIGURE 5 \| Twist/BRD4 induces IL31RA expression. (A) The gene expression correlation of IL31RA and Twist was analyzed based on three GEO datasets. Correlation coefﬁcients and corresponding p-values are shown. (B) Bio-informatic analyses were performed to measure IL31RA mRNA level in breast cancer subtypes. (C) Real-time PCR experiments were conducted in a series of breast cell lines to measure the mRNA levels of Twist and IL31RA. Data presented are representative of three experiments performed as the mean ± SD. (D) Vector-T47D and Twist-T47D cells were treated with or without JQ1 (1 µM), real-time PCR and western blot analyses were performed to determine the expression of IL31RA (E) Twist and/or BRD4 were knocked down by siRNA in MDA-MB-231 and FIGURE 5 \| Twist/BRD4 induces IL31RA expression. (A) The gene expression correlation of IL31RA and Twist was analyzed based on three GEO datasets. Correlation coefﬁcients and corresponding p-values are shown. (B) Bio-informatic analyses were performed to measure IL31RA mRNA level in breast cancer subtypes. (C) Real-time PCR experiments were conducted in a series of breast cell lines to measure the mRNA levels of Twist and IL31RA. Data presented are representative of three experiments performed as the mean ± SD. (D) Vector-T47D and Twist-T47D cells were treated with or without JQ1 (1 µM), real-time PCR and western blot analyses were performed to determine the expression of IL31RA. (E) Twist and/or BRD4 were knocked down by siRNA in MDA-MB-231 and MDA-MB-157 cells; the expression of IL31RA mRNA was measured by real-time PCR. Data presented are representative of three experiments performed as the mean ± SD. (F) BLBC cells were treated with JQ1 (1 µM), the protein levels of IL31RA and p-STAT3 were detected by western blot. FIGURE 5 \| Twist/BRD4 induces IL31RA expression. (A) The gene expression correlation of IL31RA and Twist was analyzed based on three GEO datasets. Correlation coefﬁcients and corresponding p-values are shown. (B) Bio-informatic analyses were performed to measure IL31RA mRNA level in breast cancer subtypes. IL31RA Is a Target Gene of Twist/BRD4 Complex (C) Real-time PCR experiments were conducted in a series of breast cell lines to measure the mRNA levels of Twist and IL31RA. Data presented are representative of three experiments performed as the mean ± SD. (D) Vector-T47D and Twist-T47D cells were treated with or without JQ1 (1 µM), real-time PCR and western blot analyses were performed to determine the expression of IL31RA. (E) Twist and/or BRD4 were knocked down by siRNA in MDA-MB-231 and MDA-MB-157 cells; the expression of IL31RA mRNA was measured by real-time PCR. Data presented are representative of three experiments performed as the mean ± SD. (F) BLBC cells were treated with JQ1 (1 µM), the protein levels of IL31RA and p-STAT3 were detected by western blot. May 2020 \| Volume 10 \| Article 816 Frontiers in Oncology \| www.frontiersin.org Frontiers in Oncology \| www.frontiersin.org 8 He et al. IL31RA Promotes BLBC Progression FIGURE 6 \| IL31RA is a target gene of Twist/BRD4 complex. (A) The IL31RA gene promoter luciferase activity was measured when this construct was co-expressed with Twist and BRD4 plasmids in HEK293T cells for 24 h. Statistical data (mean ± SD) for fold change are shown. (B) The promoter luciferase activity was measured when BLBC cells were treated with JQ1 (1 µM) for 6 h. Statistical data (mean ± SD) for fold change are shown. (C) The promoter luciferase activity was measured when Twist and/or BRD4 was knocked down in BLBC cells. Statistical data (mean ± SD) for fold change are shown. Data presented are representative of three experiments. (D) Chromatin immunoprecipitation assay was performed by anti-Twist and anti-BRD4 antibodies when MDA-MB-231 cells were treated with or without JQ1 (1 µM) for 6 h, or when Twist was knocked down. Results were analyzed by real-time PCR. Statistical data (mean ± SD) for fold change are shown. FIGURE 6 \| IL31RA is a target gene of Twist/BRD4 complex. (A) The IL31RA gene promoter luciferase activity was measured when this construct was co-expressed with Twist and BRD4 plasmids in HEK293T cells for 24 h. Statistical data (mean ± SD) for fold change are shown. (B) The promoter luciferase activity was measured when BLBC cells were treated with JQ1 (1 µM) for 6 h. Statistical data (mean ± SD) for fold change are shown. (C) The promoter luciferase activity was measured when Twist and/or BRD4 was knocked down in BLBC cells. IL31RA Is a Target Gene of Twist/BRD4 Complex Statistical data (mean ± SD) for fold change are shown. Data presented are representative of three experiments. (D) Chromatin immunoprecipitation assay was performed by anti-Twist and anti-BRD4 antibodies when MDA-MB-231 cells were treated with or without JQ1 (1 µM) for 6 h, or when Twist was knocked down. Results were analyzed by real-time PCR. Statistical data (mean ± SD) for fold change are shown. high rate of early-stage metastasis and lack of eﬀective targeted therapies, it is urgent to reveal the underlying signaling molecules that determine the progression and metastasis of basal-like breast cancer cells. Our results show that the expression level of IL31RA is up-regulated in BLBC in comparison with other breast cancer subtypes. Silencing of IL31RA suppresses the cancer stem cell- like properties, migration, and invasion of BLBC cells in vitro as well as tumor growth and metastasis in vivo. These data suggest that any chemicals or antibodies that target IL-31/IL31RA axis may confer therapeutic beneﬁt to treatment of BLBC. Recently, CIMM331 humanized anti-human IL31RA antibody was tested in a phase I/Ib study for patients of atopic dermatitis (20). We will try this antibody or screen other small inhibitors for blockade of IL-31 signaling and treatment of BLBC. enhanced the IL31RA promoter luciferase activity (Figure 6A). Knockdown of Twist and/or BRD4 greatly repressed the luciferase activity in BLBC cells (Figure 6B). BET inhibitor JQ1, which is able to disrupt the interaction between Twist and BRD4, inhibited the luciferase activity in BLBC cells as well (Figure 6C). Moreover, chromatin immunoprecipitation assay showed that Twist and BRD4 proteins associated with the IL31RA gene promoter; JQ1 disrupted not only the binding of BRD4 also the interaction of Twist on IL31RA promoter; Consistently, silencing of Twist weakened the binding of BRD4 to the IL31RA gene promoter (Figure 6D). All above data indicate that Twist/BRD4 directly regulate IL31RA gene transcription. Frontiers in Oncology \| www.frontiersin.org REFERENCES 14. Yang MH, Hsu DS, Wang HW, Wang HJ, Lan HY, Yang WH, et al. Bmi1 is essential in Twist1-induced epithelial-mesenchymal transition. Nat Cell Biol. (2010) 12:982–92. doi: 10.1038/ncb2099 1. Polyak K. Heterogeneity in breast cancer. J Clin Invest. (2011) 121:3786– 8. doi: 10.1172/JCI60534 1. Polyak K. Heterogeneity in breast cancer. J Clin Invest. (2011) 121:3786– 8. doi: 10.1172/JCI60534 15. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. (2008) 133:704–15. doi: 10.1016/j.cell.2008.03.027 2. Denkert C, Liedtke C, Tutt A, von Minckwitz G. Molecular alterations in triple-negative breast cancer-the road to new treatment strategies. Lancet. (2017) 389:2430–42. doi: 10.1016/S0140-6736(16)32454-0 2. Denkert C, Liedtke C, Tutt A, von Minckwitz G. Molecular alterations in triple-negative breast cancer-the road to new treatment strategies. Lancet. (2017) 389:2430–42. doi: 10.1016/S0140-6736(16)32454-0 16. Shi J, Wang Y, Zeng L, Wu Y, Deng J, Zhang Q, et al. Disrupting the interaction of BRD4 with diacetylated Twist suppresses tumorigenesis in basal- like breast cancer. Cancer Cell. (2014) 25:210–25. doi: 10.1016/j.ccr.2014. 01.028 3. Harbeck N, Gnant M. Breast cancer. Lancet. (2017) 389:1134– 50. doi: 10.1016/S0140-6736(16)31891-8 3. Harbeck N, Gnant M. Breast cancer. Lancet. (2017) 389:1134– 50. doi: 10.1016/S0140-6736(16)31891-8 4. Matsumoto H, Koo SL, Dent R, Tan PH, Iqbal J. Role of inﬂammatory inﬁltrates in triple negative breast cancer. J Clin Pathol. 68:506–10. doi: 10.1136/jclinpath-2015-202944 17. Ip WK, Wong CK, Li ML, Li PW, Cheung PF, Lam CW. Interleukin-31 induces cytokine and chemokine production from human bronchial epithelial cells through activation of mitogen-activated protein kinase signalling pathways: implications for the allergic response. Immunology. (2007) 122:532– 41. doi: 10.1111/j.1365-2567.2007.02668.x 5. Marotta LL, Almendro V, Marusyk A, Shipitsin M, Schemme J, Walker SR, et al. The JAK2/STAT3 signaling pathway is required for growth of CD44(+)CD24(-) stem cell-like breast cancer cells in human tumors. J Clin Invest. (2011) 121:2723–35. doi: 10.1172/JCI44745 18. Ferretti E, Tripodo C, Pagnan G, Guarnotta C, Marimpietri D, Corrias MV, et al. The interleukin (IL)-31/IL-31R axis contributes to tumor growth in human follicular lymphoma. Leukemia. (2015) 29:958–67. doi: 10.1038/leu.2014.291 6. Burugu S, Asleh-Aburaya K, Nielsen TO. Immune Inﬁltrates in the Breast Cancer Microenvironment: Detection, Characterization and Clinical Implication. Breast Cancer. (2017) 24:3-15. doi: 10.1007/s12282-016-0698-z 7. Gangemi S, Quartuccio S, Casciaro M, Trapani G, Minciullo PL, Imbalzano E. Interleukin 31 and skin diseases: a systematic review. Allergy Asthma Proc. (2017) 38:401–8. doi: 10.2500/aap.2017.38.4080 19. DISCUSSION These ﬁndings also identify the transcription regulation manner of IL31RA gene. Our previous study indicated that the Twist/BRD4 complex represents a druggable target for treating BLBC (16). BET-speciﬁc BD inhibitors disrupt the Twist-BRD4 interaction, leading to inhibition of tumorigenicity of BLBC cells in vitro and in vivo. However, BET inhibitors are able to block the interaction of BRD4 with several other acetylated transcriptional factors or inhibit the biological functions of other BET family members, such as BRD2 and BRD3. Therefore, it is rational to develop the ways for inhibition of speciﬁc downstream signaling of Twist/BRD4 complex. Studies of Drosophila mesoderm development indicated that Twist is a master transcriptional factor which governs multiple gene transcriptional activation (21). Our microarray analysis has Plenty of studies have demonstrated the pathogenic role for IL-31 in atopic dermatitis and allergic asthma (17). In recent years, a few literatures implicated the involvement of the IL- 31/IL31RA axis in cancer. The expression of IL-31 and IL31RA was found to be higher in lymph nodes from follicular lymphoma patients with grade IIIa compared with grade I/II. The elevation of IL-31/IL31RA signaling was indicated to be responsible for primary follicular lymphoma cell proliferation (18). Patients with endometrial carcinoma displayed high serum levels of IL- 31, which may represent promising disease biomarkers (19). Intriguingly, the pathological roles of IL-31/IL31RA signaling in tumor progression remain largely unknown. Owing to the May 2020 \| Volume 10 \| Article 816 9 He et al. IL31RA Promotes BLBC Progression ETHICS STATEMENT implicated that IL31RA is transcriptionally active in Twist- overexpressing cells, suggesting it is likely a target gene of Twist (16). In this study, by promoter luciferase and chromatin immunoprecipitation evidences, we identify that IL31RA transcription is directly modulated by Twist/BRD4 complex. Collectively, these data implicate that BLBC cells might employ Twist/BRD4 transcription complex to induce IL31RA expression, facilitating the utilization of IL-31 pro-oncogenic signaling and promoting the tumorigenicity of BLBC. Our study may explain how tumor cells utilize the pro-oncogenic signals derived from inﬂammatory microenvironment and further clarify the working mechanism of Twist/BRD4 complex. The animal study was reviewed and approved by Animal Care and Use Committee of Southern Medical University. AUTHOR CONTRIBUTIONS JS conceived and designed the project, and wrote the manuscript. YH, XZ, WP, and FT carried out the experiments. LL gave advice on the manuscript. DATA AVAILABILITY STATEMENT This research was supported by National Natural Science Foundation of China (81672629, 81872168), and Science and Technology Program of Guangzhou, China (201707010331) to JS. The analyzed data sets generated during the present study are available from the corresponding author on reasonable request. REFERENCES Zeng X, Zhang Z, Gao QQ, Wang YY, Yu XZ, Zhou B, et al. Clinical signiﬁcance of serum interleukin-31 and interleukin-33 levels in patients of endometrial cancer: a case control study. Dis Markers. (2016) 2016:9262919. doi: 10.1155/2016/9262919 8. Rabenhorst A, Hartmann K. Interleukin-31: a novel diagnostic marker of allergic diseases. Curr Allergy Asthma Rep. (2014) 14:423. doi: 10.1007/s11882-014-0423-y 20. Ferretti E, Corcione A, Pistoia V. The IL-31/IL-31 receptor axis: general features and role in tumor microenvironment. J Leukoc Biol. (2017) 102:711– 7. doi: 10.1189/jlb.3MR0117-033R 9. Dillon SR, Sprecher C, Hammond A, Bilsborough J, Rosenfeld- Franklin M, Presnell SR, et al. Interleukin 31, a cytokine produced by activated T cells, induces dermatitis in mice. Nat Immunol. (2004) 5:752–60. doi: 10.1038/ni1084 21. Sandmann T, Girardot C, Brehme M, Tongprasit W, Stolc V, Furlong EE. A core transcriptional network for early mesoderm development in Drosophila melanogaster. Genes Dev. (2007) 21:436–49. doi: 10.1101/gad.15 09007 10. Feld M, Shpacovitch VM, Fastrich M, Cevikbas F, SteinhoﬀM. Interferon- gamma induces upregulation and activation of the interleukin-31 receptor in human dermal microvascular endothelial cells. Exp Dermatol. (2010) 19:921– 3. doi: 10.1111/j.1600-0625.2010.01147.x 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. 11. Shi J, Cao J, Zhou BP. Twist-BRD4 complex: potential drug target for basal-like breast cancer. Curr Pharmaceut Design. (2015) 21:1256– 61. doi: 10.2174/1381612821666141211153853 Copyright © 2020 He, Zhang, Pan, Tai, Liang and Shi. 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. 12. Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C, et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell. (2004) 117:927–39. doi: 10.1016/j.cell.2004.06.006 13. Eckert MA, Lwin TM, Chang AT, Kim J, Danis E, Ohno-Machado L, et al. Twist1-induced invadopodia formation promotes tumor metastasis. Cancer Cell. (2011) 19:372–86. doi: 10.1016/j.ccr.2011.01.036 May 2020 \| Volume 10 \| Article 816 Frontiers in Oncology \| www.frontiersin.org 10
https://openalex.org/W2129984811	https://europepmc.org/articles/pmc3814001?pdf=render	English	null	Intrinsic disorder within an AKAP-protein kinase A complex guides local substrate phosphorylation	eLife	2,013	cc-by	13,185	elife.elifescienc elife.elifescienc RESEARCH ARTICLE elife.elifesciences.org Intrinsic disorder within an AKAP-protein kinase A complex guides local substrate phosphorylation F Donelson Smith1†, Steve L Reichow2†, Jessica L Esseltine1, Dan Shi2, Lorene K Langeberg1, John D Scott1, Tamir Gonen2 1Department of Pharmacology, Howard Hughes Medical Institute, University of Washington, Seattle, United States; 2Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, United States Abstract Anchoring proteins sequester kinases with their substrates to locally disseminate intracellular signals and avert indiscriminate transmission of these responses throughout the cell. Mechanistic understanding of this process is hampered by limited structural information on these macromolecular complexes. A-kinase anchoring proteins (AKAPs) spatially constrain phosphorylation by cAMP-dependent protein kinases (PKA). Electron microscopy and three-dimensional reconstructions of type-II PKA-AKAP18γ complexes reveal hetero-pentameric assemblies that adopt a range of flexible tripartite configurations. Intrinsically disordered regions within each PKA regulatory subunit impart the molecular plasticity that affords an ∼16 nanometer radius of motion to the associated catalytic subunits. Manipulating flexibility within the PKA holoenzyme augmented basal and cAMP responsive phosphorylation of AKAP-associated substrates. Cell-based analyses suggest that the catalytic subunit remains within type-II PKA-AKAP18γ complexes upon cAMP elevation. We propose that the dynamic movement of kinase sub-structures, in concert with the static AKAP-regulatory subunit interface, generates a solid-state signaling microenvironment for substrate phosphorylation. DOI 10 7554/ Lif 01319 001 For correspondence: scottjdw@u.washington.edu (JDS); gonent@janelia.hhmi.org (TG) †These authors contributed equally to this work Competing interests: The authors declare that no competing interests exist. Funding: See page 16 Received: 01 August 2013 Accepted: 26 September 2013 Published: 05 November 2013 Reviewing editor: Roger Davis, University of Massachusetts Medical School, United States Copyright Smith et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. For correspondence: scottjdw@u.washington.edu (JDS); gonent@janelia.hhmi.org (TG) †These authors contributed equally to this work Competing interests: The authors declare that no competing interests exist. DOI: 10.7554/eLife.01319.001 †These authors contributed equally to this work Introduction Protein kinase A is a family of enzymes that contains both ordered and disordered regions, with the ordered sections being involved in phosphorylation, a chemical process that is widely used for communication within cells. However, in order to initiate phosphorylation, these kinases must be anchored to a rigid substrate nearby, so a second group of proteins called AKAPs–which is short for A-kinase anchoring proteins–hold the kinases in place by binding to their disordered regions. These AKAPs also help the kinases to dock with other molecules involved in phosphorylation. A full structural picture of how the kinases induce phosphorylation has yet to be obtained, partly because it is extremely difficult to determine the structure of the disordered regions within the kinases. Moreover, the AKAPs are also disordered, which makes it difficult to work out how the kinases are held in position. Smith, Reichow et al. have used electron microscopy to reveal that the disordered region has two important roles: it determines how far away from the anchoring protein that the active region of the kinase can operate, and it influences how efficiently the kinase can bind to its target molecule in order to induce phosphorylation. Future challenges include investigating how the inherent flexibility of AKAP complexes contribute to the efficient phosphorylation of physiological targets. DOI 10 7554/ Lif 01319 002 2004). AKAPs also organize higher-order macromolecular signaling complexes through their association with G-protein coupled receptors, GTPases and additional protein kinases. Likewise, AKAP-associated phosphatases and phosphodiesterases act to locally terminate these signals. While physiological roles for AKAPs that sequester enzymatic activity with ion channels, cytoskeletal components and regulatory enzymes have been well established, the structural mechanisms involved in these protein–protein interactions have been difficult to characterize. 2004). AKAPs also organize higher-order macromolecular signaling complexes through their association with G-protein coupled receptors, GTPases and additional protein kinases. Likewise, AKAP-associated phosphatases and phosphodiesterases act to locally terminate these signals. While physiological roles for AKAPs that sequester enzymatic activity with ion channels, cytoskeletal components and regulatory enzymes have been well established, the structural mechanisms involved in these protein–protein interactions have been difficult to characterize. Currently, structural details on PKA anchoring are limited because most AKAPs are large, intrinsi cally disordered macromolecules that lack recognizable structural domains. An exception is the crystal structure of the central domain of AKAP18γ that bears homology to bacterial 2H phosphoesterase domains (Gold et al., 2008). Introduction Intrinsically disordered regions of proteins are widespread in nature, yet the mechanistic roles they play in biology are underappreciated. Such disordered segments can act simply to link functionally coupled structural domains or they can orchestrate enzymatic reactions through a variety of allosteric mechanisms (Dyson and Wright, 2005). The regulatory subunits of protein kinase A provide an example of this important phenomenon where functionally defined and structurally conserved domains are connected by intrinsically disordered regions of defined length with limited sequence identity (Scott et al., 1987). In this study, we show that this seemingly paradoxical amalgam of order and disorder permits fine-tuning of local protein phosphorylation events. Received: 01 August 2013 Accepted: 26 September 2013 Received: 01 August 2013 Accepted: 26 September 2013 Published: 05 November 2013 Reviewing editor: Roger Davis, University of Massachusetts Medical School, United States Phosphorylation of proteins is a universal means of intracellular communication that is tightly controlled within the spatial context of the cell. A variety of stimuli trigger these events, which are catalyzed by numerous protein kinases and reversed by phosphoprotein phosphatases (Hunter, 1995). A classic example is production of the second messenger cyclic AMP (cAMP), which stimulates a cAMP-dependent protein kinase (PKA) to phosphorylate a range of cellular targets (Taylor et al., 2012). The PKA holoenzyme is a tetramer composed of two regulatory subunits (R) and two autoinhibited catalytic subunits (PKAc). Binding of cAMP to each R subunit is believed to liberate active kinase and phosphorylation ensues. The local action of PKA is dictated by A-kinase anchoring proteins (AKAPs) that impose spatial constraint by tethering this kinase in proximity to substrates (Wong and Scott, Smith et al. eLife 2013;2:e01319. DOI: 10.7554/eLife.01319 1 of 19 1 of 19 Research article Research article Biochemistry \| Biophysics and structural biology eLife digest It was once thought that proteins needed to have structures that were both ordered and stable, but this view was changed by the discovery that certain proteins contain regions that are disordered and flexible. In some cases these regions of intrinsic disorder help the protein to function by linking more stable regions that are active. However, in other proteins the disordered regions are themselves biologically active and can, for example, function as enzymes. Results and discussion Macromolecular assemblies were formed when purified human γ isoform of AKAP18 (AKAP18γ) was incubated with the PKA holoenzyme (PKAholo), formed by the RIIα and PKAc subunits (Figure 1A, ‘Materials and methods’). Following separation by size-exclusion chromatography, fractions from the front of the elution peak (indicated by arrow in Figure 1B) were analyzed by SDS-PAGE (Figure 1C, left). The subunit composition was confirmed by immunoblotting (Figure 1C, mid). Native electrophoresis established that a majority of this material migrated as a single species with an apparent molecular weight in excess of 240 kD (Figure 1C, right). This molecular mass is consistent with a hetero-penta meric complex composed of a single AKAP18γ molecule anchored to an RIIα subunit dimer and two PKAc subunits. The structure of the AKAP18γ–PKAholo complex was resolved by electron microscopy of negatively stained particles since these complexes are too small to be imaged in vitrified ice (Figure 1D). Single particle analysis revealed clusters of three densities, each approximately 60–100 Å in size resembling beads on a string (Figure 1D,E). Closer inspection of individual particles established that these tripartite structures adopted a range of conformations (Figure 1E). In solution, these complexes may adopt many more configurations and flattening on the carbon support of the EM grid likely captured only a subset of the possible topologies. Approximately 7,000 particles were selected from electron micrographs for structural analysis. Projection averages were derived by classifying particles of similar orientation and structural conformation using reference-free multivariate statistical analysis (van Heel et al., 1996), and iterative stable alignment and clustering procedures (Yang et al., 2012) (Figure 1F, ‘Materials and methods’). Class averages revealed a remarkable variety of configurations (Figure 1F; Video 1). These ranged from a tightly packed pseudo-symmetric triangular configuration (Figure 1F, left panel) to a fully extended linear configuration of the three densities (Figure 1F, right panel). In either case, the central density was consistently smaller (∼60 Å) than the two peripheral densities (85 × 100 Å). Similar size differences between the central and peripheral lobes were observed at the single particle level in raw micrographs (Figure 1D,E). Results and discussion Affinity-labeling of strep-tagged AKAP18γ with a 5 nm gold particle targeted the smaller central density, indicating that this element includes the anchoring protein (Figure 1D, inset).i Three-dimensional (3D) reconstructions for the triangular and linear configurations of the AKAP18γ–PKAholo complex were determined at 35 Å resolution from a tilted-series dataset (Figure 2A, Figure 2—figure supplements 1 and 2). Negative staining in EM can cause flattening of particles, which might lead to apparent structural distortions. However, inspection of particles at various tilt angles showed that particles of linear and triangular conformations remained clearly distinguishable, even at high tilt angles (Figure 2—figure supplement 1). In both 3D reconstructions, a central mass with dimensions of 60 × 60 × 80 Å (corresponding to the site of AKAP18γ as demonstrated by affinity gold labeling, Figure 2A, black triangle and Figure 1D, inset) was flanked on either side by larger densities of 100 × 100 × 85 Å (Figure 2A). These flanking densities can each accommodate a sub-complex of RIIα and PKAc. In the triangular conformation, the two peripheral densities are oriented at a 100° angle with respect to the central density and exhibit an end-to-end length of ∼300 Å (Figure 2A, top). The end-to-end length increases to ∼385 Å in the extended linear configuration (Figure 2A, bottom). Back-projections calculated from the final 3D maps compare well with the experimental class averages (Figure 2—figure supplement 2). Moreover, this back-projection analysis demonstrated that when the triangular model is tilted completely on its edge (where it may appear more linear in projection) its dimensions (max length = 300 Å) are significantly smaller than the maximum end-to-end length obtained for the linear reconstruction (max length = 385 Å). Hence, we conclude that the linear and triangular conformations are structurally distinct.i Pseudo-atomic models of the pentameric protein assembly in both configurations were constructed by fitting Protein Data Bank coordinates for regions of AKAP18γ and PKAholo subunits (Knighton et al., 1991, 1992; Gold et al., 2006, 2008; Wu et al., 2007) (Figure 2A, ‘Materials and methods’). A model for AKAP18γ (residues 88–317) was derived by connecting the central domain (residues 88–290) (Gold et al., 2008) via a short linker to the PKA anchoring helix (AKAP(helix), residues 301–317) (Gold et al., 2006) (Figure 2A, yellow , ‘Materials and methods’). Introduction Insights from these studies may be broadly applicable to the understanding of other intrinsically disordered proteins and anchoring complexes that organize enzymatic action in the cell. Introduction Likewise, high-resolution crystallographic structures of the catalytic subunit (PKAc) when free and in complex with the C-terminal autoinhibitory and cAMP binding domains of the type I or type II regulatory subunits of PKA (RI and RII) have provided details on the mechanisms of catalysis and autoinhibition (Knighton et al., 1991, 1992; Gold et al., 2006, 2008; Wu et al., 2007). Yet, despite decades of effort, a complete structural picture of the PKA holoenzyme is lacking. This is presumably due to the presence of long flexible intrinsically disordered linker regions within the R subunit that tether this complex. NMR spectroscopy and X-ray crystallographic studies show that the N-terminal domains of RI and RII homodimerize through a four-helix bundle docking and dimerization interface (D/D) (Newlon et al., 2001; Gold et al., 2006; Kinderman et al., 2006; Sarma et al., 2010). The D/D creates a high-affinity binding groove for a canonical amphipathic helix on each AKAP that forms the reciprocal binding surface (Gold et al., 2006). Although the physiological consequences of anchored PKA phosphorylation events have been established in a variety of cellular contexts, we have yet to discern how the individual protein components are assembled and operate within AKAP complexes (Scott and Pawson, 2009). Even more elusive is a mechanistic role for intrinsically disordered domains within these macromolecular assemblies. For example, does internal flexibility within these signaling assemblies modulate other aspects of enzyme action in addition to sequestering PKA at specific regions of the cell? Here we have used electron microscopy (EM) to evaluate the topological arrangement of the fully assembled type IIα PKA holoenzyme when anchored to AKAP18γ. This analysis unveils a remarkable level of conformational plasticity that resides within the AKAP–PKA complex. In vitro and cell-based structure-function approaches reveal an unexpected functional requirement for intrinsically disordered regions within RIIα. These regions not only define a radius of action of the anchored catalytic subunit but also modulate the enzymatic efficiency 2 of 19 Smith et al. eLife 2013;2:e01319. DOI: 10.7554/eLife.01319 2 of 19 Research article Research article Biochemistry \| Biophysics and structural biology Biochemistry \| Biophysics and structural biology of the kinase. Insights from these studies may be broadly applicable to the understanding of other intrinsically disordered proteins and anchoring complexes that organize enzymatic action in the cell. of the kinase. Results and discussion When AKAP18γ is docked to residues 1–43 of RIIα (RII(D/D)), the resulting sub-structure fits within the central density of maps for both the triangular and extended configurations (Figure 2A, yellow). This tallies with the single particle affinity-labeling Smith et al. eLife 2013;2:e01319. DOI: 10.7554/eLife.01319 3 of 19 3 of 19 Research article Research article Biochemistry \| Biophysics and structural biology di h AKAP18 hi i (Fi 1D i ) I i il gure 1. Purification and electron microscopy of the AKAP18γ–PKAholo complex. (A) SDS-PAGE and Coomassie staining of purif mponents. (B) Size-exclusion chromatography (SEC) trace for purification of the assembled AKAP18γ–PKAholo complex. Fractio the peak (indicated by gray bar) were chosen for further analysis. (C) SDS-PAGE (left), western blot (middle) and native gel ele tained from the SEC peak elution fraction (arrow in B). (D) Electron micrograph of the negatively stained AKAP18γ–PKAholo co angles indicate the three major densities of the AKAP18γ–PKAholo complex. Inset, shows labeling with a gold nanoparticle (arr AKAP18γ-streptavidin moiety (arrow). (E) Left, enlarged images of individual AKAP18γ–PKAholo complexes (denoted by asteris ghlighted outline (yellow) of particle shapes. (F) Projection averages of the AKAP18γ–PKAholo complex classified into distinct AC (Yang et al., 2012). Unlabeled scale bars represent 25 nm. OI: 10.7554/eLife.01319.003 gure 1. Purification and electron microscopy of the AKAP18γ–PKAholo complex. (A) SDS-PAGE and Coomassie staining of puri omponents. (B) Size-exclusion chromatography (SEC) trace for purification of the assembled AKAP18γ–PKAholo complex. Fracti the peak (indicated by gray bar) were chosen for further analysis. (C) SDS-PAGE (left), western blot (middle) and native gel ele btained from the SEC peak elution fraction (arrow in B). (D) Electron micrograph of the negatively stained AKAP18γ–PKAholo co iangles indicate the three major densities of the AKAP18γ–PKAholo complex. Inset, shows labeling with a gold nanoparticle (arr n AKAP18γ-streptavidin moiety (arrow). (E) Left, enlarged images of individual AKAP18γ–PKAholo complexes (denoted by asteri ghlighted outline (yellow) of particle shapes. (F) Projection averages of the AKAP18γ–PKAholo complex classified into distinct AC (Yang et al., 2012). Unlabeled scale bars represent 25 nm. OI: 10 7554/eLife 01319 003 Figure 1. Purification and electron microscopy of the AKAP18γ–PKAholo complex. (A) SDS-PAGE and Coomassie staining of purified individual complex components. (B) Size-exclusion chromatography (SEC) trace for purification of the assembled AKAP18γ–PKAholo complex. Fractions at the leading edge of the peak (indicated by gray bar) were chosen for further analysis. Results and discussion AKAP18γ–RII(D/D) sub-complex to each RIIα-PKAc unit through a flexible chain corresponding to residues 44–90 of RIIα (Figure 2A, ‘Materials and methods’). The complete pseudo-atomic model of AKAP18γ–PKAholo complex is presented in Figure 2B. Our model of the anchored PKA complex implies that an intrinsically disordered flexible linker region within RIIα supports the array of con formations that were observed in the raw micro graphs and the projection averages (Figures 1 and 2C). To quantitatively assess the distribution of conformations assumed by this complex, we measured the end-to-end distance between the two large peripheral densities of 223 individual particles (Figure 2D). This population of structures followed a Gaussian distribution (Figure 2D, green trace) with a mean particle length of 275 ± 65 Å (n = 223). We propose that conformational plas ticity observed in these analyses is facilitated by this intrinsically disordered region between resi dues 44 and 90 of RIIα, a linker that connects the AKAP docking site (D/D domain) to the cAMP-responsive transduction domains. This notion is further substantiated by a primary sequence analysis of RIIα orthologs, showing that the linker regions are of similar length but exhibit low amino acid identity (Figure 3A).l Video 1. Conformational dynamics of the wild-type AKAP18γ–PKA holoenzyme complex. A montage of projection averages obtained for the wild-type AKAP18γ–PKA holoenzyme complex displays the variety of topological configurations sampled by the dynamic signaling particle. DOI: 10.7554/eLife.01319.004 Video 1. Conformational dynamics of the wild-type AKAP18γ–PKA holoenzyme complex. A montage of projection averages obtained for the wild-type AKAP18γ–PKA holoenzyme complex displays the variety of topological configurations sampled by the dynamic signaling particle. / f Video 1. Conformational dynamics of the wild-type AKAP18γ–PKA holoenzyme complex. A montage of projection averages obtained for the wild-type AKAP18γ–PKA holoenzyme complex displays the variety of topological configurations sampled by the dynamic signaling particle. DOI: 10.7554/eLife.01319.004 dues 44 and 90 of RIIα, a linker that connect the AKAP docking site (D/D domain) to the cAMP-responsive transduction domains. This notion further substantiated by a primary sequence analysis of RIIα orthologs, showing that the linker region are of similar length but exhibit low amino acid identity (Figure 3A). We reasoned that if the linker region in RIIα contributes to conformational flexibility of the holoenzyme, altering its length could affect the structure and function of the anchored kinase. Results and discussion This structural postulate was tested by producing modified AKAP18γ–PKAholo complexes in which the linker region of RIIα was deleted (RIIα Δ44–86) or replaced with an extended sequence of 60 residues found in zebrafish (RIIα ZeChimera; Figure 3A,B). Formation of modified AKAP–PKA complexes (assembled as described above) was monitored by Coomassie blue staining and immunoblot detection of the component proteins (Figure 3C). The conformations of these modified AKAP18γ–PKAholo complexes were analyzed by electron microscopy as previously described (Figure 3D–G). Single-particle EM and class averages of the assemblies formed with RIIα Δ44–86 yielded uniform complexes exclusively in a compact triangular configuration (Figure 3D,F). In contrast, complexes formed with RIIα ZeChimera resembled the array of conformations observed for the wild-type AKAP18γ–PKAholo assembly (Figure 3E,G). Quantitative analysis of the differing linker lengths was assessed by measuring the radius of each particle, defined by the center of the AKAP18γ subunit to the distal end of each PKA subunit. Particle radii for the RIIα Δ44–86 complexes ranged in length from 55 to 125 Å, (mean value of 87 ± 13 Å, n = 142; Figure 3H) whereas the assemblies formed with the RIIα ZeChimera were extended with lengths ranging from 100 to 265 Å (mean value of 168 ± 33 Å, n = 296; Figure 3H). These latter measurements are similar to the parameters of the native complex, (160 ± 29 Å, n = 216; Figures 2D and 3D). Thus we conclude that a flexible linker in RIIα is responsible for the conformational plasticity of the AKAP18γ–PKAholo assemblies. One mechanistic ramification of our structural analyses is that flexibility within PKAholo complex could permit precise orientation of the anchored catalytic subunit toward substrates. This would be particularly true for substrates that are physically associated with AKAPs. For example, the type 4 phosphodiesterase isoforms PDE4D3 and PDE4D5 associate with the central domain of AKAP18γ and are phosphorylated on two sites by PKA (Sette and Conti, 1996; Carlisle Michel et al., 2004; Stefan et al., 2007). We confirmed this protein–protein interaction upon co-expression of the compo nents in HEK293 cells (Figure 4A). PDE4Ds co-precipitate with AKAP18γ and the RII subunits of PKA (Figure 4A, lane 1) but not with GFP controls (Figure 4A, lane 2). Accordingly, cAMP phosphodies terase activity was enriched 3.27 ± 0.48-fold (n = 5) in AKAP18γ–GFP immune complexes as compared to GFP controls or samples treated with the PDE4 selective inhibitor rolipram (Figure 4B). Results and discussion (C) SDS-PAGE (left), western blot (middle) and native gel electrophoresis (right) obtained from the SEC peak elution fraction (arrow in B). (D) Electron micrograph of the negatively stained AKAP18γ–PKAholo complexes (circles). Triangles indicate the three major densities of the AKAP18γ–PKAholo complex. Inset, shows labeling with a gold nanoparticle (arrow) conjugated to an AKAP18γ-streptavidin moiety (arrow). (E) Left, enlarged images of individual AKAP18γ–PKAholo complexes (denoted by asterisks in D). (E) Right, highlighted outline (yellow) of particle shapes. (F) Projection averages of the AKAP18γ–PKAholo complex classified into distinct conformations using SAC (Yang et al., 2012). Unlabeled scale bars represent 25 nm. DOI: 10.7554/eLife.01319.003 Figure 1. Purification and electron microscopy of the AKAP18γ–PKAholo complex. (A) SDS-PAGE and Coomassie staining of purified individual complex components. (B) Size-exclusion chromatography (SEC) trace for purification of the assembled AKAP18γ–PKAholo complex. Fractions at the leading edge of the peak (indicated by gray bar) were chosen for further analysis. (C) SDS-PAGE (left), western blot (middle) and native gel electrophoresis (right) obtained from the SEC peak elution fraction (arrow in B). (D) Electron micrograph of the negatively stained AKAP18γ–PKAholo complexes (circles). Triangles indicate the three major densities of the AKAP18γ–PKAholo complex. Inset, shows labeling with a gold nanoparticle (arrow) conjugated to an AKAP18γ-streptavidin moiety (arrow). (E) Left, enlarged images of individual AKAP18γ–PKAholo complexes (denoted by asterisks in D). (E) Right, highlighted outline (yellow) of particle shapes. (F) Projection averages of the AKAP18γ–PKAholo complex classified into distinct conformations using ISAC (Yang et al., 2012). Unlabeled scale bars represent 25 nm. DOI: 10.7554/eLife.01319.003 studies that map AKAP18γ to this region (Figure 1D, inset). In a similar manner, the peripheral densities each accommodate a PKA sub-structure consisting of one catalytic subunit (Figure 2A, blue) in complex with the cAMP-binding domain of RIIα, residues 91–392 (Figure 2A, green) (Knighton et al., 1991; Wu et al., 2007). Finally these models were completed by connecting the central 4 of 19 Smith et al. eLife 2013;2:e01319. DOI: 10.7554/eLife.01319 Research article Research article Video 1. Conformational dynamics of the wild-type AKAP18γ–PKA holoenzyme complex. A montage of projection averages obtained for the wild-type AKAP18γ–PKA holoenzyme complex displays the variety of topological configurations sampled by the dynamic signaling particle. DOI 10 7554/ Lif 01319 004 AKAP18γ–RII(D/D) sub-complex to each RIIα-PKAc unit through a flexible chain corresponding to residues 44–90 of RIIα (Figure 2A, ‘Materials and methods’). The complete pseudo-atomic model of AKAP18γ–PKAholo complex is presented in Figure 2B. Results and discussion These data allowed us to move to an in vitro system to study phosphorylation of PDE4D by AKAP18–PKA complexes. 5 of 19 Smith et al. eLife 2013;2:e01319. DOI: 10.7554/eLife.01319 5 of 19 Research article Research article Biochemistry \| Biophysics and structural biology Figure 2. 3D reconstructions and pseudo-atomic structure of the AKAP18γ–PKAholo complex. (A) Three-dimensional (3D) EM reconstructions and 90° rotated views of the fitted molecular models for the AKAP18γ–PKAholo complex. High-resolution structures for regions of AKAP18γ (gold), RIIα (green) and C subunit of PKA (blue) that fit within the EM densities are indicated. Models are presented in the (top) compact triangular and (bottom) extended linear conformation. (B) Pseudo-atomic model of the AKAP18γ–PKAholo complex. (C) (top) Projection averages of the AKAP18γ–PKAholo complex with structural domains fitted into the EM densities and connected by lines representing the RIIα flexible linker regions. (bottom) Projection averages were removed for clarity. Scale bar represents 25 nm. (D) Statistical analysis of individual particle lengths in angstroms (Å) displays a Gaussian distribution (green line) with a mean value of 275 Å and a standard deviation (σ) ±65 Å. DOI: 10.7554/eLife.01319.005 The following figure supplements are available for figure 2: Figure supplement 1. Tilted-series electron microscopy data. DOI: 10.7554/eLife.01319.006 Figure supplement 2. Three-dimensional EM maps of the AKAP18γ–PKAholo complex. DOI: 10.7554/eLife.01319.007 Figure 2. 3D reconstructions and pseudo-atomic structure of the AKAP18γ–PKAholo complex. (A) Three-dimensional (3D) EM reconstructions and 90° rotated views of the fitted molecular models for the AKAP18γ–PKAholo complex. High-resolution structures for regions of AKAP18γ (gold), RIIα (green) and C subunit of PKA (blue) that fit within the EM densities are indicated. Models are presented in the (top) compact triangular and (bottom) extended linear conformation. (B) Pseudo-atomic model of the AKAP18γ–PKAholo complex. (C) (top) Projection averages of the AKAP18γ–PKAholo complex with structural domains fitted into the EM densities and connected by lines representing the RIIα flexible linker regions. (bottom) Projection averages were removed for clarity. Scale bar represents 25 nm. (D) Statistical analysis of individual particle lengths in angstroms (Å) displays a Gaussian distribution (green line) with a mean value of 275 Å and a standard deviation (σ) ±65 Å. DOI: 10.7554/eLife.01319.005 The following figure supplements are available for figure 2: Figure supplement 1. Tilted-series electron microscopy data. DOI: 10.7554/eLife.01319.006 Figure supplement 2. Three-dimensional EM maps of the AKAP18γ–PKAholo complex. DOI: 10.7554/eLife.01319.007 In vitro phosphorylation studies on AKAP18-associated PDE4D were performed in two phases. Research article Biochemistry \| Biophysics and structural biology Biochemistry \| Biophysics and structural biology Figure 3. Continued Figure 3. Continued Figure 3. Continued yellow, RIIα in green and PKAc in blue. (C) Biochemical analysis of the purified AKAP18γ–RIIα-PKAc complexes assembled with the wild-type RIIα subunit, the RIIα Δ44–86 mutant where the linker region was deleted, and the RIIα ZeChimera mutant where the mouse RIIα linker region was replaced with the corresponding and extended sequence from zebrafish. Top panel shows SDS-PAGE and Coomassie blue staining of the protein components. The next three panels show western blotting for RIIα, PKAc subunit and AKAP18γ, respectively. (D) Electron micrograph of negatively stained AKAP18γ–PKAholo complexes (circles) assembled with the RIIα Δ44–86 construct. (E) Electron micrograph of negatively stained AKAP18γ–PKAholo complexes (circles) assembled with the RIIα ZeChimera construct. Insets in (D and E) show enlarged views of individual particles outlined in gold for clarity. (F) Projection averages of the AKAP18γ–PKAholo complexes assembled with a truncated RIIα Δ44–86 construct using ISAC (Yang et al., 2012). (G) Projection averages of AKAP18γ–PKAholo complex assembled an RIIα ZeChimera construct. Scale bars in (F) and (G) represent 25 nm. (H) Statistical analysis of particle radius in angstroms (Å) for each AKAP18γ–PKAholo complexes. Box plot displays second and third quartile values, tails corresponding to minimum and maximum distances, () indicates p<0.01; (**) indicates p<0.0001. DOI: 10.7554/eLife.01319.008 In the second phase, experiments were conducted using higher-order complexes formed with wild-type RIIα, RIIα Δ44–86, or the RIIα ZeChimera (assembled as described above) to investigate whether manipulating the intrinsic flexibility of PKA altered phosphorylation of anchored PDE4D (Figure 4F–G). We measured cAMP-independent phosphorylation of PDE4D at 5 min, a time point that showed sub-maximal substrate phosphorylation (Figure 4F,G, Figure 4—figure supplement 1). Basal PDE4D phosphorylation was enhanced 1.97 ± 0.18-fold (n = 6, p<0.05) in complexes formed with RIIα Δ44–86 when compared to a wild-type complex (Figure 4G, bars 1 and 4). In contrast, extension of the linker region in the context of AKAP18γ–RIIα ZeChimera PKAholo assembly had no effect as compared to wild type (Figure 4G, bars 1 and 7). Control experiments confirmed that addition of cAMP further augmented phosphorylation of PDE4D in all cases (Figure 4F, lanes 2, 5 and 8) and pretreatment with PKI inhibitor peptide abolished anchored kinase activity (Figure 4F, lanes 3, 6 and 9). Research article These data show that the AKAP can be thought of as a catalyst that physically brings the reactants together, and the flexibility within the anchored PKA holoenzyme allows for the precise orientation of the enzyme and substrate. This mechanism may be particularly relevant for cAMP-independent phosphorylation events that are believed to represent approximately 30% of PKA action (Taylor et al., 2012). Therefore, this hitherto unexplained but critical component of cellular PKA activity may be accomplished by the persistent phosphorylation of substrates embedded in higher-order AKAP signaling assemblies. To follow these in vitro studies, we tested our hypothesis that flexibility within the anchored PKA holoenzyme influences cAMP signaling in living cells. We generated a modified fluorescence resonance energy transfer (FRET) based PKA activity sensor using the A-kinase activity reporter (AKAR2) backbone (Zhang et al., 2005). Our modified sensor (AKAR-18RBS) was constructed by fusing the PKA binding helix of AKAP18 (18RBS) (Fraser et al., 1998; Gray et al., 1998) to the amino terminus of AKAR2 (Figure 5A). This genetically encoded reporter detects PKA phosphorylation in real-time by monitoring changes in the YFP/CFP emission ratio inside cells (Figure 5A). As a prelude to these studies AKAR-18RBS association with wild-type RIIα or either of the modified RIIα constructs was confirmed by co-immunoprecipitation of each complex from HEK293 cells (Figure 5B). In parallel, immunoblot and confocal fluorescent imaging analyses confirmed that mCherry tagged versions of each RIIα form were expressed to equivalent levels (Figure 5C) and uniformly distributed in HEK293 cells (Figure 5—figure supplement 1A). Initial experiments evaluated basal FRET of the AKAR-18RBS reporter using a modified Leica DMI 6000B microscope. The raw YFP/CFP emission ratio of AKAR-18RBS was elevated in unstimulated cells expressing RIIα Δ44–86 as compared to RIIα wild type or the RIIα ZeChimera (Figure 5D). These data further develop the concept introduced in Figure 4 that the RIIα linker region influences basal phosphorylation of AKAP-associated substrates. Next, real-time changes in the YFP/CFP emission ratio of the AKAR-18RBS reporter were monitored in cells expressing equivalent levels of the individual RIIα forms (Figure 5E–G). The β-adrenergic (β-AR) agonist isoproterenol (Iso) was administered after 100 s to initiate the cAMP response (Figure 5E–G). Notable increases in AKAR-18RBS FRET were evident in cells expressing RIIα wild type or the RIIα ZeChimera (Figure 5E,G). Results and discussion Our structural data predict that the C subunit of anchored PKA resides within ∼100 Å of its substrate PDE4D (shown schematically in Figure 4C). We reasoned that this tight configuration could permit cAMP-independent phosphorylation of the phosphodiesterase. Therefore, in the first phase, ‘basal’ phosphorylation of PDE4D was measured in the context of the AKAP18γ signaling complex (Figure 4D). Phosphate incorporation into PDE4D was increased 1.87 ± 0.14-fold (p<0.05) upon its tethering to AKAP18γ as assessed by autoradiography (Figure 4D, E). This suggests that the proximity to a substrate afforded by AKAP18γ augments the cAMP-independent action of PKA. This could explain why physiologically relevant PKA targets such as aquaporins or phospholamban, which require continual or instantaneous phosphorylation to fulfill their biological roles, have their own AKAP-associated pool of kinase (Henn et al., 2004; Lygren et al., 2007; Gold et al., 2012). Accordingly, a primary function of AKAPs may be to ensure basal phosphorylation of such tethered substrates. Smith et al. eLife 2013;2:e01319. DOI: 10.7554/eLife.01319 6 of 19 Research article Research article Biochemistry \| Biophysics and structural biology Biochemistry \| Biophysics and structural biology Figure 3. Flexibility within RIIα constrains the configuration of the anchored kinase assembly. (A) Amino acid sequence a RIIα that connects the conserved N-terminal D/D domain to the C-terminal autoinhibitor and cAMP binding domains Th Figure 3. Flexibility within RIIα constrains the configuration of the anchored kinase assembly. (A) Amino acid sequence alignment of the linker region in RIIα that connects the conserved N-terminal D/D domain to the C-terminal autoinhibitor and cAMP binding domains. This region shows low sequence homology and is likely to be structurally disordered. (B) Schematic representations of modified AKAP18γ–PKAholo complexes with AKAP18γ depicted in Figure 3. Continued on next page Figure 3. Flexibility within RIIα constrains the configuration of the anchored kinase assembly. (A) Amino acid sequence alignment of the linker region in RIIα that connects the conserved N-terminal D/D domain to the C-terminal autoinhibitor and cAMP binding domains. This region shows low sequence homology and is likely to be structurally disordered. (B) Schematic representations of modified AKAP18γ–PKAholo complexes with AKAP18γ depicted in Figure 3. Continued on next page Smith et al. eLife 2013;2:e01319. DOI: 10.7554/eLife.01319 7 of 19 Research article Research article Figure 4. Continued Figure 4. Continued immunocomplexes as in (A) were used for phosphodiesterase activity assays. Inclusion of the small molecule rolipram (10 μM, bar 3) inhibited associated PDE4D activity. (C) Schematic of AKAP18–PKA holoenzyme-PDE4D3/5 complexes used in subsequent in vitro substrate phosphorylation assays. Based on our structural data, the PKA catalytic subunit is expected to be positioned within ∼100 Å of its substrate PDE4D. (D) Anchoring of PKA and PDE4D stimulates cAMP-independent phosphorylation of the phosphodiesterase. (Top panel) Basal 32P incorporation into PDE4D (detected by autoradiograph) is shown in the absence or presence of AKAP18γ. (Bottom panels) Levels of PDE4D, RIIα, PKAc and AKAP18γ were assessed by immunoblot. (E) Densitometeric quantification of phospho-PDE4D in panel (D), n = 4 (p<0.05). (F–G) Deletion of the flexible linker augments cAMP-independent phosphorylation of PDE4D by 1.97 ± 0.18-fold (p<0.05). (F) (Top panel) Autoradiograph showing incorporation of 32P into PDE4D in each complex. (Bottom panel) Coomassie blue staining of the SDS-PAGE gel showing components of the assay. The PDE4D is at the top, while the different complexes are shown at the bottom. (G) Densitometric quantification of phospho-PDE4D levels in (F), n = 6. DOI: 10.7554/eLife.01319.009 The following figure supplements are available for figure 4: The following figure supplements are available for figure 4: Figure supplement 1. Phosphorylation of PDE4D is time-dependent. DOI: 10.7554/eLife.01319.010 RIIα Δ44–86 form responded more rapidly and robustly to isoproterenol with a maximal FRET response at 200 s (Figure 5F,H, green trace). The RIIα Δ44–86 effect was abolished when control experiments were performed with a proline modified AKAR-18RBS derivative that is unable to anchor PKA (Fraser et al., 1998; Gray et al., 1998; Figure 5—figure supplement 1). Other control experiments established that application of isoproterenol did not stimulate an AKAR-18RBS derivative (T/A) where the threonine phospho-acceptor was replaced with alanine (Figure 5H, inset, red trace). RIIα Δ44–86 form responded more rapidly and robustly to isoproterenol with a maximal FRET response at 200 s (Figure 5F,H, green trace). The RIIα Δ44–86 effect was abolished when control experiments were performed with a proline modified AKAR-18RBS derivative that is unable to anchor PKA (Fraser et al., 1998; Gray et al., 1998; Figure 5—figure supplement 1). Other control experiments established that application of isoproterenol did not stimulate an AKAR-18RBS derivative (T/A) where the threonine phospho-acceptor was replaced with alanine (Figure 5H, inset, red trace). Figure 4. Continued Scatter plot representation of the Iso stimulated FRET responses at 200 s reveal that AKAR-18RBS-PKA complexes formed with RIIα or the RIIα ZeChimera responded with similar magnitudes (Figure 5I, black and orange). In contrast, the normalized FRET responses of the more compact and less flexible AKAR-18RBS-PKA complexes formed with RIIα Δ44–86 were significantly higher (Figure 5I, green, p<0.001). Taken together, these cell-based studies indicate that removal of the flexible linker region in RIIα augments cAMP-responsive PKA phosphorylation of AKAR-18RBS inside the cells. These findings complement the interpretation of our EM reconstructions and in vitro phosphorylation studies. These observations argue that compact and rigid AKAP–PKA assemblies enhance kinase action within the immediate vicinity of the substrate. Local activation of PKA is believed to involve dissociation of the C subunits from the R subunit dimer. Yet, cAMP binds to the R subunits avidly (KD 6–8 nM), and its rate of release is so slow that it is not readily apparent how the cAMP-binding sites turnover within the cell (Poppe et al., 2008). Additionally, there is a report that cAMP can activate PKA without C subunit release (Yang et al., 1995). Therefore, we evaluated whether cAMP stimulation altered the composition of PKA holoenzymes associated with the AKAR-18RBS reporter. Interestingly, both the RII and C subunits of PKA were present in AKAR-18RBS immune complexes, even after stimulation of cAMP production by isoproterenol (Figure 5J, lanes 1 and 2). The isoproterenol-stimulated activation of PKA was validated by immunoblot detection of phosphorylated substrates with a phospho-PKA site antibody (Figure 5J, bottom panel). Control experiments confirmed that PKA subunits did not associate with the proline-modified AKAR-18RBS derivative, whereas the AKAR-18RBS-T/A reporter retained the ability to anchor PKA (Figure 5J, lanes 3–6). This AKAR-18RBS reporter data raised the question of whether anchoring can stabilize the PKA holoenzyme even in the presence of cAMP. To further test this hypothesis, co-precipitation experi ments were repeated with full-length AKAP18γ. Again the C subunit was retained within the AKAP complex upon isoproterenol stimulation (Figure 5K, lanes 1 and 2). This occurred regardless of whether the PKA holoenzymes were formed with wild-type RIIα, RIIα Δ44–86, or RIIα ZeChimera (Figure 5K, top panel). These results confirm that the increased agonist-stimulated activity of the AKAR-18RBS–RIIα Δ44–86 complex shown in Figure 5H,I is not due to enhanced dissociation of the PKA catalytic subunit. Research article In both the cases the normalized FRET ratio was maximal at 200 s and gradually declined over the remainder of the time course (Figure 5H, black and orange traces). This latter phenomenon may be attributed to either desensitization of the β-AR system or dephosphorylation of the reporter by phosphoprotein phosphatases (Bouvier et al., 1987; Pitcher et al., 1992; Violin et al., 2003). Importantly, cells expressing the more compact Smith et al. eLife 2013;2:e01319. DOI: 10.7554/eLife.01319 8 of 19 Research article R Biochemistry \| Biophysics and structural biology Figure 4. RIIα linker length influences basal PKA phosphorylation of associated substrates. (A) PDE4D isoforms associate with AK cells expressing AKAP18–GFP or GFP alone along with PDE4D and RIIα were subjected to immunoprecipitation with anti-GFP an Immunocomplexes were separated by SDS-PAGE and immunoblotted for PDE4D and RIIα. AKAP18 was detected by RII overlay a Figure 4. Continued on next page Figure 4. RIIα linker length influences basal PKA phosphorylation of associated substrates. (A) PDE4D isoforms associate with AKAP18 in cells. HEK293 cells expressing AKAP18–GFP or GFP alone along with PDE4D and RIIα were subjected to immunoprecipitation with anti-GFP antibodies. Immunocomplexes were separated by SDS-PAGE and immunoblotted for PDE4D and RIIα. AKAP18 was detected by RII overlay analysis. (B) Similar Figure 4. Continued on next page Smith et al. eLife 2013;2:e01319. DOI: 10.7554/eLife.01319 9 of 19 Research article Research article Research article Biochemistry \| Biophysics and structural biology Biochemistry \| Biophysics and structural biology Figure 4. Continued Research article Biochemistry \| Biophysics and structural biology Biochemistry \| Biophysics and structural biology Figure 5. Continued Figure 5. Continued Figure 5. Continued adjacent Forkhead homology-associated (FHA) phospho-Thr binding domain. Subsequent rearrangement brings together the ECFP and YFP (citrine) moieties to produce an increase in FRET signal as readout of kinase activity. (B) Co-immunoprecipitation of AKAR-18RBS-PKA complexes with holoenzymes composed of three different RII forms. AKAR-18RBS was immunoprecipitated with anti-GFP antibodies and bound PKA subunits were detected by western blotting. (C) Lysates from cells transfected with the AKAR-18RBS reporter and each of the three RIIαforms were immunoblotted to confirm similar expression levels in each cohort. (D) Basal (unstimulated) raw FRET signals from cells expressing RIIα (gray), RIIα Δ44–86 (green) and RIIα ZeChimera (orange). (E and G) Time course of AKAR-18RBS activation in response to the β-adrenergic agonist isoproterenol in cells co-expressing the FRET reporter with (E) RIIα wild type, (F) RIIα Δ44–86, or (G) RIIα ZeChimera. Isoproterenol (Iso, 10 μM) was added at t = 100 s and FRET was recorded for 5 min post-stimulation. Warmer colors indicate increasing phosphorylation as shown in the pseudo-color scale. (H) Amalgamated traces of the Iso stimulated changes in FRET in each cohort (0–400 s). Data are normalized to unstimulated basal FRET level for each respective RIIα form. Changes in the AKAR-18RBS normalized FRET ratio are shown from cells expressing RIIα wild type (black), RIIα Δ44–86 (green) and RIIα ZeChimera (orange). Mutation of the phosphoacceptor threonine in the FRET reporter to create AKAR-18RBS–T/A blocks phosphorylation and abolishes FRET in response to Iso treatment (Inset). (I) Scatter plot representation of peak FRET signals from all cells at 200 s. This plot indicates that AKAR-18RBS-PKA complexes formed with RIIα wild type (black) or the RIIα ZeChimera (orange) had similar peak responses, while complexes formed with RIIα Δ44–86 displayed significantly greater peak FRET responses (p<0.001). This plot also shows that in all cases, some cells fail to respond entirely; these non-responders are included in the amalgamated data analysis presented in (H). (J) C subunit of PKA association with AKAR-18RBS, AKAR-18RBS–pro or AKAR-18RBS–T/A complexes. AKAR-18RBS and AKAR-18RBS–T/A co-precipitate RIIα and endogenous PKA catalytic subunit; AKAR-18RBS–pro, a mutant that cannot bind RII subunits, fails to co-precipitate PKA complexes (top panels). Iso treatment (1 μM, 5 min) does not cause dissociation of PKAc from the AKAR-18RBS or AKAR-18RBS–T/A complexes (top panel, lanes 1–2 and 5–6). Research article Control immunoblots show equivalent levels of AKAR-18RBS, AKAR-18RBS–pro and AKAR-18RBS–T/A in immunoprecipitates as well as RIIα and PKAc expression in cell lysates (middle panels). Immunoblotting for phospho-PKA substrates (R-X-X-pS/T motif) confirms that Iso treatment activates endogenous β-ARs and initiates downstream phosphorylation events (bottom panel). (K) Immunoprecipitation of full-length AKAP18 complexes following Iso treatment. Cells expressing AKAP18γ and RIIα variants were treated with vehicle or Iso (1 μM, 5 min) and AKAP18γ complexes were immunoprecipitated. Immunoblotting shows that Iso has no effect on the amount of PKA catalytic subunit in complexes formed with RIIα wild type, RIIα Δ44–86, or RIIα ZeChimera. DOI: 10.7554/eLife.01319.011 The following figure supplements are available for figure 5: Figure supplement 1. Comparison of AKAR-18RBS and AKAR-18RBS–pro anchoring and FRET controls. DOI: 10.7554/eLife.01319.012 substrate. Moreover, the more compact AKAP-PKAΔ44-86-substrate assembly may augment kinase activity by inhibiting the actions of signal termination enzymes. This could involve the steric exclusion of cellular protein phosphatases that catalyze the removal of phosphate or protection from phospho diesterase activity that metabolizes cAMP. Deletion of the flexible linker between residues 44–86 of RIIα not only enhances basal phosphorylation of these anchored substrates, but also supplements the catalytic efficiency of the anchored PKA holoenzyme in this cellular context. However, this enhanced anchored kinase action may be an undesirable feature in vivo. Hence, we propose that the evolution of flexible linkers in RII may be a means to ensure bi-directional regulation of phosphorylation events by signal termination enzymes. substrate. Moreover, the more compact AKAP-PKAΔ44-86-substrate assembly may augment kinase activity by inhibiting the actions of signal termination enzymes. This could involve the steric exclusion of cellular protein phosphatases that catalyze the removal of phosphate or protection from phospho diesterase activity that metabolizes cAMP. Deletion of the flexible linker between residues 44–86 of RIIα not only enhances basal phosphorylation of these anchored substrates, but also supplements the catalytic efficiency of the anchored PKA holoenzyme in this cellular context. However, this enhanced anchored kinase action may be an undesirable feature in vivo. Hence, we propose that the evolution of flexible linkers in RII may be a means to ensure bi-directional regulation of phosphorylation events by signal termination enzymes. It is noteworthy that extending the number of residues in the RIIα ZeChimera linker had little effect on the overall structure of the AKAP-PKA complex, or on the activity of PKA monitored by our in vitro and cell-based assays. Figure 4. Continued A broader implication is that release of the C subunit from anchored PKA holoenyzmes may not be required for the phosphorylation of nearby substrates. Under this scenario, association with AKAPs limits and thereby defines the range of kinase action within the cell. This underscores the central role of AKAPs in the governance of cAMP responsive phosphorylation events. A combination of steric effects and environmental factors may explain the enhanced catalytic efficiency of this condensed AKAP–PKA assembly inside cells. For example, the less flexible RIIα Δ44–86 dimer may constrain the PKAc subunits in a manner that minimizes the distance to their target A combination of steric effects and environmental factors may explain the enhanced catalytic efficiency of this condensed AKAP–PKA assembly inside cells. For example, the less flexible RIIα Δ44–86 dimer may constrain the PKAc subunits in a manner that minimizes the distance to their target Smith et al. eLife 2013;2:e01319. DOI: 10.7554/eLife.01319 10 of 19 Research article Research article Biochemistry \| Biophysics and structural biology Figure 5. Flexibility within the anchored PKA holoenzyme impacts cAMP responsive signaling inside cells. (A) Schemati FRET reporter used in these studies. The PKA RII binding site common to all AKAP18 isoforms was fused to the N-term FRET-based kinase activity sensor that anchors type II PKA holoenzyme. Phosphorylation of a consensus site Thr by PKA Figure 5. Continued on next page Figure 5. Flexibility within the anchored PKA holoenzyme impacts cAMP responsive signaling inside cells. (A) Schematic of the modified AKAR-1 FRET reporter used in these studies. The PKA RII binding site common to all AKAP18 isoforms was fused to the N-terminus of AKAR2 to create a FRET-based kinase activity sensor that anchors type II PKA holoenzyme. Phosphorylation of a consensus site Thr by PKA induces recruitment of t Figure 5. Continued on next page Figure 5. Flexibility within the anchored PKA holoenzyme impacts cAMP responsive signaling inside cells. (A) Schematic of the modified AKAR-18RBS FRET reporter used in these studies. The PKA RII binding site common to all AKAP18 isoforms was fused to the N-terminus of AKAR2 to create a FRET-based kinase activity sensor that anchors type II PKA holoenzyme. Phosphorylation of a consensus site Thr by PKA induces recruitment of the Figure 5. Continued on next page Smith et al. eLife 2013;2:e01319. DOI: 10.7554/eLife.01319 11 of 19 Research article Protein expression and purification p pi RIIα and PKAc were expressed in BL21(DE3)pLysS. Escherichia coli was transformed with pET28b-RIIα- 6xHis (encoding full-length RIIα, accession NM_008924) or pET15a-6xHis-mPKAc (Addgene plasmid 14921; www.addgene.org), encoding full-length mouse PKA catalytic subunit (Narayana et al., 1997) and grown to an OD600 of 0.6. Protein expression was induced with 1 mM IPTG, and cells were grown with shaking for 16 hr at 28°C (for RIIα-His) or 24 hr at 16°C (for PKAc). The cells were harvested by centrifugation at 5,000×g for 10 min at 4°C. Pellets containing induced protein were stored at −20°C until use. For purification, the cells were thawed in 50 mM NaPhosphate, pH 7.5, 100 mM NaCl, 10 mM Imidazole, 2 mM MgCl2 containing 4 mg/ml lysozyme, 1 mM AEBSF, 50 μg/ml soybean trypsin inhibitor, 2 μg/ml leupeptin, 16 μg/ml benzamidine, and 25 U/ml benzonase. After resuspension, Triton X-100 was added to 0.5% and lysates were rocked at 4°C for 30–60 min. When the lysate was no longer viscous, debris was pelleted by centrifugation at 37,000×g for 30 min at 4°C. Cleared lysates were added to 1/25 vol Ni+–NTA beads (bed volume; GE Healthcare, Pittsburg, PA) and rocked for 1 hr at 4°C. The bead slurry was then added to a column and allowed to settle. Beads were washed with >10 bed volumes of 50 mM NaPhosphate, pH 7.5, 500 mM NaCl, 20 mM Imidazole. Purified protein was eluted by collecting 2 × 2 bed volume fractions of 50 mM NaPhosphate, pH 7.5, 500 mM NaCl, 50 mM Imidazole followed by 4 × 2 bed volumes 50 mM NaPhosphate, pH 7.5, 500 mM NaCl, 300 mM Imidazole. Fractions were analyzed by SDS-PAGE and Coomassie staining. The fractions containing the protein of interest were pooled, concentrated and applied to a Superdex 16/600 S200 gel filtration (GF) column. The column was run in 25 mM HEPES, pH 7.5, 200 mM NaCl, 1 mM EDTA, 1 mM TCEP. Peak fractions were pooled, concentrated, brought to 10% glycerol and frozen in liquid N2 for storage at −80°C. AKAP18γ (accession NM_016377 [Note: This sequence was updated during the course of these studies and corresponds to the longer AKAP18δ isoform; the construct used in these studies encodes the γ isoform starting at Met23 of the referenced ORF]) was expressed with an N-terminal strep-II tag and a C-terminal 6x-His tag in High Five insect cells (Trichopulsia ni; Invitrogen). Research article enhance kinase access to multiple phosphorylation sites on the same substrate, or, alternatively, orient each catalytic subunit toward distinct substrate proteins within the vicinity of the scaffolding site. Our structural and cellular characterization of higher-order AKAP assemblies emphasizes how static protein– protein interactions act in concert with the dynamic movement of sub-structures to orchestrate local phosphorylation events. We believe that these combined molecular approaches are broadly applicable for the investigation of related enzyme complexes that are spatially organized within the cell. Protein expression and purification Sf-9 cells were transfected with a Bacmid containing the expression cassette from pFastBac-C-His-strep-AKAP18γ. After two rounds of viral amplification, High Five cells were infected with baculovirus (at a cell density of ∼2 × 106 cells/ml) and grown for 72 hr at 26°C shaking at 105 rpm. Cells were harvested by centrifugation (10 min, 700×g) and frozen at −20°C. Protein was purified as above using Ni+–NTA Sepharose, except lysozyme was omitted from the lysis buffer. Gel filtration chromatography was performed as described above. MBP-PDE4D3-8xHis was expressed in BL21(DE3)pLysS transformed with the vector pMAL-C5P2- PDE4D3-8His, which was constructed by PCR and InFusion (Clontech) cloning into a modified pMAL vector (NEB) containing an 8xHis tag prior to MBP and a PreScission protease cleavage site after MBP. Protein was purified using Ni+–NTA Sepharose, and gel filtration chromatography was performed as described above. Research article According to our structural analysis, this construct samples a range of radial conformations that are very similar to wild-type particles (Figure 3H). Moreover, this observation is consistent with theoretical analyses of random coiled chain models that demonstrate the average end-to-end length of random coil scales by the square root of the number of amino acids in the chain (average length ∼ N0.5, where N is the number of residues) (Flory, 1975). This statement implies that the average length separating the ends of a random coil reaches a plateau, or maximum, that is only marginally altered upon the inclusion of more residues. Consequently extending the linker in the context of the RIIα ZeChimera may explain why this affords minimal benefit to the overall structure and dynamics of this seemingly more extended and malleable anchored enzyme complex. Hence, evolutionary constraint of the RIIα linker domain to 45–47 residues may ensure appropriate separation between the anchoring and transduction domains of anchored mammalian PKA holoenzymes. Another key element of our model is that the array of conformations adopted by the anchored RII subunit dimer defines a ‘radius of action’ for the tethered catalytic subunit. Not only does this structural plasticity explain why an intact PKA holoenzyme structure has eluded X-ray crystallographers for over 20 years but also suggests that the ‘built in’ flexibility within this protein complex sets a dynamic range for basal and cAMP-dependent phosphorylation of nearby substrates. This latter concept could have profound implications for PKA and other kinases, where enzyme activity is constrained within anchored complexes or enzyme scaffolds. The conformational variability of the tethered complex could Smith et al. eLife 2013;2:e01319. DOI: 10.7554/eLife.01319 12 of 19 Research article Research article Biochemistry \| Biophysics and structural biology Biochemistry \| Biophysics and structural biology enhance kinase access to multiple phosphorylation sites on the same substrate, or, alternatively, orient each catalytic subunit toward distinct substrate proteins within the vicinity of the scaffolding site. Our structural and cellular characterization of higher-order AKAP assemblies emphasizes how static protein– protein interactions act in concert with the dynamic movement of sub-structures to orchestrate local phosphorylation events. We believe that these combined molecular approaches are broadly applicable for the investigation of related enzyme complexes that are spatially organized within the cell. Molecular biology, cloning, and mutagenesis Constructs for deleted and extended linker forms of mRIIα were created using standard cloning methods. Briefly, to create the linker deletion, AscI restriction sites were introduced into the cDNA by site-directed mutagenesis. The resulting plasmid was digested with AscI and then re-ligated closed. To create the Zebrafish linker chimera, a minigene encoding the Zebrafish linker from NCBI Reference sequence NM_212958.1 was synthesized with flanking AscI sites (Integrated DNA Technologies). This minigene was digested out of the carrier vector and inserted into the AscI sites in the modified RIIα cDNA. Clones were screened for orientation and verified by sequencing (Genewiz). Mammalian expression constructs encoding RII variants were created by PCR cloning into Gateway DONR vectors (Invitrogen) followed by LR cloning into pcDNA-DEST40. Vectors that express AKAR-18RBS, the AKAR-18RBS–T/A and the AKAR-18RBS–pro (L9P, A13P in the AKAP18 anchoring helix) mutants were 13 of 19 Smith et al. eLife 2013;2:e01319. DOI: 10.7554/eLife.01319 Research article Research article Biochemistry \| Biophysics and structural biology created upon insertion of a cDNA encoding the PKA binding site from AKAP18 into pcDNA3-AKAR2.2 (Zhang et al., 2005) followed by site directed mutagenesis (QuikChange XL II, Stratagene). created upon insertion of a cDNA encoding the PKA binding site from AKAP18 into pcDNA3-AKAR2.2 (Zhang et al., 2005) followed by site directed mutagenesis (QuikChange XL II, Stratagene). AKAP complex formation AKAP18γ–PKA complexes were assembled using purified proteins. RIIα and PKAc were mixed at a 1:1.2 molar ratio, and AKAP18γ was added in molar excess to favor the isolation of stoichiometric complexes by gel filtration. Complexes were dialyzed against 15 mM MOPS, pH 6.5, 100 mM NaCl, 1 mM MgCl2, 1 mM TCEP, 2% glycerol for >4 hr at 4°C in 0.5 ml Slide-A-Lyzer MINI dialysis cups (88401; Thermo Scientific). The complexes were then loaded onto a Superdex 16/600 S200 gel filtration column on an AKTA Purifier (GE Healthcare) and separated at a flow rate of 0.5 ml/min in 25 mM HEPES, pH 7.5, 200 mM NaCl, 1 mM EDTA, 1 mM TCEP. Peak fractions were analyzed by SDS-PAGE and Coomassie staining. The fractions containing approximately stoichiometric complex components were pooled, concentrated, brought to 10% glycerol and frozen in liquid N2 for storage at −80°C. RIIα-PKAc holoenzyme complexes were assembled as above, omitting AKAP18γ. Phosphodiesterase assays PDE assays were performed as described (Hill et al., 2005). Briefly, after washing, immunocomplexes were suspended in 50 μl KHEM (50 mM KCl, 50 mM HEPES-KOH, pH 7.2, 10 mM EGTA, 1.92 mM MgCl2). Next, 50 μl of PDE assay buffer (20 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 2 μM cAMP, 100 nM okadaic acid) containing ∼50,000 cpm [3H]-cAMP was added, and the samples were incubated at 30°C for 20 min with shaking. The samples were boiled for 3 min and incubated on ice for 15 min. Snake venom (25 μl of a 1 mg/ml solution) was added, and the samples were incubated at 30°C for 10 min with shaking. Dowex AG 1×8 (200–400 mesh, CL form, washed and used as a 1:1:1 slurry of resin:water:ethanol) ion exchange resin (400 μl) was added, and the samples were incubated on ice for 15 min. The resin was centrifuged for 3 min at 14,000×g, and 150 μl of the supernatant was mixed with 1 ml scintillation fluid for counting. All assays were performed in duplicate. Western blotting Duplicate samples were separated on 10% Tris-Glycine-SDS gels. Proteins were transferred to nitrocellulose membranes and blocked with 5% NFDM, 1% BSA in TBST. Membranes were probed with either anti-AKAP18 rabbit polyclonal antiserum (VO57), mouse anti-PKAc or anti-RIIα monoclonal antibodies (BD Biosciences) overnight at 4°C. The membranes were washed, incubated with appropriate secondary antibodies, washed again and protein was detected using ECL (Super-signal Pico, Thermo Pierce) on an AlphaInnotech Multiimager III. Native PAGE AKAP18γ–PKA complexes were analyzed by native PAGE using precast 3–8% Tris-Acetate gels (Invitrogen). The samples were mixed with Invitrogen’s 2X native sample buffer and run in 50 mM Tris pH 8.3, 50 mM Tricine, along with native MW markers (Invitrogen). Protein was detected by silver staining (SilverQuest, Invitrogen). Kinase assays PKA holoenzyme was incubated in the absence or presence of molar equivalent of purified AKAP18γ. These pre-formed complexes (1 μg) were mixed with excess substrate MBP-PDE4D3 (2 μg) in 40 mM Tris, pH 7.5, 10 mM MgOAc, 0.1 mM EGTA, 100 μM IBMX. Mg2+-ATP containing 1 μCi 32P-ATP was added to a final concentration of 10 μM. The samples were incubated at 30°C with rapid shaking for 5 min. Reactions were stopped by the addition of 5X Laemmli sample buffer and boiling for 5 min. The samples were separated by SDS-PAGE, and gels were either stained with Coomassie blue or transferred to nitrocellulose for India ink staining. Phosphorylation of PDE4D3 was detected by auto radiography. Films were scanned for quantification by densitometry, followed by analysis using GraphPad Prism. Analysis of AKAP18γ–PKA complexes containing different RIIα variants was performed and analyzed similarly. In these assays, PKI was used at a final concentration of 10 μM and added to appropriate samples 10 min prior to the start of the assay. Prior to initiation of the assay, cAMP was added to some samples to a final concentration of 30 μM. Immunoprecipitations and immunoblotting HEK293 cells were transiently transfected (TransIT LT1; Mirus) with vectors encoding various proteins according to figure legends. After 40–48 hr and prior to harvesting, the cells were serum starved in DMEM for 1 hr at 37°C. The cells were then treated with vehicle or Isoproterenol for 5 min at 37°C and harvested in lysis buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 2% glycerol, 0.3% Triton X-100) containing protease inhibitors. AKAP18γ or AKAR-18RBS complexes were immunoprecipitated with anti-GFP rabbit IgG (Invitrogen) and protein A agarose for 2 hr at 4°C. Beads were washed 1 × 1 ml in lysis buffer containing 350 mM NaCl, 1 × 1 ml wash buffer (lysis buffer with no detergent) containing 350 mM NaCl and 2 × 1 ml wash buffer. Proteins were separated on 4–12% gradient gels (Invitrogen) and transferred to nitrocellulose membranes. Primary antibodies (PKA catalytic mAb [BD Biosciences], 1:1000; RIIα mAb [BD Biosciences], 1:2000; GFP mAb [Santa Cruz Biotechnology], 1:2000; phospho-PKA substrates rabbit mAb [Cell Signaling Technology], 1:1000) were incubated with membranes overnight at 4°C in TBST/Blotto. The membranes were washed extensively in TBST, incubated with secondary antibodies, washed again and developed using ECL (Thermo Pierce) on an Alpha Innotech MultiImage III with FluoroChem Q software. FRET measurements HEK293 cells were transiently transfected with cDNAs encoding AKAR-18RBS and individual RII subunits, and cultured on 35-mm glass coverslip dishes (MatTek Corporation). 48 hr after transfection, 14 of 19 Smith et al. eLife 2013;2:e01319. DOI: 10.7554/eLife.01319 Research article Research article Biochemistry \| Biophysics and structural biology the cells were imaged in HEPES-Buffered Saline Solution (HBSS; 116 mM NaCl, 20 mM HEPES, 11 mM Glucose, 5 mM NaHCO3, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, pH 7.4). The cells were stimulated by isoproterenol (10 μM). Fluorescence emission was acquired using a DMI6000B inverted microscope (Leica), an EL6000 component (fluorescent light source, filter wheel, ultra fast shutter, Leica) and a CoolSnap HQ camera (Photometrics), all controlled by MetaMorph 7.6.4 (Molecular Devices). Dual-emission images were obtained simultaneously through a Dual-View image splitter (Photometrics) with S470/30 and S535/30 emission filters and 505 dcxr dichroic mirror (Chroma). Exposure time was 200 ms with an image interval of 10 s. FRET changes within a region of interest were calculated as the ratio of measured fluorescent intensities from two channels (Mdonor, MindirectAcceptor) after subtraction of background signal. FRET ratio (YFP/CFP) changes were normalized to the average FRET ratio value before stimulation. Confocal imaging Confocal microscopy was performed using a Zeiss LSM-510 META laser scanning confocal microscope equipped with a Zeiss 63 × g, 1.4 numerical aperture, oil immersion lens. For live cell imaging, HEK293 cells expressing AKAR-18RBS and individual RII-mCherry constructs were imaged in HBSS. Colocalization studies were performed using dual excitation (488, 543 nm) and emission (bandpass 505–530 nm and long-pass 560 nm for YFP and mCherry, respectively) filter sets. Electron microscopy The wild-type and mutant AKAP18γ–PKA holoenzyme complexes were prepared similarly for electron microcopy studies. Freshly purified samples were diluted 1:25 times to a final concentration of 5 μg/ml with EM buffer containing 25 mM HEPES (pH—7.4), 200 mM NaCl, 0.5 mM EDTA and 1 mM dithioth reitol (DTT). For gold-labeling studies, a 0.05% solution of streptavidin-conjugated gold particles (5 nm, Nanocs) was diluted 1:100 with EM buffer and mixed 1:1 (vol/vol) with the wild-type AKAP18γ– PKA complex and allowed to incubate for 1 hr at 4°C. 2 μl of specimen samples were applied to carbon coated EM grids and negatively stained with 0.75% (wt/vol) uranylformate. Wild-type particles were visualized on a 120 kV TEM (FEI), and images were recorded at a nominal magnification of 52,000 × at the specimen level on a 4 k × 4 k CCD (Gatan). Serial tomographic images were obtained using automated data collection software (Xplore3D, FEI) collected at tilt angles of 0°, 20°, 40° and 50° (Figure 2—figure supplement 1). The mutant complexes and gold-labeled particles were visualized on a 120 kV TEM (FEI) and recorded at a nominal magnification of 68,000 × at the specimen level on a 4 k × 4 k CMOS-based camera (Tietz). For image processing, micrographs were binned two times yielding final pixel sizes of 4.2 Å per pixel and 3.0 Å per pixel for the wild-type dataset and mutant datasets, respectively. Thon rings in the power spectra were used to select only those micrographs free of drift or significant astigmatism. The contrast transfer function (CTF) parameters were determined for each micrograph using the program CTFTILT (Mindell and Grigorieff, 2003). ∼7,000 wild-type particles and 1,000 mutant particles were hand selected from micrographs using Ximdisp (Smith, 1999). Two-dimensional (2D) projection averages were generated using reference-free multivariate statistical analysis methods in IMAGIC Smith et al. eLife 2013;2:e01319. DOI: 10.7554/eLife.01319 15 of 19 Research article Research article Biochemistry \| Biophysics and structural biology (van Heel et al., 1996) and with the Iterative Stable Alignment and Clustering routines implemented using the ISAC program (Yang et al., 2012). Initial three-dimensional (3D) reconstructions were gener ated from 0°, 20°, 40° and 50° tilted particle class-average datasets displaying either linear or bent conformations using angular reconstitution routines in IMAGIC (van Heel et al., 1996). Refinement of the initial 3D density maps was performed using individual particle images in FREALIGN (Grigorieff, 2007) without symmetry constraints. Modeling of the AKAP18γ–PKA holoenzyme complex g γ y p Structural models of the AKAP18γ–PKA holoenzyme complex were constructed to represent the triangular and extended conformations determined by the 3D reconstructions. A model of AKAP18γ (residues 88–317) was initially constructed as follows. The atomic coordinates of the AKAP– PKA binding helix in complex with the RIIα docking and dimerization domain (RIID/D-AKAPhelix; PDB 2IZX) (Gold et al., 2006) was used as a template to model the corresponding region in AKAP18γ, residues 301–317, by computational mutation and minimization in COOT (Emsley and Cowtan, 2004). The AKAPhelix domain was oriented with the N-terminus (residue 301) placed in close proximity to the C-terminus of the AKAP18γ central domain (PDB 2VFL) (Gold et al., 2008), residues 88–290. The relative orientations were guided by their corresponding fit into the 3D density map. The two AKAP18γ domains were connected by a 9-residue linker corresponding to the AKAP18γ sequence 291–299 using COOT (Emsley and Cowtan, 2004). This model of the linked AKAP18γ structure (residues 88–317) bound to the RIID/D domain was placed within the central density of the density map, and the fit was refined by computational minimization in Chimera (Pettersen et al., 2004). The central density was determined to be the site of AKAP18γ localization by affinity gold labeling studies (see above). The atomic coordinates corresponding to the mouse PKA catalytic domain (PKAc) bound to the RIIα cAMP binding domain (PDB 2QVS) (Wu et al., 2007) were placed into each of the peripheral EM densities, and their fits were independently minimized in Chimera (Pettersen et al., 2004). Each RIIα regulatory domain (residues 91–392) was connected to a corresponding RIID/D domain (residues 3–43) by a 46-residue linker region (corresponding to residues 44–90 from mouse) using the modeling program COOT (Emsley and Cowtan, 2004). The RIIα linker regions were modeled as unstructured polypeptides connecting the sub-domains of the AKAP18γ-PKA holoenzyme complex oriented in the triangular and linear conformations determined by EM. Figures were prepared in Adobe Photoshop. Protein structures were visualized and images captured in UCSF chimera version 1.6. Acknowledgements The authors would like to thank Nikolaus Grigorieff (Janelia Farm Research Campus, Howard Hughes Medical Institute) for critically reading this manuscript, as well as Alexis Rohou, Don Olbris, Mark Bolstad, and Sean Murphy (Janelia Farm Research Campus, Howard Hughes Medical Institute) and Katherine Forbush (University of Washington) for technical assistance. The authors also thank Patrick J Nygren (University of Washington) for valuable discussions and insight. EM maps have been deposited in the EM Data Bank. Atomic coordinates of composite models for the AKAP18γ-PKAholo complex have been deposited in the Protein Data Bank. Additional information Electron microscopy The final 3D density maps were filtered to 35 Å resolution as suggested by Fourier shell correlation (FSC) analysis. Back-projection analysis was carried out in SPIDER (Frank et al., 1996). Statistical analysis of individual particle dimensions was obtained by measuring particle lengths on unbinned micrographs using ImageJ (Schneider et al., 2012) and evaluated by the two-tailed t test. Additional information Funding Funder Grant reference number Author Howard Hughes Medical Institute F Donelson Smith, Steve L Reichow, Jessica L Esseltine, Dan Shi, Lorene K Langeberg, John D Scott, Tamir Gonen Grant reference number 16 of 19 16 of 19 Smith et al. eLife 2013;2:e01319. DOI: 10.7554/eLife.01319 Biochemistry \| Biophysics and structural biology Funder Grant reference number Author National Institutes of Health HL088366, GM48231 F Donelson Smith, Jessica L Esseltine, Lorene K Langeberg, John D Scott The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Research article Research article Biochemistry \| Biophysics and structural biology Additional files The following datasets were generated: Author(s) Year Dataset title Dataset ID and/or URL Database, license, and accessibility information Smith FD, Reichow SL, Esseltine JL, Shi D, Langeberg LK, Scott JD, Gonen T 2013 3D EM reconstruction of the AKAP18-PKA complex in a bent conformation. (corresponding with PDB 3J4Q) EMD-5755; http://www. ebi.ac.uk/pdbe/entry/ EMD-5755 Publicly Available at Electron Microscopy Data Bank (EMDB). Smith FD, Reichow SL, Esseltine JL, Shi D, Langeberg LK, Scott JD, Gonen T 2013 3D EM reconstruction of the AKAP18-PKA complex in a linear conformation. (corresponding with PDB 3J4R) EMD-5756; http://www. ebi.ac.uk/pdbe/entry/ EMD-5756 Publicly Available at Electron Microscopy Data Bank (EMDB). Smith FD, Reichow SL, Esseltine JL, Shi D, Langeberg LK, Scott JD, Gonen T 2013 Pseudo-atomic model of the AKAP18-PKA complex in a bent conformation derived from electron microscopy 3J4Q; http://www.rcsb. org/pdb/explore/ explore.do? structureId=3J4Q Publicly Available at Protein Data Bank (PDB). Smith FD, Reichow SL, Esseltine JL, Shi D, Langeberg LK, Scott JD, Gonen T 2013 Pseudo-atomic model of the AKAP18-PKA complex in a linear conformation derived from electron microscopy 3J4R; http://www.rcsb. org/pdb/explore/ explore.do? structureId=3J4R Publicly Available at Protein Data Bank (PDB). 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https://openalex.org/W4360801169	https://zenodo.org/records/7766860/files/15.pdf	Hindi	null	BHAGWAN BUDDHA KI AMULTA DHARODHAR: VIPAKSHATA DHYAN	Zenodo (CERN European Organization for Nuclear Research)	2,015	cc-by	1,670	www.aarhat.com/vol II Issues I/July 112 2 दीघतनकाय, महापरितनब्बानसुत्तं, 2.3, 221 3 धम्मपद, मग्गवग्गो, 277 Educreator Research Journal (ERJ) ISSN : 2394-8450 िगवान बुद्ध की अमूल्य धरोहर : ववपश्यना ध्यान भिव प्रसाद पााँचे , पी चडी, िोधार्ी बौद्ध अध्ययन , ववभाग ग्वायि हॉल , हदल्ली ववश्वववद्यालय हदल्ली -110007 Educreator Research Journal (ERJ) ISSN : 2394-8450 िगवान बुद्ध की अमूल्य धरोहर : ववपश्यना ध्यान भिव प्रसाद पााँचे , पी चडी, िोधार्ी बौद्ध अध्ययन , ववभाग ग्वायि हॉल , हदल्ली ववश्वववद्यालय हदल्ली -110007 Educreator Research Journal (ERJ) ISSN : 2394-8450 िगवान बुद्ध की अमूल्य धरोहर : ववपश्यना ध्यान भिव प्रसाद पााँचे , पी चडी, िोधार्ी बौद्ध अध्ययन , ववभाग ग्वायि हॉल , हदल्ली ववश्वववद्यालय हदल्ली -110007 भिव प्रसाद पााँचे , पी चडी, िोधार्ी बौद्ध अध्ययन , ववभाग ग्वायि हॉल , हदल्ली ववश्वववद्यालय हदल्ली -110007 आज से लगभग 2600 वर्ष पूवष ससद्धार्ष गौतम ने अपने अर्क परिश्रम के द्वािा प्रकृतत में लुप्त ववपश्यना ध्यान साधना ववधध का पुन: अनुसन्धान कि बुद्धत्व को प्राप्त ककया तर्ा बुद्ध कहलाये. औि वैश्श्वक सवष कल्याण की भावना से प्रेरित लगाताि 45 वर्ो तक सिक्षा बाेते िहे तर्ा ेसे जनमानस के सल व्यवहारिक रूप प्रदान ककया. अर्ाषत - सभक्खुओं, बहुत जनों के हहत के सल , बहुत जनों के सुख के सल , लोक पि दया किने के सल ववचिण किो ! ््र्ं .ेस ध्यान साधना ववधध के सतत प्रयोग से अतीत से लेकि वतषमान तक लाखो-किोडो लोग दुक्खों के संताप से मुक्त हु । अब ेस ववद्या को धािण कि हमे भी दुुःख मुक्त होना है। वस्तुत: “ ववपश्यना” दो िब्दों से समलकि बना है, वव + पश्यना , यहााँ ‘वव’ का अर्ष वविेर् या गहन रूप से तर्ा ‘पश्यना’ का अर्ष है देखना. अर्ाषत ििीि पि होने वाली पल-पल की घेनाओ को वविेर् रूप से देखना। ये घेनाये संवेदना ं के रूप में घहेत होती है. संवेदना अर्ाषत वेदना, पाली भार्ा में श्जसका अर्ष होता है अनुभूतत. यातन ििीि पि नाना प्रकाि के संवेदनाये हि पल होती ही िहती है जैसे- कभी सुखद, कभी दुखद, तो कभी अदुुःखद-असुखद ेत्याहद. पि जब साधक ववपश्यना किते समय सजग िहकि ेन संवेदनाओ के प्रतत प्रततकिया नही किता औि समतापूवषक िह किके संवेदनवाओ के अतनत्य स्वाभाव को देखता है, तब उसी संवेदना के आधाि पि िाग-द्वेर्, मोह, तृष्णाहद दुख उपजता है. तर्ा ेन्ही संवेदनाओ को आधाि बनाकि देखने पि दुुःख का अतनत्य बोध होता है. ेस तिह दुुःख का क्षिण होता है . ेस तिह धीिे-धीिे जैसे -जैसे दुखो का क्षिण होता है वैसे-वैसे भीति मानस में www.aarhat.com/vol II Issues I/July 111 1 ववनयवपेक , महावग्ग, 32, मािकर्ा www.aarhat.com/vol II Issues I/July 111 1 ववनयवपेक , महावग्ग, 32, मािकर्ा उपझित्वा ननरुिंती, तेसं वुपसमो सुखो ।। 2 अर्ाषत सचमुच! सािे संस्काि अतनत्य ही तो है, उत्पन्न होनेवाली सभी श्स्र्ततयां, वस्तु, व्यश्क्त, अतनत्य ही तो है। उत्पन्न होना औि नष्े हो जाना, यह तो ेनका धमष ही है. स्वाभाव ही है. ववपश्यना साधना के अभ्यास द्वािा उत्पन्न होकि तनरूद्द होने वाले ेस प्रपंच का जब पूणषतया उपिमन हो जाता है. पुन: उत्पन्न होने का िम समाप्त हो जाता है, उसी का नाम पिम सुख है, वही तनवाषण-सुख है। ्् अर्ाषत सचमुच! सािे संस्काि अतनत्य ही तो है, उत्पन्न होनेवाली सभी श्स्र्ततयां, वस्तु, व्यश्क्त, अतनत्य ही तो है। उत्पन्न होना औि नष्े हो जाना, यह तो ेनका धमष ही है. स्वाभाव ही है. ववपश्यना साधना के अभ्यास द्वािा उत्पन्न होकि तनरूद्द होने वाले ेस प्रपंच का जब पूणषतया उपिमन हो जाता है. पुन: उत्पन्न होने का िम समाप्त हो जाता है, उसी का नाम पिम सुख है, वही तनवाषण-सुख है। यातन ििीि औि धचत्त के संयोजन से जो भी संवेदना का तनमाषण होती है वह कुछ समय बाद समाप्त हो जाती है. यही उसका तनत्य गुणधमष है. ेस प्रकाि साधक ववपश्यना का अभ्यास किके वतषमान क्षण में श्स्र्त होकि संवेदनाओ के प्रतत प्रततकिया न किके उसके उदय-व्यय स्वाभाव को जानकि सािे दुखो-संस्कािों से मुक्त हो जाताहै ेससल भगवाननेकहा यातन ििीि औि धचत्त के संयोजन से जो भी संवेदना का तनमाषण होती है वह कुछ समय बाद समाप्त हो जाती है. यही उसका तनत्य गुणधमष है. ेस प्रकाि साधक ववपश्यना का अभ्यास किके वतषमान क्षण में श्स्र्त होकि संवेदनाओ के प्रतत प्रततकिया न किके उसके उदय-व्यय स्वाभाव को जानकि सािे दुखो-संस्कािों से मुक्त हो जाता है. ेससल भगवान ने कहा- “सब्बे संखारा अननच्चानत, यदा पञ्ञाय पस्सनत । अथ ननम्ब्बन्दनत दुक्खे, एस मग्गो ववसुवद्धया ।।‘’ 3 Educreator Research Journal (ERJ) ISSN : 2394-8450 प्रिांतत महसूस होती है. ेस तिह ही वतषमान में घहेत संवेदनाओ को यर्ाभूत जानकि, प्रज्ञा जगाकि, उसके अतनत्य भाव का बोध कि दुखो का नाि किता है, यही जीवन जीने की असली कला है. चूाँकक प्रत्येक संवेदना का गुणधमष होता है - " उदय-व्यय" यातन अतनत्य। भगवान बुद्ध कहते है:- प्रिांतत महसूस होती है. ेस तिह ही वतषमान में घहेत संवेदनाओ को यर्ाभूत जानकि, प्रज्ञा जगाकि, उसके अतनत्य भाव का बोध कि दुखो का नाि किता है, यही जीवन जीने की असली कला है. चूाँकक प्रत्येक संवेदना का गुणधमष होता है - " उदय-व्यय" यातन अतनत्य। भगवान बुद्ध कहते है:- आरोग्यपरमा लािा, सन्तुहिपरमं धनं। ववस्सासपरमा ञानत,ननब्बानं परमं सुखं।। 4 अर्ाषत आिोग्य िहना पिम लाभ है. संतुष्ठ िहना पिम धन है. ववश्वास ही पिम बंधु है औि तनवाषण पिम सुख है. ववपश्यना साधना के व्यवहारिक प्रयोग से व्यश्क्त सांसारिक जीवन में िागववहीन, द्वेर्ववहीन, मोहववहीन होकि सम्पूणष दुखो का नाि किके, सुखद जीवन जी कि अपने उत्तम लक्ष्य को साध सकता है। भगवान बुद्ध ने अपनी अंततम वाणी में भी कहा है- ्् Educreator Research Journal (ERJ) ISSN : 2394-8450 भगवान बुद्ध के अमूल्य साधना ववधध ववपश्यना द्वािा साधक तृष्णा को जड़ से समाप्त कि लौककक (सांसारिक गृहस्र् जीवन) औि पािलौककक सुखो (तनब्बाना) को प्राप्त कि सकता है. भगवान ने कहे- “सब्बे संखारा अननच्चानत, यदा पञ्ञाय पस्सनत । अथ ननम्ब्बन्दनत दुक्खे, एस मग्गो ववसुवद्धया ।।‘’ 3 “सब्बे संखारा अननच्चानत, यदा पञ्ञाय पस्सनत । अथ ननम्ब्बन्दनत दुक्खे, एस मग्गो ववसुवद्धया ।।‘’ 3 अर्ाषत संस्काि /संस्कृत ( बनी हुई) चीजे जो दुुःख देने वाले है, वह अतनत्य है। औि साधक ववपश्यना ्््र्ञ््ध अर्ाषत संस्काि /संस्कृत ( बनी हुई) चीजे जो दुुःख देने वाले है, वह अतनत्य है। औि साधक ववपश्यना के अभ्यास द्वािा प्रज्ञा जगाकि सभी दुखो से मुक्त हो जाता है. ऐसा है ये वविुवद्ध का मागष। चूाँकक ेन्ही संवेदनाओ के माध्यम से व्यश्क्त िाग- द्वेर्, मोह, तृष्णा, अहंकाि आहद का संपोर्ण किता है. औि मनवांतछत फल प्राप्त न होने पि दुखी हो जाता है. फलत: सभन्न-सभन्न दुष्कृत्य दुखो को जन्म देता है. वस्तुत: दुख जीवन जगत की सच्चाई है. औि हि ककसी के जीवन में दुुःख व्याप्त है। चाहे वह िाजा हो या िंक, अमेरिकी हो या भाितीय, देिीय हो या ववदेिी आहद । दुुःख के भी अनेक आयाम है जैसे-िािीरिक दुुःख, मानससक दुुःख, आधर्षक ववपन्नताओ का दुुःख, ेच्छाओं की पूततष न होने वाला दुुःख ेत्याहद। अत व दुुःख का मूल कािण है "तृष्णा"। चूाँकक व्यश्क्त वतषमान में श्स्र्त न होकि वह भूतकाल या भववष्यकाल आधारित तृष्णा के बािे में प्रततकिया किके दुखी हो जाता है. जबकक भूतकाल तो बीत चूका होता है. तर्ा भववष्यकाल तो आने वाला काल्पतनक चीज होता है. वस्तुतुः वतषमान क्षण में िहना सबसे उत्तम औि जीवंत होता है. क्योकक व्यश्क्त को वतषमान में ही जीना होता है औि यही फल का तनमाषण किता है, यह 'तथ्य' है. www.aarhat.com/vol II Issues I/July 114 8 धम्मपद, बुद्धवग्गो, 183 Educreator Research Journal (ERJ) ISSN : 2394-8450 Educreator Research Journal (ERJ) ISSN : 2394-8450 "वयधम्मा संखारा, अप्पमादेन सम्पादेथ " 5 "वयधम्मा संखारा, अप्पमादेन सम्पादेथ " 5 अर्ाषत सभी संस्काि व्यय-धमष है. नािवान है। प्रमाद िहहत होकि अपने लक्ष्य को प्राप्त किो.औि आगे भी भगवान बुद्ध ने सािनार् में अपने प्रर्म उपदेि में चार आयय सत्य श्जसमे दुुःख है, दुुःख का कािण होता है, दुुःख का तनवािण तर्ा दुख तनवािण का उपाय, श्जसे आयय अष्ांगगक मागय भी कहा जाता है, को ववस्ताि से उपदेसित ककया।6 श्जसमे सम्यक दृश्ष्े, सम्यक वाणी, सम्यक संकल्प, सम्यक कमाांत, सम्यक आजीववका, सम्यक वायाम, सम्यक सजगता (सतत ), सम्यक समाधी आहद का सम्यक रूप से अनुपालन किके संपूणष दुखो का नाि कि के जीवन के सवोत्तम आध्याश्त्मक सोपान यर्ाुः-तनवाषण, अहषत, बुद्धत्व की अवस्र्ा को प्राप्त कि सकता है, जो कक अमृत, अतुलनीय, अमापतनय है. ऐसा धचिकालीन सुख देने वाला बुद्ध की व्यवहारिक सिक्षा है "ववपश्यना " । ववपश्यना ध्यान साधना अर्ाषत जीवन जीने की कला. अभी तक ेस साधना पध्दतत से लाखो- किोडो लोगो ने मुश्क्त पाई है। अब हम भी ेस पावन अवसि पि ेस सम्प्रदायववहीन, पन्र्ववहीन, जातत-धमष के पिे ेस सिक्षा को आत्मसात किे तर्ा अपने जीवन को मंगल बनाये ! ेस ववपश्यना ध्यान का व्यवहारिक पक्ष् को जानने-संमझने के सल ववपश्यना वविोधन ववन्यास7 या www.vri.dhamma.org पि संपकष ककया जा सकता है. अंतत: भगवान बुद्ध ने अपनी सिक्षा का मूलसाि संक्षक्षप्त में ेस तिह कहा- 4 धम्मपद, सुखवग्गो, 197 www.aarhat.com/vol II Issues I/July 113 5 महापरितनब्बानसुत्तं 6 धम्मचक्कपवत्तनसुत्तं 7 ववपश्यना वविोधन ववन्यास(VRI), ेगतपुिी, नाससक www.aarhat.com/vol II Issues I/July 113 5 महापरितनब्बानसुत्तं 6 धम्मचक्कपवत्तनसुत्तं 7 ववपश्यना वविोधन ववन्यास(VRI), ेगतपुिी, नाससक सब्ब पापस्स अकरणं, कुसलस्स उपसम्पदा । सगचत्तपररयोदपनं, एतं बुद्धानं सासनं ।। 8 अर्ाषत सभी पाप कमो (अकुिल कमो) को न किना, कुिल कमो (पुण्य कमो) का संपादन किना। अपने धचत्त (मन) को ववपश्यना के द्वािा िुद्ध किते िहना, यही बुद्धों की सिक्षा है. ्र्ध््ष्ं््र्ञ अर्ाषत सभी पाप कमो (अकुिल कमो) को न किना, कुिल कमो (पुण्य कमो) का संपादन किना। अपने धचत्त (मन) को ववपश्यना के द्वािा िुद्ध किते िहना, यही बुद्धों की सिक्षा है. ेस प्रकाि हम भगवान बुद्ध की व्यावहारिक सिक्षा ववपश्यना को जीवन में उतािकि िील, समाधध, तर्ा प्रज्ञा काअभ्यासकिके समलदक्खोकाप्रहाणकिजीवनके सवोत्तमलक्ष्यतनवाषण, मोक्ष, मश्क्त, पिमानन्द, अर्ाषत सभी पाप कमो (अकुिल कमो) को न किना, कुिल कमो (पुण्य कमो) का संपादन किना। अपने धचत्त (मन) को ववपश्यना के द्वािा िुद्ध किते िहना, यही बुद्धों की सिक्षा है. ्र्ध््ष्ं््र्ञ ्् ु्ध ु्धं्ष ेस प्रकाि हम भगवान बुद्ध की व्यावहारिक सिक्षा ववपश्यना को जीवन में उतािकि िील, समाधध, तर्ा प्रज्ञा का अभ्यास किके समूल दुक्खो का प्रहाण कि जीवन के सवोत्तम लक्ष्य तनवाषण, मोक्ष, मुश्क्त, पिमानन्द, पिमसत्य को प्राप्त कि सकते है. Copyrights @ भिव प्रसाद पााँचे.Thi Copyrights @ भिव प्रसाद पााँचे.This is an open access reviewed article distributed under the creative common attribution license which permits unrestricted use, distribution and reproduction in any medium, provide the original work is cited. www.aarhat.com/vol II Issues I/July 114 8 धम्मपद, बुद्धवग्गो, 183
https://openalex.org/W3200076698	https://pubs.rsc.org/en/content/articlepdf/2021/na/d1na00358e	English	null	Cation non-stoichiometry in Fe:SrTiO<sub>3</sub> thin films and its effect on the electrical conductivity	Nanoscale advances	2,021	cc-by	13,505	aInstitute of Chemical Technologies and Analytics, TU Wien, Getreidemarkt 9-164/EC, 1060 Vienna, Austria. E-mail: markus.kubicek@tuwien.ac.at bCatalonia Institute for Energy Research (IREC), Jardins de Les Dones de Negre 1, 08930 Sant Adria del Besos, Barcelona, Spain cInstitute of Radiation Physics, Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Bautzner Landstraße 400, 01328 Dresden, Germany dFachbereich R¨ontgenzentrum, TU Wien, Getreidemarkt 9, 1060 Vienna, Austria eICREA, 23 Passeig Lluis Companys, Barcelona 08010, Spain † Electronic supplementary information (ESI) available. See DOI: 10.1039/d1na00358e Cation non-stoichiometry in Fe:SrTiO3 thin ﬁlms and its eﬀect on the electrical conductivity† The interplay of structure, composition and electrical conductivity was investigated for Fe-doped SrTiO3 thin ﬁlms prepared by pulsed laser deposition. Structural information was obtained by reciprocal space mapping while solution-based inductively-coupled plasma optical emission spectroscopy and positron annihilation lifetime spectroscopy were employed to reveal the cation composition and the predominant point defects of the thin ﬁlms, respectively. A severe cation non-stoichiometry with Sr vacancies was found in ﬁlms deposited from stoichiometric targets. The across plane electrical conductivity of such epitaxial ﬁlms was studied in the temperature range of 250–720 C by impedance spectroscopy. This revealed a pseudo-intrinsic electronic conductivity despite the substantial Fe acceptor doping, i.e. conductivities being several orders of magnitude lower than expected. Variation of PLD deposition parameters causes some changes of the cation stoichiometry, but the ﬁlms still have conductivities much lower than expected. Targets with signiﬁcant Sr excess (in the range of several percent) were employed to improve the cation stoichiometry in the ﬁlms. The use of 7% Sr-excess targets resulted in near-stoichiometric ﬁlms with conductivities close to the stoichiometric bulk counterpart. The measurements show that a ﬁne-tuning of the ﬁlm stoichiometry is required in order to obtain acceptor doped SrTiO3 thin ﬁlms with bulk-like properties. One can conclude that, although reciprocal space maps give a ﬁrst hint whether or not cation non-stoichiometry is present, conductivity measurements are more appropriate for assessing SrTiO3 ﬁlm quality in terms of cation stoichiometry. Received 17th May 2021 Accepted 10th September 2021 DOI: 10.1039/d1na00358e rsc.li/nanoscale-advances © 2021 The Author(s). Published by the Royal Society of Chemistry Nanoscale Advances Open Access Article. Published on 10 September 2021. Downloaded on 10/24/2024 6:21:48 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online View Journal \| View Issue Cite this: Nanoscale Adv., 2021, 3, 6114 Received 17th May 2021 Accepted 10th September 2021 DOI: 10.1039/d1na00358e rsc.li/nanoscale-advances PAPER pen Access Article. Published on 10 September 2021. Downloaded on 10/24/2024 6:21:48 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Cation non-stoichiometry in Fe:SrTiO3 thin ﬁlms and its eﬀect on the electrical conductivity† Maximilian Morgenbesser,a Stefanie Taibl, a Markus Kubicek, *a Alexander Schmid, a Alexander Viernstein,a Niklas Bodenm¨uller,a Christopher Herzig, a Federico Baiutti, b Juan de Dios Sirvent, b Maciej Oskar Liedke, c Maik Butterling, c Andreas Wagner, c Werner Artner,d Andreas Limbeck, a Albert Tarancon be and J¨urgen Fleig a 1. Introduction and composition depends on the deposition parameters, particularly on the laser uence12–15 and on the oxygen partial pressure.14 The combination of diﬀerent eﬀects such as scat- tering of elements in the plasma plume and incongruent abla- tion may cause substantial cation non-stoichiometries leading to either Ti-rich thin lms at higher uences (due to preferential ablation of Ti species) or, opposite, Sr-rich thin lms at low uences (due to enhanced scattering of Ti).15 Stoichiometric thin lms are reported when using a laser uence of 1.1 J cm2.15 Overall, the existing literature clearly indicates that the preparation of truly stoichiometric lms by pulsed laser deposition is far from trivial. SrTiO3 (STO) single crystals and thin lms are currently a cornerstone for the study of multiple phenomena such as resistive switching in memristive devices,1–4 photoconductivity,5 increased oxygen incorporation under UV light,6,7 photovoltages in bulk SrTiO3,8,9 and interfacial conductivity, especially with LaAlO3.10,11 However, SrTiO3 thin lms are not fully understood yet and show great variability in properties depending on the fabrication conditions. For instance, thin lms prepared by pulsed laser deposition (PLD) showed that the lm structure Despite the numerous studies performed so far, there is still only a limited understanding of the structure–composition– property relations in SrTiO3 thin lms. For example, it remains unclear how the crystal structure, the defect structure and the electrical properties of SrTiO3 thin lms are correlated and how much those properties diﬀer from the values found for single crystals. Particularly, charge transport properties might be very sensitive to changes in the point defect chemistry (e.g. due to cation non-stoichiometry) and thus pulsed laser deposited thin © 2021 The Author(s). Published by the Royal Society of Chemistry 6114 \| Nanoscale Adv., 2021, 3, 6114–6127 6114 Nanoscale Advances View Article Online Paper Paper lms may behave electrically very diﬀerent compared to bulk samples. For example, a correlation between conductivity and lattice constant was found for Nb:SrTiO3 thin lms.16,17 Other studies on nominally 0.4 mol% Fe doped SrTiO3 thin lms (100 to 413 nm thickness) showed conductivities with values being orders of magnitude lower than those of the corresponding targets.18,19 Interestingly, these conductivities were very close to those expected for hypothetical ultrapure electronically intrinsic SrTiO3. 1. Introduction Similarly, conductivities of Nb-donor doped SrTiO3 thin lms have been varied by orders of magnitude simply by modifying the laser uence in PLD deposited layers, which was correlated with diﬀerent lattice expansion and, thus, cation non-stoichiometries.20 Also the properties of conducting SrTiO3/LaAlO3 heterointerfaces have been proved to be strongly aﬀected by small variations of the LaAlO3 composition.21,22 Table 1 Pulsed laser deposition (PLD) parameters used for the preparation of the Fe-doped SrTiO3 thin ﬁlms. Temperature and substrate material are the same for all samples, only frequenc and – most importantly – laser ﬂuence were varied. As a substrate, Nb:SrTiO3 (0.5 wt% Nb) was used for electrochemical characterization and positron lifetime annihilation spectroscopy, Mg and STO were used for XRD and RSM, respectively, and MgO was used as a substrate for chemical analysis Parameter set I II III IV V VI VII VIII IX X Fluence [J cm2] 0.55 0.74 0.825 1.1 1.375 1.1 1.1 1.1 1.1 1.1 1.1 Frequency [Hz] 5 5 5 5 5 1 1 5 5 5 5 Pressure [mbar O2] 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 Temperature [C] 650 650 650 650 650 650 650 650 650 650 650 Target Stoichiometric polycrystal (PC) Single crystal (SC) PC 3% Sr excess PC 5% Sr excess PC 7% Sr excess PC 11% Sr excess P hor(s). Published by the Royal Soc Owing to this sensitivity of properties to the exact composi- tion and/or structure of the thin lms, it is mandatory to have sensitive tools for quantitatively evaluating the quality of PLD lms and to control the deposition parameters for a proper tuning of the layers. A common tool for evaluating the lm quality is the analysis of out-of-plane lattice constants in high resolution X-ray diﬀraction (HR-XRD) measurements.14,23–25 In these measurements, additional reections (indicating diﬀerent out-of-plane lattice parameters of epitaxially grown lms) can be interpreted in terms of the presence of cation vacancies,14,26,27 revealing the existence of severe cation non- stoichiometries, i.e. Sr or Ti vacancies. Regarding the control of the layers, a common approach for the optimization of the lm composition or lm structure is the modication of deposition parameters (laser uence, oxygen partial pressure, etc.) such that deviations of lattice constants vanish. In this work, we study Fe-doped SrTiO3 lms deposited using diﬀerent parameters and target stoichiometries to elucidate their eﬀect on the resulting layers (cation stoichiometry, struc- ture and conductivity). 1. Introduction The cation non-stoichiometry in 2% Fe doped SrTiO3 thin lms is studied using HR-XRD, inductively coupled plasma optical emission spectroscopy (ICP-OES), positron annihilation lifetime spectroscopy (PALS) and by measuring across plane conductivities by means of electro- chemical impedance spectroscopy (EIS). Film properties are modied by varying the PLD deposition parameters (laser u- ences ranging from 0.55 J cm2 to 1.375 J cm2) and by using non-stoichiometric targets (ranging from 3% to 11% Sr excess). The latter approach turns out to be highly eﬃcient when aiming at stoichiometric Fe:SrTiO3 lms with conductivities close to those of macroscopic bulk Fe:SrTiO3. The sensitivity of the diﬀerent tools for quantitative evaluation of the cation non- stoichiometry is compared. 2.5 Structural characterization Diﬀerent types of X-ray measurements were used to get infor- mation on the phase purity of the target (measurement in Bragg–Brentano geometry, BB), phase purity of the thin lms (measurement in grazing incidence geometry, GID) and on the lattice mismatch of SrTiO3 layers compared to the substrate (reciprocal space mapping, RSM). For the phase identication of SrTiO3 targets, a PANalytical XPert Pro (MPD) powder diﬀractometer was used. A 2q angle range of 15 to 90 was scanned, allowing a clear identication of SrTiO3. GID thin lm measurements and reciprocal space mapping (RSM) were done on an Empyrean multipurpose diﬀractometer (PANalytical, Germany). The diﬀractometer was equipped with a Cu-anode operating at 45 kV and 40 mA providing a wavelength of l ¼ 1.5406 ˚A (Cu K Alpha 1) and l ¼ 1.5444 ˚A (Cu K Alpha 2). A hybrid monochromator (2xGe(220)) with a 1 2 divergence slit and a 2 mm mask was located within the incident beam path. On the detector side, a PIXcel 3D detector in scanning mode with an anti-scatter slit of 7.5 mm and a 0.04 rad soller slit was used. For the gracing incidence measurements, again a range between 15 and 90 was mapped. Reciprocal space maps were conducted for the (002) reex (2q ¼ 46.4752) and the (113) reex (2q ¼ 81.7327). The scan range around these two reexes was 1 2q with a step size of u of 0.0015 and a continuous scan of 2q, resulting in a measuring time of around 12 h. 2.2 Polycrystalline targets The polycrystalline targets were prepared by a mixed oxide route, starting with SrCO3, TiO2 and Fe2O3 (Sigma Aldrich, Germany). Stoichiometric as well as nonstoichiometric (3, 5, 7, and 11% Sr excess compared to the sum of Fe and Ti) amounts of the solids were accurately weighed, thoroughly mixed in an agate mortar and calcined at 1000 C for 2 h under ambient conditions. For a stoi- chiometric cation composition (i.e. for a Sr amount corresponding to the amount of Ti + Fe), this preparation step already led to phase pure SrTiO3 powders. Aerwards the powders were crushed, cold isostatic pressed and sintered at 1200 C for 5 h in air, resulting in targets used for PLD. The nonstoichiometric powders were calcined under the same conditions, but showed several diﬀerent phases aer the 1000 C calcination. Nonetheless, the powders were crushed, pressed and sintered at 1200 C for 5 h (X-ray character- ization still showed diﬀerent additional phases) and at 1400 C for 4.5 h, yielding a target material that consisted of SrTiO3 with some Sr3Ti2O7 as an additional phase, see Results. As no futile compounds are expected to form, no change in composition is expected. 2.4 Chemical characterization of the SrTiO3 lms The information on the elemental composition became acces- sible by dissolving the lms and subsequent analysis by means of solution based ICP-OES (iCAP 6500, Thermo Fisher Scientic, USA). The SrTiO3 lms on MgO were dissolved in 3% v/v HNO3 and 0.3% v/v HF for 30 min at 25 C. Quantication was done via external calibration with liquid standard solutions. For a more detailed description, see ESI 1.† 2.3 Substrate material Fe:SrTiO3 thin lms for conductivity measurements were grown on 5 5 0.5 mm3 Nb-doped SrTiO3 (Nb:STO) single crystals (0.5 wt% Nb content, CrysTec GmbH, Germany). Owing to the high conductivity of Nb:SrTiO3, this substrate acts as an electrode and allows across plane conductivity measurements of the thin lms. Moreover, slightly doped Nb:SrTiO3 has the advantage of a very similar lattice parameter compared to undoped or slightly Fe-doped SrTiO3,2,20,28 which favours an epitaxial growth of the layer. Films grown on Nb:SrTiO3 were also used for the reciprocal space mapping (RSM) measurements. For positron annihilation lifetime spectroscopy, 10 10 0.5 mm3 Nb:SrTiO3 substrates were used in order to get information on the same substrate-thin lm combination used in electrical measurement without any possible inuence of a change in substrate. Fe:SrTiO3 thin lms grown on 10 10 0.5 mm3 MgO (CrysTec GmbH, Germany), on the other hand, were used for additional gracing incident X-ray measure- ments and 5 5 0.5 mm3 MgO (CrysTec GmbH, Germany) 2.1 Thin lm preparation Fe-doped SrTiO3 (Fe:STO) thin lms were prepared on diﬀerent substrates by pulsed laser deposition (PLD). The laser source was a 248 nm KrF excimer laser (COMPex Pro 201F, Coherent, The Netherlands) with a pulse duration of 25 ns. The system was operated at two diﬀerent pulse frequencies, 5 and 1 Hz, Nanoscale Adv., 2021, 3, 6114–6127 \| 6115 View Article Online View Article Online Paper View Article Online Nanoscale Advances Paper respectively. Laser uences ranging from 0.55 J cm2 to 1.375 J cm2 were achieved by using nominal laser energies between 200 and 500 mJ. In the following, unless specied diﬀerently, a combination of 5 Hz and 1.1 J cm2 is used (see ref. 15). In all cases, substrate temperature and oxygen partial pressure were 650 C and 0.15 mbar, respectively, with a substrate to target distance of 55 mm. Not surprisingly the lm thickness changes with changing laser uence, laser frequency or target compo- sition. In this study, deposition times were adapted such that all resulting lms were of similar thickness for the impedance measurements, i.e. in the thickness range of about 150 to 350 nm for electrochemical measurements. Film thicknesses were measured by means of prolometry. Polycrystalline Fe doped SrTiO3 with 2% Fe per SrTiO3 unit cell was used as target material for most lms, see below. Deposition parameters are summarized in Table 1. substrates were used for the chemical analysis by ICP-OES. The MgO substrate leads to polycrystalline thin lms, allowing a clear identication of the SrTiO3 phase in XRD. Moreover, the chemical analysis of the lms can be performed in a standard manner, i.e. by dissolving the layer without quantication problems due to some substrate dissolution. For comparison, also 5 5 0.5 mm3 yttria stabilized zirconia (YSZ, CrysTec GmbH, Germany) were used as a reference substrate for chemical analysis. Please note that in all cases the substrate size equals the deposited area. During deposi- tion, custom-made sample holders were used to prevent deposition on the sides and backside of the samples. The point of cross- comparison of data obtained for diﬀerent substrates is addressed in ESI 8.† Open Access Article. Published on 10 September 2021. Downloaded on 10/24/2024 6:21:48 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 2.6 Positron annihilation lifetime spectroscopy Variable energy positron annihilation lifetime spectroscopy (VEPALS) measurements were conducted on Fe:SrTiO3 samples at the Mono-Energetic Positron Source (MePS) beamline at HZDR, Germany.29 A digital lifetime CrBr3 scintillator detector [51 mm diameter (200) and 25.4 mm length (100)] coupled to a Hamamatsu R13089-100 PMT was utilized. The detector was © 2021 The Author(s). Published by the Royal Society of Chemistry 6116 \| Nanoscale Adv., 2021, 3, 6114–6127 Nanoscale Advances View Article Online Paper Fig. 1 Sketch of the sample consisting on the Nb:SrTiO3 (Nb:STO) substrate, the Fe:SrTiO3 (Fe:STO) thin ﬁlm and a porous Pt counter electrode as well as circular Pt microelectrodes on top. corresponding relative intensities reect to a large extend the concentration of each defect type (size) as long as the size of compared defects is in the similar range. In general, positron lifetime is directly proportional to defects size, i.e., the larger is the open volume, the lower is the probability and longer it takes for positrons to be annihilated with electrons.31,32 The positron lifetime and its intensity have been probed as a function of positron implantation energy Ep, which is proportional to the positron implantation depth. Fig. 1 Sketch of the sample consisting on the Nb:SrTiO3 (Nb:STO) substrate, the Fe:SrTiO3 (Fe:STO) thin ﬁlm and a porous Pt counter electrode as well as circular Pt microelectrodes on top. Fig. 1 Sketch of the sample consisting on the Nb:SrTiO3 (Nb:STO) substrate, the Fe:SrTiO3 (Fe:STO) thin ﬁlm and a porous Pt counter electrode as well as circular Pt microelectrodes on top. 2.7 Electrical characterization The electrical characterization was performed by means of electrochemical impedance spectroscopy (EIS) on Fe:SrTiO3 lms deposited on conducting Nb:SrTiO3. A sketch of the set-up is given in Fig. 1. Pt top layers were sputtered using a high voltage magnetron coating device (BAL-TEC MED 020, Ger- many) and microelectrodes with diameters in the range of 100– 300 mm were prepared by li-oﬀphotolithography. Pt paste (Tanaka Precious Metals, Japan) was brushed on the bottom side of the sample as a counter electrode. The Pt paste was pre- dried and then sintered at 850 C for 2 h, resulting in a porous Pt electrode (see Fig. 1). Pt/Ir needles were used to contact the microelectrodes for impedance measurements. a m-metal shielded and housed inside a solid Au casing. For the data acquisition a homemade soware was employed, executed from a multi-channel digitizer (SPDevices ADQ14DC-2X) with 14 bit vertical resolution and 2GS/s horizontal resolution. The time resolution of about 0.210 ns was achieved. The resolution function required for spectra analysis uses two Gaussian func- tions with distinct shis and intensities, which depend on the positron implantation energy, Ep. All spectra contained at least 1 107 counts. Typical lifetime spectrum N(t) is described by N(t) ¼ S(1/si)Ii exp(t/si), where si and Ii are the positron life- time and intensity of the i-th component, respectively (SIi ¼ 1). All the spectra were deconvoluted using the non-linearly least- squared based package PALSt tting soware30 into two discrete lifetime components, which directly evidence localized annihilation at 2 diﬀerent defect types (sizes; s1 and s2). The a m-metal shielded and housed inside a solid Au casing. For the data acquisition a homemade soware was employed, executed from a multi-channel digitizer (SPDevices ADQ14DC-2X) with 14 bit vertical resolution and 2GS/s horizontal resolution. The time resolution of about 0.210 ns was achieved. The resolution function required for spectra analysis uses two Gaussian func- tions with distinct shis and intensities, which depend on the positron implantation energy, Ep. All spectra contained at least 1 107 counts. Typical lifetime spectrum N(t) is described by N(t) ¼ S(1/si)Ii exp(t/si), where si and Ii are the positron life- time and intensity of the i-th component, respectively (SIi ¼ 1). All the spectra were deconvoluted using the non-linearly least- squared based package PALSt tting soware30 into two discrete lifetime components, which directly evidence localized annihilation at 2 diﬀerent defect types (sizes; s1 and s2). © 2021 The Author(s). Published by the Royal Society of Chemistry 2.7 Electrical characterization The Electrical measurements were then performed in a homoge- neously heated furnace33 within a temperature range of 250 C to 720 C (though meaningful conductivities could be deduced for some lms only in a limited temperature range). Impedance Fig. 2 (a) Impedance spectrum of an Fe:SrTiO3 thin ﬁlm deposited at 1.1 J cm2 from a stoichiometric polycrystal with 2% Fe measured at 551 C. Fitting was done with the transmission line model of a mixed conductor (b), consisting of an electronic rail, a blocked ionic rail and the displacement rail. The conductivity calculated from the DC resistance (¼electronic resistance Reon) is plotted in an Arrhenius diagram in (c) and compared with the bulk conductivity of a polycrystalline pellet (2% Fe) as well as the calculated electronically intrinsic (ce ¼ ch) conductivity.40 Temperature dependent geometrical and chemical capacitance values for the 2% Fe doped ﬁlm (370 nm thick) are shown in (d). Fig. 2 (a) Impedance spectrum of an Fe:SrTiO3 thin ﬁlm deposited at 1.1 J cm2 from a stoichiometric polycrystal with 2% Fe measured at 551 C. Fitting was done with the transmission line model of a mixed conductor (b), consisting of an electronic rail, a blocked ionic rail and the displacement rail. The conductivity calculated from the DC resistance (¼electronic resistance Reon) is plotted in an Arrhenius diagram in (c) and compared with the bulk conductivity of a polycrystalline pellet (2% Fe) as well as the calculated electronically intrinsic (ce ¼ ch) conductivity.40 Temperature dependent geometrical and chemical capacitance values for the 2% Fe doped ﬁlm (370 nm thick) are shown in (d). © 2021 The Author(s). Published by the Royal Society of Chemistry © 2021 The Author(s). Published by the Royal Society of Chemistry Nanoscale Adv., 2021, 3, 6114–6127 \| 6117 View Article Online View Article Online Nanoscale Advances Paper Fig. 3 Electronic and ionic conductivities of SrTiO3 thin ﬁlms depos- ited at 1.1 J cm2 from 2% Fe doped stoichiometric polycrystals. Both are deduced from the transmission line ﬁts. For comparison, ionic conductivities are also shown for other laser ﬂuences. The intrinsic conductivity seon was calculated using ref. 40. somewhat distorted main arc, a well-separated much smaller arc appears at high frequencies. The permittivities deduced from the capacitance of the high frequency arc are very close to the expected SrTiO3 lm bulk capacitances while the capaci- tance of the main arc is substantially larger (see also below). 2.7 Electrical characterization Typically, two separated arcs may either result from two serial processes, e.g. bulk transport and space charges at interfaces34 or from two parallel processes such as electron and ion conduction in a mixed conducting material, provided one of the two processes (paths) is blocked at the electrodes.35 SrTiO3 is known to be a mixed conductor36 and our set-up leads to a blocking of ions at one or both electrodes. Thus, use of a parallel path model is realistic here. More specically, we employed the transmission line impedance model suggested in literature for such materials.37–39 This model consists of an electronic rail (Reon) and an ionic rail (Rion), which are con- nected via the chemical capacitance (Cchem), and a displace- ment rail (Cgeom). The ionic rail is blocked by an interfacial capacitance (Cint), as sketched in Fig. 2b. (In order to avoid over- parameterization, the same Cint is used for both electrodes.) This model excellently ts the measurement data of our 2% Fe- doped SrTiO3 lms (see Fig. 2a). The number of tting param- eters is 6, namely Rion, Reion, CPEint (i.e. Tint, Pint), Cchem, and Cgeom. As a comparison, a parametrization using a serial t with two R-CPE elements also results in 6 tting parameters (R1, T1, P1, R2, T2, P2). For a discussion of the validity of the t as well as a discussion on the error of the measurements as well as the t used, see ESI 9–11.† Furthermore, three arguments considering the DC resistance as well as the resulting capacitances shown in Fig. 2d support the validity of the model and the corresponding data interpretation. Those are discussed in the ESI 2.† Open Access Article. Published on 10 September 2021. Downloaded on 10/24/2024 6:21:48 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 3 Electronic and ionic conductivities of SrTiO3 thin ﬁlms depos- ited at 1.1 J cm2 from 2% Fe doped stoichiometric polycrystals. Both are deduced from the transmission line ﬁts. For comparison, ionic conductivities are also shown for other laser ﬂuences. The intrinsic conductivity seon was calculated using ref. 40. spectra were obtained for frequencies from 0.9 MHz to 1 Hz with a resolution of 10 point per frequency decade, measured by an Alpha-A High Resolution Analyzer (Novocontrol, Germany). To ensure the probing in a linear regime, an AC rms amplitude of 20 mV was applied. 2.7 Electrical characterization Aer electrical characterization, the lms were checked for cracks or changes in the surface structure using optical microscopy. Furthermore, such Fe:SrTiO3 thin lms were investigated using atomic force microscopy (AFM) and trans- mission electron microscopy (TEM) in previous studies.18 An Arrhenius-type diagram of the resulting DC conductivity (i.e. electronic conductivity) of the thin lms is shown in Fig. 2c. For comparison purposes, the electronic bulk conductivity of the corresponding 2% target material is also shown in Fig. 2c (the polycrystalline target leads to a bulk and a grain boundary arc in the complex impedance plane and only the bulk arc is considered here). As clearly visible in Fig. 2c, values of the 2% Access Article. Published on 10 September 2021. Downloaded on 10/24/2024 6:21:48 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Ionic conductivities of the same lms are plotted in Fig. 3 showing one to two orders of magnitude larger values than the pseudo-intrinsic electronic conductivity. Here, an activation energy of 1.33 0.01 eV is found. For a detailed discussion of activation energy for ionic conductivity, see below. Fig. 3 further displays variations of the ionic conductivities for lms prepared with other uences, see below. Diﬀerent ionic defects are most probably also the reason behind diﬀerences in spectra shapes found between our 2% doped lms and 0.4% Fe doped lms.18,19 The yet not interpreted distortion of the spectra for 0.4% Fe thin lms in ref. 18 and 19 thus probably comes from the mixed conduction. This is detailed in ESI 3.† Fe-doped SrTiO3 thin lm are three to ve orders of magnitude lower than the expected ones (depending on temperature). Moreover, in accordance with literature data on 0.4% Fe doped lms,18 the electronic conductivity of the 2% Fe lms and its temperature dependence t very well to that of intrinsic SrTiO3, calculated for the materials parameters from literature,40 i.e. a band gap Eg of 3.3 eV 6.0 104 eV T, a hole mobility of 8.9 105 (T/K)2.36 cm2 V1 s1, an electron mobility of 4.5 105 (T/K)2.2 cm2 V1 s1 and K0 ¼ 7.67 1042 cm6. Here, “intrinsic” means identical hole and electron concentrations (ce, ch) as for a pure semiconductor without any further defect, i.e. ce ¼ ch ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ K0eEg=kT p (k ¼ Boltzmann constant, T ¼ temperature). Please note that electron and hole concentrations at 400 C are thus in the range of 3 1010 cm3 and in a usual situation this requires purity levels of semiconductors in the 0.01 ppb range. An activation energy of 1.75 0.01 eV is found, 3.1 Pseudo-intrinsic conductivity of lms from stoichiometric targets Published by the Royal Society of Chemistry 6118 \| Nanoscale Adv., 2021, 3, 6114–6127 Nanoscale Advances View Article Online Nanoscale Advances View Article Online Nanoscale Advances View Article Online Fe-doped SrTiO3 thin lm are three to ve orders of magnitude lower than the expected ones (depending on temperature). Moreover, in accordance with literature data on 0.4% Fe doped lms,18 the electronic conductivity of the 2% Fe lms and its temperature dependence t very well to that of intrinsic SrTiO3, calculated for the materials parameters from literature,40 i.e. a band gap Eg of 3.3 eV 6.0 104 eV T, a hole mobility of 8.9 105 (T/K)2.36 cm2 V1 s1, an electron mobility of 4.5 105 (T/K)2.2 cm2 V1 s1 and K0 ¼ 7.67 1042 cm6. Here, “intrinsic” means identical hole and electron concentrations (ce, ch) as for a pure semiconductor without any further defect, i.e. ce ¼ ch ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ K0eEg=kT p (k ¼ Boltzmann constant, T ¼ temperature). Please note that electron and hole concentrations at 400 C are thus in the range of 3 1010 cm3 and in a usual situation this requires purity levels of semiconductors in the 0.01 ppb range. An activation energy of 1.75 0.01 eV is found, Table 2 Composition of Fe:SrTiO3 thin ﬁlms prepared from stoi- chiometric targets by diﬀerent deposition parameters and measured by ICP-OES. A pronounced Sr deﬁciency was found for all samples prepared from stoichiometric targets with 2% Fe and from the single crystal (SC) with 0.16% Fe Sr Ti Fe Sr/(Ti + Fe) PC, 2% Fe, 0.55 J cm2, 5 Hz 0.94 1.01 0.051 0.88 PC, 2% Fe, 0.825 J cm2, 5 Hz 0.94 1.04 0.023 0.88 PC, 2% Fe, 1.1 J cm2, 5 Hz 0.94 1.02 0.035 0.90 PC, 2% Fe, 1.375 J cm2, 5 Hz 0.95 1.03 0.027 0.90 PC, 2% Fe, 1.1 J cm2, 1 Hz 0.94 1.02 0.014 0.89 SC, 1.1 J cm2, 1 Hz 0.96 1.03 0.008 0.92 Paper This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 3.1 Pseudo-intrinsic conductivity of lms from stoichiometric targets Fig. 2 shows a typical spectrum for SrTiO3 thin lms prepared from 2% Fe doped stoichiometric targets. In addition to the Fig. 4 Reciprocal space mapping measurements for Fe:SrTiO3 standard thin ﬁlms deposited from 2% Fe doped stoichiometric targets on Nb:SrTiO3 substrate material (PLD parameters [laser ﬂuence, frequency, temperature, pressure, time]: 1.1 J cm2, 5 Hz, 650 C, 0.15 mbar and 200). The out-of-plane measurement in close proximity of the (002) reﬂex is shown in (a). Arrows point at the substrate reﬂex as well as the thin ﬁlm reﬂex. The diﬀerences indicating a lattice expansion Dc are shown. The same sample was also measured around the (113) reﬂex (in-plane measurement). The resulting reciprocal space map is shown in (b). For clariﬁcation, arrows and dotted boxes indicate the direction of the diﬀraction vectors Qh002i and Qh113i, respectively. Fig. 4 Reciprocal space mapping measurements for Fe:SrTiO3 standard thin ﬁlms deposited from 2% Fe doped stoichiometric targets on Nb:SrTiO3 substrate material (PLD parameters [laser ﬂuence, frequency, temperature, pressure, time]: 1.1 J cm2, 5 Hz, 650 C, 0.15 mbar and 200). The out-of-plane measurement in close proximity of the (002) reﬂex is shown in (a). Arrows point at the substrate reﬂex as well as the thin ﬁlm reﬂex. The diﬀerences indicating a lattice expansion Dc are shown. The same sample was also measured around the (113) reﬂex (in-plane measurement). The resulting reciprocal space map is shown in (b). For clariﬁcation, arrows and dotted boxes indicate the direction of the diﬀraction vectors Qh002i and Qh113i, respectively. Fig. 4 Reciprocal space mapping measurements for Fe:SrTiO3 standard thin ﬁlms deposited from 2% Fe doped stoichiometric targets on Nb:SrTiO3 substrate material (PLD parameters [laser ﬂuence, frequency, temperature, pressure, time]: 1.1 J cm2, 5 Hz, 650 C, 0.15 mbar and 200). The out-of-plane measurement in close proximity of the (002) reﬂex is shown in (a). Arrows point at the substrate reﬂex as well as the thin ﬁlm reﬂex. The diﬀerences indicating a lattice expansion Dc are shown. The same sample was also measured around the (113) reﬂex (in-plane measurement). The resulting reciprocal space map is shown in (b). For clariﬁcation, arrows and dotted boxes indicate the direction of the diﬀraction vectors Qh002i and Qh113i, respectively. © 2021 The Author(s). Paper Table 2 Composition of Fe:SrTiO3 thin ﬁlms prepared from stoi- chiometric targets by diﬀerent deposition parameters and measured by ICP-OES. A pronounced Sr deﬁciency was found for all samples prepared from stoichiometric targets with 2% Fe and from the single crystal (SC) with 0.16% Fe tting reasonably well to half of the band-gap energy, which is expected for an intrinsic material. An exact mechanistic expla- nation of this pseudo-intrinsic behaviour of Fe:SrTiO3 thin lms, seemingly independent of the dopant concentration in the range between 0.4 to 2% Fe doping, is beyond the scope of this paper, but the following XRD and chemical analysis gives important empirical information on possible reasons. Sr Ti Fe Sr/(Ti + Fe) PC, 2% Fe, 0.55 J cm2, 5 Hz 0.94 1.01 0.051 0.88 PC, 2% Fe, 0.825 J cm2, 5 Hz 0.94 1.04 0.023 0.88 PC, 2% Fe, 1.1 J cm2, 5 Hz 0.94 1.02 0.035 0.90 PC, 2% Fe, 1.375 J cm2, 5 Hz 0.95 1.03 0.027 0.90 PC, 2% Fe, 1.1 J cm2, 1 Hz 0.94 1.02 0.014 0.89 SC, 1.1 J cm2, 1 Hz 0.96 1.03 0.008 0.92 3.2 Structural, chemical and defect analysis of pseudo- intrinsic SrTiO3 lms 6 Composition of diﬀerent SrTiO3 thin ﬁlms on MgO measured by ICP-OES highlighting diﬀerences in the Sr content for diﬀerent deposition parameters (targets without Sr excess: 2% Fe in PC, 0.16% Fe in SC) and for diﬀerent Sr excess in the target. A stoichiometric thin ﬁlm is only obtained by using a target with a 7% Sr overstoichiometry. At 11% Sr excess, a slight Sr excess is present in the thin ﬁlm. In all other cases, a Sr deﬁciency can be seen. Fe is counted as B site ion in all cases. Fig. 6 Composition of diﬀerent SrTiO3 thin ﬁlms on MgO measured by ICP-OES highlighting diﬀerences in the Sr content for diﬀerent deposition parameters (targets without Sr excess: 2% Fe in PC, 0.16% Fe in SC) and for diﬀerent Sr excess in the target. A stoichiometric thin ﬁlm is only obtained by using a target with a 7% Sr overstoichiometry. At 11% Sr excess, a slight Sr excess is present in the thin ﬁlm. In all other cases, a Sr deﬁciency can be seen. Fe is counted as B site ion in all cases. Hence, we nd a lm growth with in-plane lattice constants as for the substrate, but signicantly diﬀerent out of plane lattice constants of substrate and lm. This phenomenon is well-known in literature12,13,44 and is commonly attributed to the existence of cation vacancies, i.e. to lms with a Sr/(Ti + Fe) ratio diﬀering from one.27,45–47 For example, Wicklein et al. report that a broad range of diﬀerent target materials lead to thin lms showing either a reduced Sr-content, meaning that eihter Sr vacancies or Ti vacancies are present in the thin lms.15 diﬀraction vector Qh002i normal to the surface is indicated. The detector streak (DS) as well as the monochromator streak (MS) can be attributed to the measurement set-up. The streak normal to the qk axis is the so-called crystal truncation rod (CTR), which is a diﬀraction phenomenon originating from the fact that the sample has nite dimensions. Fig. 7 Positron annihilation lifetime spectroscopy measurements on Fe:SrTiO3 thin ﬁlms deposited from 2% Fe doped targets with diﬀerent cation stoichiometry on Nb:SrTiO3 substrates (5 Hz, 1.1 J cm2). The thin ﬁlm deposited from a stoichiometric target (labeled 0% Sr excess) clearly shows a positron lifetime which ﬁts excellently to Sr vacancies. For larger implantation energies, transition to bulk values is seen. 3.2 Structural, chemical and defect analysis of pseudo- intrinsic SrTiO3 lms In the following, results from the structural, chemical and defect analysis of these pseudo-intrinsic SrTiO3 lms are shown. First, we consider the XRD data of the lms. Fig. 4 displays results from reciprocal space mapping (RSM) measurements on a thin lm deposited with a repetition rate of 5 Hz and a uence of 1.1 J cm2. This high-resolution X-ray diﬀraction method can resolve deviations in the microstruc- ture between the thin lm and the substrate material.41 The Fig. 5 u–2q scans for various thin ﬁlm samples. In (a), a comparison of SrTiO3 ﬁlms (2% Fe doped stoichiometric targets) prepared with diﬀerent laser ﬂuences is shown (all other deposition parameters are held constant). The HR-XRD measurements were conducted around the (002) reﬂex. A black line indicates that the substrate reﬂex is in the same position for every sample. As reference the pure Nb:SrTiO3 substrate was measured. Arrows indicate the substrate reﬂex and illustrate the shift of these reﬂexes for changing deposition parameters. In (b), the laser ﬂuence was kept constant at 1.1 J cm2. Varying laser frequency (1 and 5 Hz) as well as target material (polycrystalline, 2% Fe doped, stoichiometric and single crystalline targets) result in thin ﬁlms with a distinctly decreased lattice expansion. (Shifting of the layer reﬂex towards the substrate reﬂex.) The four RSM images belong to the four curves in (b). Fig. 5 u–2q scans for various thin ﬁlm samples. In (a), a comparison of SrTiO3 ﬁlms (2% Fe doped stoichiometric targets) prepared with diﬀerent laser ﬂuences is shown (all other deposition parameters are held constant). The HR-XRD measurements were conducted around the (002) reﬂex. A black line indicates that the substrate reﬂex is in the same position for every sample. As reference the pure Nb:SrTiO3 substrate was measured. Arrows indicate the substrate reﬂex and illustrate the shift of these reﬂexes for changing deposition parameters. In (b), the laser ﬂuence was kept constant at 1.1 J cm2. Varying laser frequency (1 and 5 Hz) as well as target material (polycrystalline, 2% Fe doped, stoichiometric and single crystalline targets) result in thin ﬁlms with a distinctly decreased lattice expansion. (Shifting of the layer reﬂex towards the substrate reﬂex.) The four RSM images belong to the four curves in (b). Nanoscale Adv., 2021, 3, 6114–6127 \| 6119 © 2021 The Author(s). 3.2 Structural, chemical and defect analysis of pseudo- intrinsic SrTiO3 lms Published by the Royal Society of Chemistry Paper View Article Online Paper View Article Online View Article Online Nanoscale Advances Fig. 6 Composition of diﬀerent SrTiO3 thin ﬁlms on MgO measured by ICP-OES highlighting diﬀerences in the Sr content for diﬀerent deposition parameters (targets without Sr excess: 2% Fe in PC, 0.16% Fe in SC) and for diﬀerent Sr excess in the target. A stoichiometric thin ﬁlm is only obtained by using a target with a 7% Sr overstoichiometry. At 11% Sr excess, a slight Sr excess is present in the thin ﬁlm. In all other cases, a Sr deﬁciency can be seen. Fe is counted as B site ion in all cases. er a Creative Commons Attribution 3.0 Unported Licence. The measurement around the symmetric (002) reex (Fig. 4a) reveals a small deviation of the layer reex in comparison to the substrate reex. The substrate can be identied by the reex position, which gives a lattice constant of 3.906 ˚A, in very good agreement with literature data for undoped SrTiO3.28,42,43 Moreover, the substrate reex shows the highest count inten- sity. The thin lm reex (smaller count intensity) is slightly below the substrate reex, indicating an expansion Dc of the out-of-plane lattice constant c. A relocation of the measuring spot on the asymmetric (113) reex gives the reciprocal space map shown in Fig. 4b. Again, an arrow indicates the diﬀraction vector Qh113i. The measurement was performed using a gracing incident angle giving negative values for qk. The lattice expansion of the thin lm normal to the layerjsubstrate interface is conrmed by this measurement. In case of the asymmetric measurement, additional informa- tion on the in-plane lattice constant can be extracted. For the sample in Fig. 4b, the lattice parameter of the thin lm parallel to the surface is perfectly matching the substrate, i.e. the layer reex is located at the position r ¼ 0. This indicates that the layer undergoes a pseudomorphic growth on top of the single crystalline SrTiO3 substrate. A relocation of the measuring spot on the asymmetric (113) reex gives the reciprocal space map shown in Fig. 4b. Again, an arrow indicates the diﬀraction vector Qh113i. The measurement was performed using a gracing incident angle giving negative values for qk. The lattice expansion of the thin lm normal to the layerjsubstrate interface is conrmed by this measurement. Fig. 3.2 Structural, chemical and defect analysis of pseudo- intrinsic SrTiO3 lms For larger implantation energies, we see the transition to bulk properties. For lms with 5% Sr excess, the lm related positron lifetime decreases close to a literature value of s1 ¼ 225 ps indicating the emergence of a new smaller dominant defect type, arguably a complex of Ti vacancies and oxygen vacan- cies.12,51 (Please note that a Sr vacancy is a larger and stronger positron trap). Concomitantly a drop in I1 intensity reects a larger chance for positrons to diﬀuse, as a consequence of a lower number of vacancy traps, and to annihilate with surface states. Open Access Article. Published on 10 September 2021. Downloaded on 10/24/2024 6:21:48 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The estimated cation non-stoichiometry of the lm was quantied by chemical analysis of the lms. The quantitative data of the ICP-OES analysis of a standard lm on MgO is shown in Table 2 and Fig. 6. The lm shows a drastically reduced Sr content. Assuming that Fe occupies the B-site, we nd an A/B site ratio in the lm of 0.90 and thus a Sr deciency in the range of 10%. Here, the error is well below 1% for main components (Sr, Ti) and in the range of 5% for the Fe dopant, e.g. 0.02 0.001. Accordingly, the cation vacancies detected by RSM can be attributed to the A-site. In literature, Sr deciencies in this range and similar out of plane deviations of lattice parameters were reported.27,45–47 Interestingly, also the Fe content detected in the lms was consistently too high, indicating that the PLD process led to an enrichment in Fe, at least in the centre part of the plasma plume used to deposit the lms. Measurements showed that indeed an inhomogeneous cation composition is expected in the PLD plasma used for SrTiO3 deposition and thus deviation of cation compositions compared to the target are not surprising.15,44 Similar non-stoichiometries introduced by the PLD process are reported in literature for other materials as well.52–62 In addition, positron annihilation lifetime spectroscopy (PALS) was performed and the implantation energy dependent positron lifetime s1 is shown in Fig. 7. The positron lifetimes for lower energies probe the thin lm and reveal Sr vacancies as predominant cation based point defect species for the thin lm deposited from a stoichiometric target. 3.2 Structural, chemical and defect analysis of pseudo- intrinsic SrTiO3 lms For ﬁlms prepared from targets with 5% Sr excess, VTiVO clusters are found as the predominant defect type present in the respective thin ﬁlm, highlighting a severe transition in defect chemistry induced by the introduction of Sr excess to the PLD targets. Fig. 7 Positron annihilation lifetime spectroscopy measurements on Fe:SrTiO3 thin ﬁlms deposited from 2% Fe doped targets with diﬀerent cation stoichiometry on Nb:SrTiO3 substrates (5 Hz, 1.1 J cm2). The thin ﬁlm deposited from a stoichiometric target (labeled 0% Sr excess) clearly shows a positron lifetime which ﬁts excellently to Sr vacancies. For larger implantation energies, transition to bulk values is seen. For ﬁlms prepared from targets with 5% Sr excess, VTiVO clusters are found as the predominant defect type present in the respective thin ﬁlm, highlighting a severe transition in defect chemistry induced by the introduction of Sr excess to the PLD targets. Fig. 7 Positron annihilation lifetime spectroscopy measurements on Fe:SrTiO3 thin ﬁlms deposited from 2% Fe doped targets with diﬀerent cation stoichiometry on Nb:SrTiO3 substrates (5 Hz, 1.1 J cm2). The thin ﬁlm deposited from a stoichiometric target (labeled 0% Sr excess) clearly shows a positron lifetime which ﬁts excellently to Sr vacancies. For larger implantation energies, transition to bulk values is seen. For ﬁlms prepared from targets with 5% Sr excess, VTiVO clusters are found as the predominant defect type present in the respective thin ﬁlm, highlighting a severe transition in defect chemistry induced by the introduction of Sr excess to the PLD targets. © 2021 The Author(s). Published by the Royal Society of Chemistry 6120 \| Nanoscale Adv., 2021, 3, 6114–6127 Nanoscale Advances View Article Online Paper Furthermore, adding Fe to the target material can cause a very diﬀerent ablation. The ablation behaviour changes due to an increased thermal conductance of the target,48,49 which then causes an increased heat penetration depth and a diﬀerent dissipation of introduced laser energy.49 This leads to less material being ablated when Fe is present in the target material. Further mechanistic reasons for this non-stoichiometric depo- sition are discussed in ref. 48–50. In accordance with all the known literature we thus conclude that also our lms exhibit a cation non-stoichiometry. for Sr vacancies.13 At the same time, the relative intensity I1 is close to 100% indicating that Sr vacancies are the most abun- dant defect type, a typical scenario of large defect concentra- tions. Open Access Article. Published on 10 September 2021. Downloaded on 10/24/2024 6:21:48 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 10 September 2021. Downloaded on 10/24/2024 6:21:48 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. All together, the complementary tools used here (conduc- tivity measurements, RSM, chemical analysis by ICP-OES, and PALS) directly or indirectly reveal the strongly non-ideal char- acter of the grown SrTiO3 lms (non-ideal in the sense of non- bulk SrTiO3 like). In the following, we discuss how this non- ideality can be reduced or even eliminated and how sensitive the tools are to monitor cation non-stoichiometry of the lm. 3.2 Structural, chemical and defect analysis of pseudo- intrinsic SrTiO3 lms Positron lifetime s1 basically overlaps with the calculated literature value of 281 ps Fig. 8 Impedance spectra of STO thin ﬁlms deposited from 2% Fe doped stoichiometric targets using diﬀerent laser ﬂuences (1.1 J cm2 and 0.74 J cm2) at 551 C and 553 C, respectively, showing a strong distortion for both semicircles (a). The dc (¼electronic) conductivities of thin ﬁlms deposited at diﬀerent laser ﬂuences are plotted in an Arrhenius plot (b) and are compared to the bulk conductivity of a polycrystal and the electronic intrinsic conductivity calculated using ref. 40. Bulk permittivity values and chemical capacitances deduced from the transmission line model are shown in (c) and (d), respectively. Fig. 8 Impedance spectra of STO thin ﬁlms deposited from 2% Fe doped stoichiometric targets using diﬀerent laser ﬂuences (1.1 J cm2 and 0.74 J cm2) at 551 C and 553 C, respectively, showing a strong distortion for both semicircles (a). The dc (¼electronic) conductivities of thin ﬁlms deposited at diﬀerent laser ﬂuences are plotted in an Arrhenius plot (b) and are compared to the bulk conductivity of a polycrystal and the electronic intrinsic conductivity calculated using ref. 40. Bulk permittivity values and chemical capacitances deduced from the transmission line model are shown in (c) and (d), respectively. Nanoscale Adv., 2021, 3, 6114–6127 \| 6121 © 2021 The Author(s). Published by the Royal Society of Chemistry View Article Online View Article Online Paper Article Online 3.3 SrTiO3 lm tuning by controlling the deposition conditions (stoichiometric targets) One common strategy for reducing the cation non- stoichiometry is the variation of the deposition parameters in PLD grown layers. It is reported that lowering the laser uence reduces the Sr vacancy concentration.14,15,23 In this work, a range of laser uences was employed, from 0.55 J cm2 to 1.375 J cm2 (Section 3.1 reports on 1.1 J cm2). Moreover, diﬀerent laser frequencies were employed (1 and 5 Hz) and also a Fe:SrTiO3 single crystal was used as the target (though with a lower Fe content of 0.16% Fe). In Fig. 5, rocking curves for thin lms deposited using diﬀerent laser uences (0.86 J cm2 to 1.375 J cm2), repetition rates (1 Hz, 5 Hz) and target materials (poly- crystal, single crystal) are shown and reveal that the higher laser uence indeed enhanced the out of plane lattice mismatch while lower laser uence lowered the mismatch. However, within the given uence range and for 5 Hz deposition rate a clear indication of a lattice mismatch remained. Reducing the frequency lead to thinner lms and thus less pronounced signals but the lattice mismatch itself remained and therefore also indication of the lm non-stoichiometry. The thin lm prepared from the 0.16% doped Fe:SrTiO3 single crystal showed least indication of a cation non-stoichiometry in RSM curves. Fig. 9 u–2q scans for various thin ﬁlm samples deposited from 2% Fe doped SrTiO3 polycrystalline targets with diﬀerent Sr over stoichiometry. The HR-XRD measurements were conducted around the (002) reﬂex. The black dashed line indicates that the substrate reﬂex is in the same position for every sample. Laser ﬂuence was kep constant at 1.1 J cm2. At the right hand side, the corresponding reciprocal space maps are shown as well. Nanoscale Advances Paper conductivity, ionic conductivity, relative permittivity and chemical capacitance were deduced. Again, the DC resistance corresponds to the electronic conductivity. The ionic conduc- tivity (Fig. 3) as well as the chemical capacitance (Fig. 8d) show some uence dependency. The moderate deviation in bulk permittivity (from Cgeom) are probably due to non-idealities of the t model. However, the electronic conductivity does not change due to the diﬀerent deposition parameters (Fig. 8b). Rather, again pseudo-intrinsic conductivity values were found for the samples investigated (0.74 and 0.825 J cm2). Activation energies for the electronic/total conductivity of 1.71 0.01 eV and 1.73 0.01 eV for 0.825 J cm2 and 0.74 J cm2 are found, which are well in line with the activation energy found in the thin lm deposited at 1.1 J cm2. For ionic conductivity, acti- vation energies of 1.57 0.01 eV and 1.58 0.01 eV are found, respectively. In literature, for ionic conductivity in undoped or slightly Fe- or Ni-doped SrTiO3 single crystals, an activation energy of 0.9 is reported,40 which is signicantly lower than the activation energies found in this study. However, interactions between dopants and oxygen vacancies are known to increase, for example, the migration enthalpy from 0.62–0.67 eV up to 1 eV.69 In our case, the extremely high amount of Sr vacancies might aﬀect oxygen transport through defect association with oxygen vacancies, thereby also aﬀecting activation energies. We consider it as most plausible that such a severe A/B site non-stoichiometry in the SrTiO3 lm is the origin of the pseudo- intrinsic lm conductivity. We think that interplay of the Fe dopant (Fe acceptor states), a deep electron trap (possibly site defects,63 e.g. Ti on the A-site64–66 acting as a donor) as well as a mid-gap state (Sr-vacancies67) causes a pinning of the Fermi level close to the centre of the band gap. Diﬀerent gap states have also been reported for nominally undoped SrTiO3 thin lms.68 However, a detailed mechanistic discussion of this phenomenon needs further investigation and is beyond the scope of this paper. 3.4 SrTiO3 lm tuning using diﬀerent target compositions 3.4 SrTiO3 lm tuning using diﬀerent target compositions Based on the knowledge that Sr deciencies (in the range of several percent) result in lms deposited by PLD, we prepared diﬀerent targets with a Sr surplus of 3 to 11% (details on the target preparation are given in the experimental part). XRD of these targets clearly revealed the Sr over-stoichiometry by reexes from a second phase, namely Sr3Ti2O7, and its amount increased with Sr content, see ESI 4.† The lms grown from these targets, however, did not show any indication of a secondary phase, neither in XRD measurements of poly- crystalline lms on MgO (see ESI 5†) nor in RSM measurements (see Fig. 9). Rather, their reections could not be distinguished from the substrate reections in rocking curves, at least for 3– 7% Sr, see Fig. 9. Only 11% Sr surplus again caused a shoulder in the RSM curve which is most probably due to Ti vacancies in this case. The impedance spectra of these lms are very diﬀerent from those of the pseudo-intrinsic standard lms. As an example, we show a spectrum for the 7% Sr excess case in Fig. 10. At 330 C, it becomes obvious that the spectrum consists of three arcs with dramatically diﬀerent sizes, a very small high frequency arc or shoulder, a medium sized medium frequency arc and a very large low frequency arc (see Fig. 10a). Hence, compared to the pseudo-intrinsic standard lms, an additional feature appears. However, the total resistance of the large low frequency arc is still much smaller than the main arc measured for the standard lms prepared from stoichiometric targets. This is illustrated by comparing spectra of the pseudo-intrinsic standard lms with the lms from 7% Sr excess targets measured at 555 C (see Fig. 10b). At this temperature, only the low and the medium frequency arcs are still visible in the measured frequency range of the 7% excess lm. The entire dc resistance of the 7% Sr excess lm (sum of three arcs) is not much larger than the high frequency arc of the pseudo-intrinsic lm. In general, the high frequency arc of the 7% Sr excess lms is orders of magnitude smaller than that of the standard lms. This strongly indicates On the basis of XRD data, all three lms with 3, 5 and 7% Sr surplus are compatible with stoichiometric compositions. Data from the chemical analysis of the lms are summarized in Table 2 and Fig. 6 and reveal that the pronounced Sr de- ciency is not much aﬀected by the diﬀerent preparation conditions. Also the lm prepared from the single crystal exhibits a slightly smaller but still severe A-site deciency. In all samples, again more Fe is found than in the target and on average the Fe content is almost twice the nominal one, most likely due to plume-related processes, such as preferential scattering, or plume-background interactions.14,15,58 The impedance spectra were similar to those of the lms in Section 3.1, but the distortion of the main arc became even more prominent in some cases (see Fig. 8a). The tting procedure for these impedance spectra was carried out as described in Section 3.1, i.e. with a transmission line based model, and the electronic Fig. 9 u–2q scans for various thin ﬁlm samples deposited from 2% Fe- doped SrTiO3 polycrystalline targets with diﬀerent Sr over- stoichiometry. The HR-XRD measurements were conducted around the (002) reﬂex. The black dashed line indicates that the substrate reﬂex is in the same position for every sample. Laser ﬂuence was kept constant at 1.1 J cm2. At the right hand side, the corresponding reciprocal space maps are shown as well. 6122 \| Nanoscale Adv., 2021, 3, 6114–6127 © 2021 The Author(s). Published by the Royal Society of Chemistry Nanoscale Advances View Article Online Paper Paper Table 3 Composition of diﬀerently prepared Fe:SrTiO3 thin ﬁlms, deposited from overstoichiometric targets and measured by ICP-OES. The Sr excess in the target material counterbalances the loss during the PLD process, leading to A : B-ratios closer to unity unity for those three lms compared to lms prepared from the stoichiometric target. However, for 3 and 5% Sr excess the nominal A/B site ratio is still only 97 or 96%, respectively (with errors well below 1%). The even slightly lower nominal value for 5% Sr excess compared to 3% Sr is within the error bar. Moreover, in these calculations, Fe is always counted as a B site ion, which is not necessarily the case if many A-site vacancies are present. The 7% Sr lm, on the other hand, showed almost exact A/B site stoichiometry in ICP-OES measurements, though still more Fe is present than in the target. For 11% Sr excess, A/B of 1.06 is measured (see Table 3), in accordance with the shoulder in the rocking curve indicating too much Sr (see Fig. 9). Please note, that most of these lms have Sr/Ti ratios which can hardly be distinguished by XPS or XRD studies with typical errors in the 5% range.14,70,71 Sr Ti Fe Sr/(Ti + Fe) 2% Fe, 0% Sr, 1.1 J cm2 0.94 1.02 0.035 0.90 2% Fe, 3% Sr, 1.1 J cm2 0.98 0.98 0.034 0.97 2% Fe, 5% Sr, 1.1 J cm2 0.98 0.98 0.043 0.96 2% Fe, 7% Sr, 1.1 J cm2 1.00 0.96 0.032 1.01 2% Fe, 11% Sr, 1.1 J cm2 1.03 0.94 0.032 1.06 Open Access Article. Published on 10 September 2021. Downloaded on 10/24/2024 6:21:48 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 3.4 SrTiO3 lm tuning using diﬀerent target compositions This conclusion, however, was only partly conrmed by the chemical analysis. Table 3 and Fig. 6 show the results from ICP-OES measurements. Indeed, the A/B site ratio was much closer to Fig. 10 (a) Impedance spectrum of a thin ﬁlm deposited from a 2% Fe doped target with 7% Sr overstoichiometry measured at 327 C. The magniﬁcation reveals a medium frequency shoulder and an additional small high frequency semicircle. Only the high frequency semicircle (RSTO) is attributed to the Fe:SrTiO3 bulk according to the geometrical capacitance. The medium frequency shoulder and the big low-frequency semicircle are attributed to space charge regions (RSCR1, RSCR2). (b) Impedance spectrum of the same thin ﬁlm measured at 556 C and the respective impedance spectrum of a thin ﬁlm deposited from a stoichiometric target as a reference (measured at 551 C), highlighting the dramatic change in absolute resistance. Fig. 10 (a) Impedance spectrum of a thin ﬁlm deposited from a 2% Fe doped target with 7% Sr overstoichiometry measured at 327 C. The magniﬁcation reveals a medium frequency shoulder and an additional small high frequency semicircle. Only the high frequency semicircle (RSTO) is attributed to the Fe:SrTiO3 bulk according to the geometrical capacitance. The medium frequency shoulder and the big low-frequency semicircle are attributed to space charge regions (RSCR1, RSCR2). (b) Impedance spectrum of the same thin ﬁlm measured at 556 C and the respective impedance spectrum of a thin ﬁlm deposited from a stoichiometric target as a reference (measured at 551 C), highlighting the dramatic change in absolute resistance. Nanoscale Adv., 2021, 3, 6114–6127 \| 6123 © 2021 The Author(s). Published by the Royal Society of Chemistry View Article Online View Article Online Paper ew Article Online Nanoscale Advances Paper lms prepared from over-stoichiometric targets are shown in Fig. 11. The lm prepared from 3% Sr excess target exhibits bulk conductivities which are higher than the electronic ones of pseudo-intrinsic lms (and similar to ionic conductivities of those), but those are still far oﬀthe expected electronic bulk conductivity of polycrystalline SrTiO3. Hence, the improved cation stoichiometry is suﬃcient to strongly reduce structural deformations (c.f. the rocking curves in Fig. 9), but defect chemical non-idealities are still so large that orders of magni- tude lower electronic conductivities are found. This also shows that XRD curves have reduced sensitivity towards oﬀ- stoichiometries, i.e. 3.4 SrTiO3 lm tuning using diﬀerent target compositions In the case of the sample prepared from the 7% Sr excess, the bulk-like behaviour of the thin lm is also in agreement with the excellent stoichiometry revealed by ICP- OES. For the 5% Sr-excess the agreement between lm and bulk pellet is even a bit surprising since the exact stoichiom- etry is still not hit: A site deciency is present with a rather large Fe content (4.3% Fe). However, those deviations (Sr deciency and Fe excess) might partly counterbalance each other for example by Fe on the A site. Transition metal ions on the A site are, for example reported also for Mn.73 In any case, the rather ideal conductivity also ts excellent to the PALS experiment, where for 5% Sr excess no Sr vacancies, but instead associates of titanium vacancies and oxygen vacancies (VTiVO) are found, compared to the Sr vacancies for the thin lm prepared from a stoichiometric target (see Fig. 7). The conductivity of the lms with 11% Sr excess is again rather low, but still larger than that of pseudo-intrinsic SrTiO3 thin lms. In terms of activation energies a trend towards lower activation energies for bulk-like thin lms is observed and values of 1.38 0.04 eV, 0.99 0.01 eV, 0.91 0.01 eV and 1.29 0.01 eV are found for thin lms deposited from targets with 3%, 5%, 7%, and 11% Sr excess. For the sample deposited from a 7% excess target, the value perfectly matches literature data for grain conductivity.74 From this we can conclude that our bulk-like thin lms indeed behave like SrTiO3 bulk samples and that any deviation from ideal stoichiometry leads to a more or less pronounced increase in activation energy up to about half of the bandgap. Based on this interpretation, the high frequency arc is used to determine the bulk conductivity of these thin lms. For the other Sr excess cases, the spectra have also a high frequency arc with bulk-like capacitance, though partly only one additional interfacial arc is visible. The same type of analysis is thus per- formed also for the other lms. These bulk conductivities of the Fig. 11 The total conductivities of Fe:SrTiO3 thin ﬁlms deposited from targets with 2% Fe and diﬀerent Sr overstoichiometry are plotted in an Arrhenius diagram and compared with the electronic bulk conductivity of a polycrystal as well as the electronic intrinsic conductivity calcu- lated using ref. 3.4 SrTiO3 lm tuning using diﬀerent target compositions they may indicate severe deviations from the desired cation stoichiometry while absence of rocking curve shoulders cannot be taken as an indication of excellent stoi- chiometry or defect chemical ideality. that also a reinterpretation of the spectra is required for these Sr-excess lms. The transmission-line based rail model does not t the data anymore, and a physical meaning must be found for the appearance of an additional feature. pp The most straight-forward tting approach is to use a serial t. A t to three serial R-CPE elements works very well and the interpretation of the spectra is done based on the correspond- ing capacitances. (Please note that 8 to 9 free parameters were used in total, i.e. R1, T1, P1 (usually xed to one), R2, T2, P2, R3, T3, and P3 with P-parameters between 0.9 and 1.) The capacitance of the high frequency arc (visible in Fig. 10a) ts well to the geometrical capacitance expected for the entire SrTiO3 lm, i.e. it leads to a very reasonable permittivity in the range of 150.72 Hence, this arc is attributed to the total bulk conductivity of the corresponding SrTiO3 thin lms. The other two capacitances are about 10 and 20 times larger than the high frequency capaci- tance and thus correspond to SrTiO3 layer regions in the 10 or 20 nm range assuming bulk permittivity. Those are most probably interfacial space charges at the two electrodes, see also ESI 6.† Those come into play since the electronic conductivity is no longer at its lowest possible value (intrinsic). Accordingly, the ionic conductivity plays no longer a role in this interpreta- tion. Hence, the serial t is not only a parametrization of the spectra, but is to our understanding physically meaningful and therefore justied. For a detailed discussion on the error of the measurement and the t, see ESI 9.† Open Access Article. Published on 10 September 2021. Downloaded on 10/24/2024 6:21:48 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The lms prepared from 5% Sr and 7% Sr excess, however, show bulk conductivities very close to the expected bulk elec- tronic conductivity of pellets. This agreement supports the interpretation that the total bulk conductivity in the thin lm is now largely electronic, as in the bulk sample. Hence, stoi- chiometry deviations occurring during the deposition process seem to be largely counter-balanced by the Sr excess in the target. © 2021 The Author(s). Published by the Royal Society of Chemistry 4. Conclusions 3 R. Muenstermann, T. Menke, R. Dittmann and R. Waser, Coexistence of lamentary and homogeneous resistive switching in Fe-doped SrTiO3 thin-lm memristive devices, Adv. Mater., 2010, 22(43), 4819–4822. Fe:SrTiO3 thin lms prepared from stoichiometric targets show severe Sr deciencies which are quantied by ICP-OES, revealing about 10% nominal A-site deciency, but also enhanced Fe content. Oﬀ-stoichiometries are also clearly visible in RSM measurements by additional reections due to diﬀerent out-of-plane lattice constants (compared to single crystalline SrTiO3). Moreover, PALS measurements show the presence of Sr vacancies. The conductivity of such layers is many orders of magnitude lower than the electronic conductivity of the polycrystalline target material and almost perfectly matches the expected electronic conduc- tivity of ultra-high purity intrinsic SrTiO3 (with mid-gap Fermi level). Variation of PLD deposition conditions aﬀects the out-of-plane lattice constant measured in XRD measurements. However, chemical composition and conductivity of these lms are not substantially changed. The use of Sr-rich targets enhances the Sr content in the deposited lms and, especially, laser ablation of 7% Sr- excess targets results in lms with the correct A/B cation stoichiometry. Moreover, these stoichiometric lms exhibit conductivities almost matching the bulk conductivity of polycrystalline Fe:SrTiO3. Activation energies range from half of the band-gap (1.6 eV) for pseudo-intrinsic samples to the literature value for bulk-like samples (0.9–1 eV). The measurement of the conductivity is thus the most sensitive tool for nding the conditions for which stoichiometric lms can be obtained. Opposite, RSM and out-of-plane lattice parameter analysis (oen employed as single char- acterization) only indicates non-idealities for rather pronounced deviations from cation stoichiometry. 4 N. Raab, C. B¨aumer and R. Dittmann, Impact of the cation- stoichiometry on the resistive switching and data retention of SrTiO3 thin lms, AIP Adv., 2015, 5(4), 047150. 5 H. Zhang, L. Yan and H. U. Habermeier, Unusual ultraviolet photoconductivity in single crystalline SrTiO3, J. Phys.: Condens. Matter, 2013, 25(3), 035802. 6 R. Merkle, R. A. De Souza and J. Maier, Optically Tuning the Rate of Stoichiometry Changes: Surface-Controlled Oxygen Incorporation into Oxides under UV Irradiation, Angew. Chem., Int. Ed., 2001, 40(11), 2126–2129. 7 A. Viernstein, M. Kubicek, M. Morgenbesser, G. Walch, G. C. Brunauer and J. Fleig, High-Temperature Photochromism of Fe-Doped SrTiO3 Caused by UV-Induced Bulk Stoichiometry Changes, Adv. Funct. Mater., 2019, 29(23), 1900196. 8 G. Walch, B. Rotter, G. C. Brunauer, E. Esmaeili, A. K. Opitz, M. Kubicek, J. Summhammer, K. Ponweiser and J. There are no conicts to declare. There are no conicts to declare. 12 D. J. Keeble, S. Wicklein, L. Jin, C. L. Jia, W. Egger and R. Dittmann, Nonstoichiometry accommodation in SrTiO3 thin lms studied by positron annihilation and electron microscopy, Phys. Rev. B: Condens. Matter Mater. Phys., 2013, 87(19), 195409. 4. Conclusions Fleig, A solid oxide photoelectrochemical cell with UV light-driven oxygen storage in mixed conducting electrodes, J. Mater. Chem. A, 2017, 5(4), 1637–1649. 9 G. C. Brunauer, B. Rotter, G. Walch, E. Esmaeili, A. K. Opitz, K. Ponweiser, J. Summhammer and J. Fleig, UV-Light-Driven Oxygen Pumping in a High-Temperature Solid Oxide Photoelectrochemical Cell, Adv. Funct. Mater., 2016, 26(1), 120–128. 10 S. A. Chambers, Understanding the mechanism of conductivity at the LaAlO3/SrTiO3(001) interface, Surf. Sci., 2011, 605(13), 1133–1140. 3.4 SrTiO3 lm tuning using diﬀerent target compositions 40. Here, the increase in conductivity of the samples with 5% and 7% Sr overstoichiometry up to electronic bulk conductivity of a polycrystal can be seen. All in all, our study reveals the importance of the deposition process in SrTiO3 thin lms and the need for a characterization beyond XRD to ensure a proper stoichiometry and, therefore, functionality since a rather small deviations from the main cation stoichiometry strongly aﬀects the conductivity. Here, we propose an eﬃcient way for preparing stoichiometric Fe:SrTiO3 lms by using Sr-rich targets (i.e. non phase-pure targets). Since Fig. 11 The total conductivities of Fe:SrTiO3 thin ﬁlms deposited from targets with 2% Fe and diﬀerent Sr overstoichiometry are plotted in an Arrhenius diagram and compared with the electronic bulk conductivity of a polycrystal as well as the electronic intrinsic conductivity calcu- lated using ref. 40. Here, the increase in conductivity of the samples with 5% and 7% Sr overstoichiometry up to electronic bulk conductivity of a polycrystal can be seen. © 2021 The Author(s). Published by the Royal Society of Chemistry 6124 \| Nanoscale Adv., 2021, 3, 6114–6127 Nanoscale Advances View Article Online Nanoscale Advances View Article Online Paper Paper it is known that PLD layers may strongly vary between diﬀerent systems, the optimal Sr-excess found here might need read- justment in other equipment. Finally, a simple methodology for properly deciding whether a stoichiometric lm has been prepared or not seems to be only found in the measurement of the conductivity at elevated temperature, yielding drastically lower conductivities even for rather small deviations from the cation stoichiometry. Conﬂicts of interest 11 A. Ohtomo and H. Y. Hwang, A high-mobility electron gas at the LaAlO3/SrtiO3 heterointerface, Nature, 2004, 427(6973), 423–426. References 1 M. Kubicek, R. Schmitt, F. Messerschmitt and J. L. M. Rupp, Uncovering Two Competing Switching Mechanisms for Epitaxial and Ultrathin Strontium Titanate-Based Resistive Switching Bits, ACS Nano, 2015, 9(11), 10737–10748. 2 C. Lenser, A. Koehl, I. Slipukhina, H. Du, M. Patt, V. Feyer, C. M. Schneider, M. Lezaic, R. Waser and R. Dittmann, Formation and Movement of Cationic Defects during Forming and Resistive Switching in SrTiO3 Thin Film Devices, Adv. Funct. Mater., 2015, 25(40), 6360–6368. Open Access Article. 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W2099934717.txt	https://bbronline.com.br/index.php/bbr/article/download/262/399	en		Composition of the board and firm value of brazilian public companies	BBR. Brazilian Business Review	2,012	cc-by	8,233	v.9, n.3 Vitória-ES, jul.-sep. 2012 p. 71 - 93 ISSN 1808-2386 DOI:http://dx.doi.org/10.15728/bbr.2012.9.3.4 Composition of the board and firm value of brazilian public companies Enalto de Oliveira Gondrige† Finance Secretariat at State of Santa Catarina Ademir ClementeΩ Federal University of Paraná – UFPR Márcia Maria dos Santos Bortolocci Espejo¥ Federal University of Paraná – UFPR ABSTRACT The purpose of this paper is to investigate the relationship between the composition of boards of directors of Brazilian public companies and the firm value. The analysis is conducted by reference to a group of 208 Brazilian companies listed on Bovespa in the year 2008. The contribution of the study is done to assess the level of adherence to the recommendations of the Brazilian Institute of Corporate Governance (IBGC) regarding the composition of the board and its relation to market value. Using a multiple regression, three variables were studied: the level of board independence (Indep), accumulation of function by the Chief Executive Officer (CeoPowerful) and board size (Nmembros). The variable Nmembros was statistically significant, indicating positive correlation between corporate value and board size. Keywords: Corporate governance; board of directors; Tobin’s Q. Received in 03/15/2011; revised in 11/17/2011; accepted in 12/14/2011; published in 08/13/2012 * Corresponding authors : †. Master in Accounting at Federal University of Paraná ‐ UFPR Affiliation: Finance Secretariat at State of Santa Catarina Address: Rod. SC 401, Km 05, 4600, Florianópolis – SC - Brazil E‐mail: enaltogondrige@hotmail.com Telephone: (47) 3643‐6926 Ω Post‐doctorate at University of London Affiliation: Professor at Federal University of Paraná ‐ UFPR Address: Av. Pref. Lothário Meissner, 632 – 1º Andar, Jardim Botânico – Curitiba – PR – Brazil E‐mail: ademir@ufpr.br Telephone: (41) 3360‐4413 ¥ Doctorate in Controllership and Accounting at University of São Paulo Affiliation: Professor at Federal University of Paraná ‐ UFPR Address: Av. Pref. Lothário Meissner, 632 – 1º Andar, Jardim Botânico – Curitiba – PR – Brazil E‐mail: : marciabortolocci@ufpr.br Telephone: (41) 3360‐4193 Editor's Note: This article was accepted by Bruno Funchal This work is licensed under the Creative Commons License – Attribution-Non-commercial use-Sharing through the same license 3.0 Unported License. 71 72 Gondrige, Clemente, Espejo 1. INTRODUCTION he recent losses recognized by some Brazilian companies - Sadia, Aracruz, Votorantim - in operations with derivative financial instruments put the controllers of companies in crisis and investors on a collision course. These facts resume the discussion of agency conflicts - widely discussed by Jensen and Meckling (1976) - and of corporate governance mechanisms. These mechanisms result from the change in the relations of ownership and control. The board is a major mechanism of governance, whose main objective is to minimize agency problems which can arise through monitoring of executives (FAMA, JENSEN, 1983a, 1983b).This monitoring carried out by members of the board of directors is necessary to protect the interests of all shareholders (JENSEN, 1993). The board occupies a prominent position in the governance of corporations, performs the critical function of monitoring and advisory of managers. Andrade et al (2009, p. 6) point out that “the board of directors is seen as an agency which has the responsibility to decide on behalf of the owners. ".Common sense suggests that a higher level of board independence allows for a more effective monitoring and consequently, improves the performance of organizations (COLES, DANIEL; NAVENN, 2008). Although this importance and theme are widely studied, to the extent the boards act in the interests of shareholders and are effective in controlling agency conflicts between managers and shareholders, a question remains: what is the relationship between board structure and corporate performance? The essence of the discussion about corporate governance is based on the possible influence that the structure of corporate governance internal and external mechanisms - may have on the results of organizations. Several studies have documented that more independent boards (mostly outsiders) result in decisions more aligned to the interests or prospective of shareholders in performing various tasks such as hiring and dismissal of the CEO (WEISBACH, 1988), adoption of antitakeover measures (BRICKLEY COLES and TERRY, 1994), and compensation policies (BYRD, HICKMAN, 1992; COTTER; SHIVDASANI; ZENNER, 1997 apud COLES, DANIEL; NAVENN, 2008). Evidences from these studies on the effectiveness of the boards are quite varied. Some studies indicate that the boards of directors whose proportion of independent members is greater make decisions more aligned with shareholders' interests (HELLAND; SYKUTA, 2003; PANASIAN; PREVOST; BHABRA, 2004; ANDRADE et al, 2009). Other studies BBR, Braz. Bus. Rev. (Engl. ed., Online) Vitória, v. 9, n. 3, Art. 4, p. 71 - 93, jul.- sep., 2012 www.bbronline.com.br Composition of the board and firm value of brazilian public companies 73 have found no relationship or found a negative relationship between performance, market value (Tobin´s Q) and the composition of the board (YERMACK, 1996; EISENBERG; SUNDGREN; WELLS, 1998; BHAGAT; BLACK, 1999). The stock market crisis , in 2008 , created a source of tension - the relationship between boards of directors, investors, and controllers - in companies that suffer most from the decline in shares and , consequently, with the decline in its market value. In private firms the board is only a dramatic play in which the protagonists only ratify the decisions of the controllers. In public companies, the board has a duty to defend the interests of the business (JENSEN, 1986), or may personally respond for damages to shareholders. In that sense, the independence of the advice is recommended and requires good governance practice (AGRAWAL; KNOEBER, 1996). Among the recommendations of IBGC through the code of best corporate governance practices (CMPGC) the structure of the board is highlighted. Regardless of its form of incorporation, every organization must have a board of directors elected by the shareholders, whose main mission is to "protect and enhance the assets and maximize return on investment" (CMPGC, 2004, GAC p.18). Among the powers of the board, the definition of strategy, election and dismissal of the chief executive officer (CEO), the approval of choice or dismissal of other executives as proposed by the CEO, monitoring management and risks are noteworthy. These activities directly affect the generation of cash flows that consequently influence the value of the shares of companies. Thus, the research question that guides this study is: Is there any relationship between the structure of the board and the value of publicly traded Brazilian companies listed on Bovespa? This study aims at investigating if there is any relation between the structure of the board and the value of publicly traded Brazilian companies listed on Bovespa. As secondary objectives this study means to analyze the evolution of corporate governance practices, regarding the independence of the dimension of councils and the accumulation of office of president by the chief executive on the board. 2. THEORETICAL REFERENCE The literature review will include reflections on the separation of ownership and control, aspects of corporate governance, as well as its mechanisms, and the board. BBR, Braz. Bus. Rev. (Engl. ed., Online) Vitória, v. 9, n. 3, Art. 4, p. 71 - 93, jul.- sep., 2012 www.bbronline.com.br 74 Gondrige, Clemente, Espejo 2.1. Separation of ownership and control Jensen and Meckling (1976, p.308) define an agency relationship as "a contract in which one or more persons (the principals) hire another person (the agent) to perform some service on their behalf, which involves delegating some authority for the decision making of the agent”. The relationship between shareholders and managers of a company with diffuse ownership is a classic example of an agency relationship. According to Ross (1973), the agency relationship is the oldest and most common form of social interaction. Essentially all forms of contracts, such as the ones between employer and employee, between government and governed, contain important elements of agency. An agency relationship exists between two or more parties when one, named as agent, on behalf or as representative of another, known as the principal decides on behalf of the principal. Indeed, one is able to observe that the principal-agent relationship does not appear only in the firm. It may appear, for example, when a political representative is elected or when someone sends an attorney. However, such relationship is not present in all contracts of the firm, for example, contracts with suppliers and customers, but only those contracts whose related parties are the owners and executive managers (IUDÍCIBUS and LOPES, 2004). Thus, the agency relationship involves some degree of delegation of power from the principal to the agent for a decision on his behalf. Such relationship may indicate the pursuit of professionalism, since the owner delegates to the executive manager the commission of managing and of maximizing his resources. Jensen and Meckling (1976) argue that, if the parties act with the purpose of maximizing their personal utilities, there is good reason to believe that the agent does not always act on behalf of the interests of the principal. Such differences of interest can be mitigated through creation of appropriate compensation policies for the agent and also providing some form of monitoring of the activities of the manager. However, when the manager is the sole owner of the capital of an enterprise, there is no separation of ownership and control and, therefore, no agency problem between the manager and owner, since the two functions are unified. In contrast, when the property is diffused among many foreign investors, as in the case of most publicly traded corporations, the separation of ownership and control leads to the potential divergence of interests between owners and managers. 2.2. Corporate Governance BBR, Braz. Bus. Rev. (Engl. ed., Online) Vitória, v. 9, n. 3, Art. 4, p. 71 - 93, jul.- sep., 2012 www.bbronline.com.br Composition of the board and firm value of brazilian public companies 75 2.2.1. Concept and Definition of Corporate Governance Corporate governance, according to Shleifer and Vishny (1997), is the mechanism by which the investor ensures the return on invested capital. And yet, a set of constraints administrators apply to themselves or that investors apply on executives, in order to reduce the misapplication of/0} ex post resources and to encourage investors to invest more ex ante resources. According to La porta et al (2000, p.4), "corporate governance is the set of mechanisms through which investors (outsiders) protect themselves against the expropriation of executives (insiders)."In Brazil, Carvalhal-da-Silva and Leal (2007, GAC, p. 23) define corporate governance as "[...] mechanisms or principles that govern the decision making process within a company. Corporate governance is a set of rules that aim at minimizing agency problems. "Such problems occur when the interests between the executive and shareholders conflict. Although the executive must always act with the goal of maximizing shareholder wealth, there are situations where this does not occur, giving rise to the emergence of opportunistic behavior of the executive. 2.2.2. Scope of Corporate Governance According to Hart (1995, GAC p. 678), corporate governance is useful in a business if two conditions are present. First, there must be an agency problem or conflict of interests between members of the organization, maybe owners, managers, workers or consumers. Second, because transaction costs are such that the agency problem cannot be resolved by contract, i.e., there is the so-called incomplete contract. The author argues that, in the absence of agency conflict, all individuals associated with the organization can be instructed in order to maximize the wealth of the owners or the value of the company or minimizing costs. Individuals would be prepared to perform their activities regardless of how organizations conducted their activities. In that situation, financial incentives would not be necessary to motivate people. Nor would a governance structure be necessary to resolve differences which would not exist. 2.2.3. Mechanisms of Corporate Governance According to Jensen (1993), there are four control forces acting on the company to resolve the problems caused by conflict between the decisions of managers and BBR, Braz. Bus. Rev. (Engl. ed., Online) Vitória, v. 9, n. 3, Art. 4, p. 71 - 93, jul.- sep., 2012 www.bbronline.com.br those 76 Gondrige, Clemente, Espejo appropriate in view of society, namely: capital markets, legal and regulatory system, competitive market forces, and internal control mechanisms. By analyzing specific mechanism of corporate governance, there actually are two main issues. First, will the mechanism in question serve to align the interests of managers and shareholders - and, if so, how? Second, does the mechanism in question t have a significant impact on the performance or value of the company? Shleifer and Vishny (1997, pages 14-15) indicate that most of the evidence that managers do not always act in the interests of their shareholders comes from the many studies of events which have been conducted. In these studies, researchers evaluate whether there is an abnormal reaction in the stock price at the announcement of a particular type of event. If the reaction is, on average, significantly negative, then, this suggests that the measure is not, in general, interest of shareholders. This allows addressing the first question about whether a particular corporate governance mechanism reduces the distance between the interests of managers and shareholders. According to Denis (2001, p. 197) "whether a particular mechanism reduces the probability of a manager carrying out any measure that reduces the value of the shareholder share, so, this mechanism seems to align the interests of managers and shareholders, at least in some extent. " In view of the author, the same can be said if the stock price reaction to a particular event is positively related to the presence of a particular governance mechanism. Considering that the interests of shareholders , with respect to their investments , whether purely financial (HE and SOMMER, 2006), theoretically , if a mechanism aligns the interests between managers and shareholders, automatically they should also result in the maximization of shareholder investments (DENIS, 2001). However , it can be difficult to find empirical evidence consistent with this statement, even if this is true. Furthermore, evidence suggests that the relationship between mechanisms of corporate governance and firm value should be interpreted with caution as they may be spurious. The great challenge of these studies is the endogeneity (BORSCH-SUPAN and KOKE, 2000; HERMALIN and WEISBACH, 2003), which is presented as a curse or as a challenge, in empirical studies on corporate governance. 2.2.4. The Board of Directors First of all, we must reflect on one question: why are there boards of directors? According to Hermalin and Weisbach (2003), one possible answer is that they are, simply, a product of legal and regulatory system, resulting from imposition of a state or imposition of BBR, Braz. Bus. Rev. (Engl. ed., Online) Vitória, v. 9, n. 3, Art. 4, p. 71 - 93, jul.- sep., 2012 www.bbronline.com.br Composition of the board and firm value of brazilian public companies 77 stock exchanges by governance codes or different levels of governance. Most companies are required to have a board that brings together a large number of requirements: must have, in Brazil, at least three members; shall meet with some regularity; it may be necessary to have several committees; and a fraction of the directors must have some level of management independence. However, the statement cannot be an absolute truth. Boards of directors are prevalent throughout the world in a variety of commercial and non-profit organizations; more importantly, the existence of these boards precedes these regulations (HERMALIN and WEISBACH, 2003). In addition, Hermalin and Weisbach (2003, p. 9) state that "If the councils exist simply to satisfy regulatory requirements, they would represent costs to companies, which subsequently, through lobbying, the regulations would be eliminated, at least in some places in the world." However, the available evidence suggest otherwise. Legislation sets a minimum number of members. However, in practice, the boards are generally much larger than required by law. In this study, it was found that in 570 publicly traded companies, more than ninety percent of them have more than three members. Thus, if the existence of councils were simply the result of legal fictions, organizations would apply the minimum required by law. Given its prevalence over time in different organizational forms, there must be an explanation for the existence of boards other than merely the result of regulation. According to Hermalin and Weisbach (2003), the most plausible hypothesis is that boards are a solution to some organizational problems, determined endogenously in organizations and that would solve common agency problems in large companies. According to CMPGC (2004), regardless of its corporate form and being a publicly traded or private company, every organization must have a board of directors elected by the members, whose primary mission is to protect and enhance the assets and maximize return on investment. The independence of the board regarding the management of companies is one of the indispensable attributes and is related to the presence of outside directors. According Carvalhal-da-Silva and Leal (2005), an independent board requires good governance practice, since this board is responsible for evaluating the directors and replacing them, if it is the interest of shareholders. In the same sense, Hart (1995, GAC p. 5) states that management control is assured by the board. From the powers of the Board, the " [...] definition of strategy, the election and dismissal of the CEO, approval or waiver of the choice or dismissal of other executives , on BBR, Braz. Bus. Rev. (Engl. ed., Online) Vitória, v. 9, n. 3, Art. 4, p. 71 - 93, jul.- sep., 2012 www.bbronline.com.br 78 Gondrige, Clemente, Espejo proposal of the CEO, monitoring of management, risk monitoring and appointment and replacement of the independent auditors are noteworthy "(CMPGC, 2004, p. 11). According to Carvalhal-da-Silva and Leal (2005), the board size is an important control mechanism, since one of the tasks is to monitor the management of the company. Likewise, Jensen (1993, p. 34), argues that the board is the main factor of success of a system of internal control, and more than that, it sets the rules for the CEO and has final responsibility for the operation of the organization. In the evaluation of Jensen (1993) and Lipton and Lorsch (1992), boards with more than seven or eight members are less efficient than smaller boards. 3. METHODOLOGY In this section, the method and research procedures are presented, in order to answer to the following research question: Is there any relationship between the structure of the board and the value of publicly traded Brazilian companies listed on Bovespa? 3.1. Definition of the Population and Sample In this study, annual information concerning the Balance Sheets and Statements of Income, obtained from Economatica® database were considered. In addition, we considered the Reports of Annual Information (IAN) and the Quarterly Information Reports, which the companies issue to the Securities and Exchange Commission (CVM). Thus, we studied 19 companies in economic sectors, which are listed in Table 1. TABLE 1 - FREQUENCY OF COMPANIES PARTICIPATING IN THE RESEARCH PER SECTOR OF THE ECONOMY Sector Frequency % Sector Frequency % Agricultural and Fisheries 4 1.9 Pulp and Paper 4 1.9 Food and Beverages 16 7.7% Oil and Gas 3 1.4 Trade 10 4.8 Chemistry 10 4.8 Construction 23 11.1 Steel & Metallurgy 16 7.7% Electronics 5 2.4 Software and Data 1 0.5% Electricity 21 10.1 Telecommunications 11 5.3 Industrial Machinery 4 1.9 Textiles 15 7.2 BBR, Braz. Bus. Rev. (Engl. ed., Online) Vitória, v. 9, n. 3, Art. 4, p. 71 - 93, jul.- sep., 2012 www.bbronline.com.br Composition of the board and firm value of brazilian public companies 79 Mining 2 1.0 Transportation Services 9 4.3 Nonmetallic Mineral 3 1.4 Vehicles and parts 10 4.8 Other 41 19.7 Total 208 100 Note: The classification of companies in sectors took place according to criteria of Economática®. TABLE 2 - FREQUENCY OF THE COMPANIES PARTICIPATING IN THE RESEARCH PER SEGMENT Sector Frequency % Sector Frequency % Traditional Bovespa 82 39.4 Level 1 29 13.9 BDR 2 1.0 Level 2 9 4.3 Traditional OTC 1 0.5% New Market 85 40.9 Total 208 100 Note: The classification of companies in sectors took place according to criteria of Economática®. In September 2008, there were 536 companies listed on the São Paulo Stock Exchange, among which, only considering the manufacturing firms with data available until the third quarter of the year studied, 227 had data in the Economatica® database. After the collection and data analysis, we proceeded to the exclusion of companies which presented data are inconsistent or incomplete, resulting in a total of 208 companies. 3.2. Theoretical and operational definition of variables This study aims to investigate if the composition of the board relates to the market value of open capital Brazilian companies. The composition of the board acts as an independent variable, while the company's value acts as a dependent variable. The choice of variables - dependent, independent and control - has, as its foundation, studies of Panasian, Bhabra and Prevost and Bhabra (2004), Bhagat and Black (1999), Fuerst and Kang (2000), Yermack (1996), Coleman and Biekpe ( 2005), Cheng (2008), Silveira Barros and Fama (2003), Silveira (2004) and Carvalhal-da-Silva (2002). 3.2.1. Theoretical and operational definition of independent variables The Brazilian Corporate Law provides that the Board of Directors is composed of at least three members. While the code of best corporate governance practices recommends that BBR, Braz. Bus. Rev. (Engl. ed., Online) Vitória, v. 9, n. 3, Art. 4, p. 71 - 93, jul.- sep., 2012 www.bbronline.com.br 80 Gondrige, Clemente, Espejo the structure of such board should have between five and nine members, depending on the profile of the company. It is also recommend that there should be no concentration of power at the expense of proper management supervision, and the accumulation of functions of the chairperson and chief executive officer (CEO) should be avoided. Thus, the structure of the board in this study is defined by the use of three independent variables:  CEO occupying the position of chairman of the board - binary variable, in which: CeoPowerful = 1 if the CEO also occupies the position of chairman of the board. CeoPowerful = 0, if the position of chairman is not occupied by the chief executive.  Level of independence of the board: proxy (ratio of external members minus the ratio of internal members). This variable is set with basis on the study by Bhagat and Black (2001), Silveira, Barros and Fama (2003). Despite other studies only considering the total of external members, this methodology is used, , for being put into a comparative framework to the proportion of internal members.   EXT   INT  Indep      TOTAL   TOTAL   (1) Indep - Level of independence of the board; Total - Total of members of the board; Ext - the number of members who do not exercise executive function; Int - number of members who exercise executive function.  Size of the board - NMembros, variable represented by the ratio between the total number of members and assets of the company. This metric is used as an attempt to relativize the size of the board and the size of the companies. 3.2.2. Theoretical and operational definition of the dependent variable This study focuses on the possible relationship between the structure of the board and the market value of Brazilian companies. In order to verify such consistency, we use the Tobin´s Q indicator. Such indicator has been used in several governance studies, for example, Agrawal and Knoeber (1996), Yermack (1996) and Bhagat and Black (1999) Cheng (2008), Panasian, Prevost and Bhabra (2004), Faleye (2007) Silveira, Barros and Fama (2003), Silveira (2004) and Carvalhal-da-Silva (2002). According to Fama and Barros (2000), the Q is defined as the ratio between the market value of a company and the replacement value of its BBR, Braz. Bus. Rev. (Engl. ed., Online) Vitória, v. 9, n. 3, Art. 4, p. 71 - 93, jul.- sep., 2012 www.bbronline.com.br Composition of the board and firm value of brazilian public companies 81 assets and expresses the value of the company in a sense of performance, making it an indicator readily comparable from company to company. In this study, the square root of Tobin's Q is used as a proxy for the value of the corporation. Such transformation is necessary to correct the non-normality and the heteroscedasticity (Hair et al, 2005). Q Valor de Mercado das Ações  Valor de Mercado das dívidas Valor de Reposição dos ativos (2) Except for the value of shares, the remaining data are not directly observable. In the absence or inability to obtain such data, approximations of Tobin's Q are used. Fama and Barros (2000) suggest as alternative to "theoretically correct" methods, but of difficult practical application, the use of simplified methods as Chung and Pruitt (1994), Shin and Stulz (2000). In this study, we chose to use the Tobin's Q estimated by the Chung and Pruitt (1994, p. 72), model who define it as: Q de Tobin  (VMAO * Qtd)  (VMAP * Qtd)  DIV AT (3) In which: VMAO - Market value of common shares; VMAP - market value of preferred shares; DIV - book value of debt and long-term minus current assets, excluding the value of stocks; AT - Total assets of the company; QTY - Quantity of shares issued. 3.2.3. Control variables These variables are included in the model for exercising some level of influence on the dependent and independent variables. The following control variables were used:  Capital Structure: defined as the degree of leverage (Alav), i.e., the total financial debt over total assets of the company at the end of each financial year. BBR, Braz. Bus. Rev. (Engl. ed., Online) Vitória, v. 9, n. 3, Art. 4, p. 71 - 93, jul.- sep., 2012 www.bbronline.com.br 82 Gondrige, Clemente, Espejo  Level of corporate governance (Ngov): this dummy variable computes 1 for each company that is listed in one of the best levels of corporate governance practices of BOVESPA and 0 for companies that are not. 3.2.4. Model Specification With the objective of investigating the possible relationship between the structure of the board and the value of publicly traded companies with shares traded on the stock exchange in Sao Paulo and assess whether these companies follow the recommendations of IBGC, research estimated the following econometric model: Qi  0  1Indepi  2 NMembrosi  3CeoPowerfuli  4 Alavi  5 Ngov  i (4) In which: Q i - proxy for the market value of companies, ratio between the market value of debt and the replacement value of assets; 0 = Is the intercept of the regression model; Indep i - level of independence of the board; NMembros i - total number of board members relativized in relation to company size; CeoPowerful i - binary variable that identifies the presence or absence of accumulation of positions by the chief executive; Alav i - ratio between total debt and total assets; Ngov i - level of corporate governance, Dummy for companies with different levels.  i - random error term of the model. 3.3. Data Collection Data collection occurred secondarily through the websites of the São Paulo Stock Exchange by the system of External Disclosure ITR / DFP / IAN, the Securities and Exchange Commission, and the Economática® software. We collected information on the composition of the boards of directors of the Bovespa database in the third quarter of 2008, latest BBR, Braz. Bus. Rev. (Engl. ed., Online) Vitória, v. 9, n. 3, Art. 4, p. 71 - 93, jul.- sep., 2012 www.bbronline.com.br Composition of the board and firm value of brazilian public companies 83 information of each company, considering only the active members. The financial information that makes up Tobin´s Q proxy are accumulated values between the months of January through September of 2008. 3.4. Treatment of Data For the execution of this study we used: a) Jarque-Bera Normality Test: confirms if a data set follows normal distribution; b) Breusch-Pagan Homoskedasticity Test: checks if the error terms of the linear regression model have the same variance; c) F and t test; d) Multiple linear regression analysis. We adopt, for the interpretation of results, significance level α, of 5%, i.e., α = 0.05.Thus, when the p-value of a hypothesis test is less than the chosen value of α, the test procedure leads to rejection of null hypothesis (HILL, GRIGGITHS, JUDGE, 2006, p.119). 4. Results Analysis 4.1. Descriptive Statistics of board compositions The information concerning the structure variables of the board are summarized in the following tables. TABLE 3 - COMPOSITION OF THE BOARD OF DIRECTORS IN 2008 Minimum Maximum Average Standard Deviation Independence 0.40 1.00 0.8574 0.13893 Independence (int ratio- ext ratio) -0.20 1.00 0.7149 0.27786 Total Members 2 17 7.0817 2.75366 Tobim´s Q adjust .34 2.00 1.0168 0.33157 External 2 16 6.1971 2.86316 Internal 0 4 .8846 0.90418 BBR, Braz. Bus. Rev. (Engl. ed., Online) Vitória, v. 9, n. 3, Art. 4, p. 71 - 93, jul.- sep., 2012 www.bbronline.com.br 84 Gondrige, Clemente, Espejo Table 3, which summarizes the information of all member companies of the sample, one is able to observe that, on average, 85.74% of directors do not perform executive activities in the organization and that, on average, when considering the independence as the difference in ratio between external members and internal members, the percentage of independence decreases to 71.49%. The total membership of the board, on average, 7.08%, is within the range between five and nine members recommended by the code of best corporate governance practices of IBGC, as well as the size suggested by Lipton and Lorsch (1992) and Jensen (1993). These results are similar to those found by He and Sommer (2006), who found , in that study , that boards , on average , have 9 members , and the percentage of outsiders (nonfamily and non-executive) is, on average, 78%.Andrade et al (2009), analyzed 147 companies, in the period from 2004 to 2006, and showed similar results about the independence of boards. It was verified that, in our sample, on average, 88% of the size of the board is formed by independent persons. Studies Yermack (1996), Eisenberg, Sundgren and Wells (1998) studies, indicate a negative association between board size and corporate performance. According to Cheng (2008), this relationship is consistent with the view that coordination and communication tend to decrease as the board increases. Lipton and Lorsch (1992) argue that, normally, directors do not criticize the policies of the managers or do not impartially evaluate corporate performance. These problems are more evident on larger boards, because the efficiency in monitoring decreases the proportion of the size of the board. According to Lipton and Lorsch (1992, p. 14), when a board has more than ten members, it becomes harder for everyone to express their ideas and opinions in the short time available. Similarly, Jensen (1993, p.44) concludes that "when a board has more than seven or eight members, they are less likely to work effectively and are easier to be controlled by the CEO."When a board is larger, it is more difficult for the company to organize meetings and even harder to reach a consensus. Consequently, larger boards are less efficient and slower in decision making. Moreover, Cheng (2008) found an inverse relationship between board size and variability of corporate performance. BBR, Braz. Bus. Rev. (Engl. ed., Online) Vitória, v. 9, n. 3, Art. 4, p. 71 - 93, jul.- sep., 2012 www.bbronline.com.br Composition of the board and firm value of brazilian public companies 85 TABLE 4 - MATRIX OF PEARSON CORRELATION BETWEEN THE STUDIED VARIABLES Tobin's Q Tobin's Q Indep 1 Sig. NMembros CeoPowerful Alav NGov - 026 .290 - 041 .275 -.097 .705 .000 .553 .000 .162 1 -.063 -.566 -.079 224 .363 .000 .257 .001 1 .130 .493 -.279 .062 .000 .000 1 .055 -.153 .426 .027 1 -.315 Indep -.026 Sig. .705 NMembros .290 -.063 Sig. .000 .363 CeoPowerful -.041 .566 .130 Sig. .553 .000 .062 Alav .275 -.079 .493 .055 Sig. .000 .257 .000 .426 NGov -.097 .224 .279** -.153 * -.315 .000 Sig. .162 .001 Significant correlation at 0.01 (bilateral) * Significant correlation at 0.05 (bilateral) .000 .027 .000 1 The results show a positive correlation between the size of the board and Tobin's Q, indicating that the higher the board, the greater the value of companies. Among the ALAV and NMembros variables, there is a moderate positive correlation, indicating that more leveraged companies have larger boards. The Ceopowerful variable showed significant negative correlation to the level of independence, indicating that the larger the board, the lower the probability of accumulation of the chief executive position as chairman of the board, result quite similar to that found by Andrade et al (2009) .The NGov variable correlates positively with the Indep and NMembros variables, indicating that companies with different levels of governance have larger boards and higher proportion of members outside the organization. Those results should be carefully analyzed, since there are methodological differences regarding the metrics of the variables under study. The most relevant results indicate no correlation between Tobin's Q and the level of independence and positive correlation between Tobin's Q and board size. As the size of the board increases, business performance increases. Table 5, presents the recommendations of CMPGP (2004), regarding the composition of boards - size of the board from five to nine members, different persons holding the positions of CEO and chairman - and the level of adherence of Brazilian member companies of the sample. BBR, Braz. Bus. Rev. (Engl. ed., Online) Vitória, v. 9, n. 3, Art. 4, p. 71 - 93, jul.- sep., 2012 www.bbronline.com.br 86 Gondrige, Clemente, Espejo TABLE 5 - RECOMMENDATION OF IBGC Category Frequency % Councils with less than five members 3 14.90 Councils between five and nine members 143 68.8 Councils with more than nine members 34 16.30 Total 208 100 Different people in the position of Chief Executive and the Council Presidency 145 69.70 These results show that most companies follow the recommendations of CMPGC (2004), regarding the size of the board. It is found that 68.8% of companies follow such recommendation. In contrast, 14.9% of companies have boards with less than five members. With respect to the accumulation of CEO and chairman positions, the Brazilian legislation allows the functions to be performed by the same person. This study, evidences that, in 31.3% of the observations, the CEO and chairman are the same person. Ventura (2000) apud Leal and Oliveira (2002, GAC p. 23) found that this occurred in 41% of companies. One is able to observe a change in relation to the recommendation of different people performing this task. 4.2. Multiple Linear Regression Analysis 4.2.1. Verification of assumptions The standard linear regression model assumes that each error term is distributed, i.e., in a more compact normally manner i ~ N( 0, 2 ) , Gujarati (2006, p.88). With the objective of verifying this premise the Jarque-Bera normality test was taken. According to Sartoris (2003: GAC p. 253) this test is based on measures of skewness and kurtosis, under the null hypothesis that the residuals are normally distributed. The JB statistics converges, asymptotically, to a χ 2 distribution with two degrees of freedom. These results indicate that residues follow a normal distribution, thereby rejecting the event of a breach of the premise of the regression model. TABLE 6 - JARQUE-BERA TEST OF NORMALITY OF RESIDUALS X-squared DF 0.3711 BBR, Braz. Bus. Rev. (Engl. ed., Online) Vitória, v. 9, n. 3, Art. 4, p. 71 - 93, jul.- sep., 2012 2 p-value 0.8306 www.bbronline.com.br Composition of the board and firm value of brazilian public companies 87 Multicollinearity is present when there is high correlation between two or more explanatory variables (SARTORIS, 2003; GUJARATI, 2006). It is noted, in Table 4, that there is high correlation between the explanatory variables. According to Sartoris (2003, p.294) and Gujarati (2006, p. 285), another way to identify multicollinearity is to obtain a highly significant F test followed by little significant t-statistics for the coefficients. According to Gujarati (2006, p. 280), "even if multicollinearity is very high, as is the case of near multicollinearity, the ordinary least square estimators still guard the property of best nontendentious linear estimators." Still, according to the author, imperfect multicollinearity does not violate any assumption of the Classical Linear Regression Model (MCRL), and would only be a serious problem if the correlation coefficients between the regressors were greater than 0.8.Considering that none of the variables exceeded 0.8, this study assumes the absence of multicollinearity. According to Gujarati (2006, p. 314), the heteroscedasticity may result from the presence of divergent data, the omission of important variables for the model and also the intrinsic characteristic phenomena of an economic nature. The Breusch-Pagan test checks the null hypothesis that the variance of non-observable effects is equal to zero. When the variance of the error terms is increasing or fluctuating, it is said that the data are heteroscedastic (Table 7). TABLE 7: BREUSCH-PAGAN TEST OF HOMOSCEDASTICITY BP df p-value 10.9551 5 0.05228 According to Gujarati (2006, p.332), the Breusch-Pagan test converges, asymptotically, for a distribution of χ 2 chi-square (1-m) degrees of freedom. Thus, the critical value at 5% is 11.0705. So, as the calculated value is smaller, the hypothesis of homoscedasticity is not rejected. 4.2.2. Analysis of Regression Results As the scope of this study was to verify if the market value of publicly traded companies can be explained by the structure of the board, we performed multiple linear regression with 208 companies for the year 2008. According to Hair et al (2005, p.144)," the multiple regression analysis, a form of general linear modeling, is a multivariate statistical technique used to examine the relationship between a single dependent variable and a set of independent variables. "Still, according to the authors, the multiple regression method is BBR, Braz. Bus. Rev. (Engl. ed., Online) Vitória, v. 9, n. 3, Art. 4, p. 71 - 93, jul.- sep., 2012 www.bbronline.com.br 88 Gondrige, Clemente, Espejo appropriate when you want to objectively assess the degree and character of the relationship between dependent and independent variables. TABLE 8 - REGRESSION RESULTS BETWEEN TOBIN'S Q AND BOARD STRUCTURE Variable Intersection Coefficient Standard Error Statistic t P-value 1.0004865 0.0909786 -0.0806525 0.0973686 0.0020238 0.0007147 -0.0829700 0.0582072 Alav 0.1328700 0.0597238 2225 0.0272* Ngov 0.0107388 0.0487916 0220 0.8260 Indep NMembros CeoPowerful R-SQUARE 0.1162 ADJUSTED R-SQUARE 0.0935 10,997 <2e16 * -0828 0.4085 2832 0.0051 -1425 0.1556 Signif. codes: 0 ‘*’ 0.001 ‘’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 The coefficient of determination or explanation adjusted R2 is the descriptive measure of quality of model adjustment. In the case of multiple regression, the value of adjusted R2 represents the combined effect of any statistical variable in the prediction. In the present study, R2 is adjusted to 0.0935, indicating that 9.35% of the variation of the dependent variable is explained by the independent variables. The multiple linear regression model used five independent variables, and was estimated by ordinary least squares procedure. From the t tests associated with the parameters shown in table 8, it is evident that the significant variables in the model are the size of the board and leverage, with positive coefficients. This finding, despite being low correlation, differs from one part of the literature (Bhagat and Black, 1999, 2001; YERMACK, 1996; EISENBERG, SUNDGREN and WELLS, 1998; SILVEIRA, BARROS and FAMA, 2003), which states no relationship between board size and value of companies or have a negative relationship. However, this result, subject to the methodological differences in the construction of variables, is convergent with the results obtained by Andrade et al (2009), wherein one of the findings is that the total amount of directors was positively related to the value of companies market. BBR, Braz. Bus. Rev. (Engl. ed., Online) Vitória, v. 9, n. 3, Art. 4, p. 71 - 93, jul.- sep., 2012 www.bbronline.com.br Composition of the board and firm value of brazilian public companies 89 4.2.3. Analysis of Variance The F test is intended to test the joint effect of the explanatory variables on the dependent variable. This means verifying if at least one of the explanatory variables of the model effectively has, influence on the dependent variable. For a level of significance α = 0.05, the value of the calculated F statistic is 5.313, greater than the tabulated value of 4.39. In that case, as the calculated value is greater than the tabulated value, H0 is rejected, concluding, with a 5% risk, that there is multiple linear regression, i.e., the structure of the board can predict and explain the proxy (Tobin's Q) of market value of Brazilian companies (Table 9). TABLE 9 - ANOVA ANOVA Gl SQ Regression MQ . 5 2645 0529 Residue 202 20,112 0100 Total 207 22,757 F Test Significance of F 5313 0.0001308 5. CONCLUSIONS The main objective of this study was to investigate whether a relationship exists between the structure of the board and the value of publicly traded Brazilian companies listed on Bovespa. The variables that showed statistical significance were the size of the board and leverage, positively related to firm value. The study identified that the most valued companies tend to have higher number of directors and, on average, a higher percentage of independent directors. A variable of degree of independence was not statistically significant, results convergent with the one found by Andrade et al (2009). The variable CeoPowerful did not present statistical significant, convergent result with the one found by Andrade et al (2009), contradicting the results of Silveira, Barros and Fama (2003), as these authors presented results that indicate a greater appreciation of the companies which positions of chairman and CEO are held by different people. The presented results, although they find evidence, definitely do not provide conclusive evidence, considering the insignificance of some coefficients between the variables of governance and market value. Possible causes of this insignificance may be due to methodological limitations, once the study was developed for a relatively short timeline. BBR, Braz. Bus. Rev. (Engl. ed., Online) Vitória, v. 9, n. 3, Art. 4, p. 71 - 93, jul.- sep., 2012 www.bbronline.com.br 90 Gondrige, Clemente, Espejo However this research points out that the boards are part of the solution to agency problems of most organizations. Visualizing the boards from this perspective is the most useful way to study how they are structured and their function. As a suggestion for future studies, it is recommended to increase the period of analysis with a panel data approach, in order to capture the evolution of levels of adherence to the recommendations of IBGC. Also other variables may be included, for example, Brazilian companies that have ADRs abroad, which allow verifying if the structure of the board of those have significant influence. REFERENCES AGRAWAL, Anup; KNOEBER, Charles R. Firm performance and mechanisms to control agency problems between managers and shareholders. The Journal of Financial and Quantitative Analysis, v. 31, n. 3, p. 377-397, set. 1996. 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https://openalex.org/W4391724798	https://link.springer.com/content/pdf/10.1007/978-3-031-49196-2_7.pdf	English	null	A Case Study on DARPA: An Exemplar for Government Strategic Structuring to Foster Innovation?	International studies in entrepreneurship	2,024	cc-by	7,233	A Case Study on DARPA: An Exemplar for Government Strategic Structuring to Foster Innovation? Rodney H. Yerger Jr Abstract Advocates for a mission economy contend that government bureaucracy can be transformed through a strategic structuring that would improve upon the dynamic capabilities necessary to pursue and direct innovation. The Defense Advanced Research Projects Agency (DARPA) is touted as a model organization of strategic structuring for inducing public sector innovation of emerging technolo- gies. Applying economic theory and employing empirical analysis, I objectively examine key factors that are attributed to DARPA’s success, such as the organiza- tion’s autonomy, small size, and limited tenure of its program managers, in order to assess the worthiness of the agency’s exemplar status of empowering a mission- oriented approach to innovation. I ﬁnd that while DARPA undoubtedly provides value for national defense and has distinct advantages over other government organizations, it falls short in representing a sustainable and scalable source of strategic structuring that would beﬁt the entrepreneurial state. Keywords Public goods · Entrepreneurship · Innovation · Political economy Keywords Public goods · Entrepreneurship · Innovation · Political economy Keywords Public goods · Entrepreneurship · Innovation · Political economy JEL Codes H41 · L26 · O31 · P16 © The Author(s) 2024 M. Henrekson et al. (eds.), Moonshots and the New Industrial Policy, International Studies in Entrepreneurship 56, https://doi.org/10.1007/978-3-031-49196-2_7 R. H. Yerger Jr (✉) Department of Economics, George Mason University, Fairfax, VA, USA e-mail: ryerger@gmu.edu Introduction Advocates for a mission-oriented directionality to innovation tout the Defense Advanced Research Projects Agency (DARPA) as one model improvement within the public sector that provides the agility and ﬂexibility to pioneer revolutionary This chapter’s contents were mostly part of the author’s dissertation Analyzing the Effectiveness of State-Guided Innovation for the degree of Doctor of Philosophy at George Mason University, USA, 2023. Reused with permission. 109 R. H. Yerger 110 technology advancement (Mazzucato 2021). The purpose of this chapter is to execute a case study analysis of the DARPA organization, exploring its origins from 1958 and detailing changes in its focus and processes over time and how those changes track with its effectiveness at Schumpeterian entrepreneurship. The bulk of the case study describes and documents the institutional mechanisms that DARPA possesses to promote innovation. Much has been espoused regarding the success of the DARPA model in the form of various attributes (Gallo 2021 and DARPA 2016), which I categorize by the following three key factors: 1) Trust and autonomy. 2) Small size and the externalization of research. 3) Limited tenure and urgency. This research objectively analyzes each factor, applying economic theory to corroborate or counter the expected outcomes from DARPA’s purported strengths and defending these assessments empirically where possible. I ﬁnd that the organi- zation’s touted autonomy is unstable over time due to political transaction costs as evidenced by increased congressional oversight, shifting focus toward incremental technology advancement to fulﬁll short-term military priorities, and a transfer of expert power to established vendors. While DARPA has distinct advantages over other government organizations, it falls short in representing a sustainable and scalable source of strategic structuring. DARPA’s History and Construct Following the Soviet Union’s success in the space race with the launch of Sputnik, the Eisenhower administration established the Advanced Research Projects Agency (ARPA) in 1958, chartered with preventing “technological surprise” (Van Atta and Windham 2019a, pp. 3–4). The agency was initially focused on large missions such as missile defense and nuclear test detection with a brief foray in space-related technology development until that function was absorbed by the standup of the National Aeronautics and Space Administration (NASA). However, within a few years, ARPA assumed an additional role in pursuing a “set of smaller, technically focused programs” in areas such as materials science, information technology, and behavioral science (Van Atta and Windham 2019a, p. 4). These pursuits led to what is typically acclaimed the agency’s two greatest contributions to innovation: the precursor to the Internet and the foundation of personal computing. In 1972, because of increased scrutiny on military spending for many reasons including the unpopularity of the Vietnam War, the agency encountered its most signiﬁcant focus change when Congress limited research efforts to only those having direct military application. This not only resulted in the name change from ARPA to DARPA but also added increased process and oversight (Fong 2019). The effects of these process changes to DARPA’s purported strengths are explored in subsequent sections. 111 A Case Study on DARPA: An Exemplar for Government Strategic Structuring. . . DARPA continued to evolve and shift focus throughout the decades following its renaming, primarily aligning with changes in national security priorities, such as the Global War on Terror in the 2000s. Despite these shifts, the underlying organiza- tional mission has remained basically the same: “to prevent and create technological surprise” (Gallo 2021, p. 5; DARPA 2016, p. 4). DARPA asserts a commitment to achieve transformative research and development (R&D) with a stress on higher risks and higher rewards over incremental advances. To accomplish its mission, DARPA adheres to a process that externalizes research through an annual budget of approximately USD 3.5 billion to fund performers primarily from industry (62 per- cent in 2020), and secondarily from universities (18 percent in 2020), federal laboratories and research centers (15 percent in 2020), other nonproﬁts (4 percent in 2020), and foreign entities (1 percent in 2020) (Gallo 2021, p. 10). DARPA’s funding levels have stayed fairly constant over time. DARPA’s History and Construct So too has the agency’s manpower footprint, which is primarily composed of approximately 100 “empowered program managers coordinating high-risk high-reward external research” (Reinhardt 2020). This feature along with special hiring and contract authorities sets DARPA apart from other government agencies in terms of its independence, which advocates claim provides ﬂexibility for both ideas generation and enhanced engagement opportunities with potential performers. These elements of the DARPA model frame my case study approach in analyzing the three key factors that purportedly promote innovation. The ﬁrst factor is trust and autonomy. Factor 1: Trust and Autonomy DARPA’s autonomy stems from its explicit separation from the larger Department of Defense (DoD) to include the military services, which allows for disruptive tech- nology pushes beyond the constraints levied by speciﬁc military requirements and missions (Gallo 2021). This uncoupling represents a mitigation of the institutional constraints that drive median results in the government domain and obstruct Schumpeterian entrepreneurship (Schnellenbach 2007). Less checks and balances, especially the avoidance of excessive oversight from Congress, provides DARPA a level of opacity that promotes speed and ﬂexibility in decision-making, garners independence in problem-solving, and incentivizes risk-taking (Miller 1992; Rein- hardt 2020). Ter Bogt (2003, p. 151) connects the “autonomization” of a public organization to transaction cost economics (TCE); speciﬁcally, DARPA represents an “internally autonomized organization,” which stakes a claim in the lowering of economic transaction costs by limiting political inﬂuence. The trust and autonomy bequeathed by the DoD and Congress to DARPA also extends to within the organization from the agency director to the aforementioned empowered program managers, who can select and terminate projects through their ability to deploy money rapidly and independently (Gallo 2021; Reinhardt 2020). Thus, DARPA’s organizational structure consists of a unique combination of cen- tralized and distributed control mechanisms. Miller (1992) stresses that causes of 112 R. H. Yerger market failure such as information asymmetry and team production externalities lead to hierarchical solutions for social dilemmas. Moreover, disadvantages of democracy such as preference instability and indecisiveness and/or manipulation in decision- making lend favor toward centralizing power (cf. Arrow 1963). In DARPA’s case, autonomy has been purposefully granted to the agency director, and other stake- holders like the military services and Congress are restrained in their decision- making authority as it pertains to DARPA’s purview. Nonetheless, a hierarchy contains its own set of issues. Central planning efforts suffer from Hayek’s knowledge problem and what Tullock (2005 [1965], pp. 148–152) refers to as “whispering down the lane,” where agile coordination is constrained by the multiple levels of superior-subordinate interactions that impede knowledge diffusion and discourage entrepreneurship alertness and discovery. Miller (1992, p. 80) argues that ﬁrms can address these issues by injecting an additional level of autonomy within the organization via delegation: “. . Factor 1: Trust and Autonomy .a dictator who needs good information and good ideas must create the basis for independence inside the hierarchy.” The DARPA equivalency is delegating real shares of decision- making authority to program managers, who are hired from industry and academia and serve as experts in their speciﬁc domains of research within the ﬁelds of science and engineering (Gallo 2021). Provided the strengths of DARPA’s unique form of independence through the combination of centralized and distributed control governance structures, theoretical counters exist to this organizational construct’s stability in maintaining autonomy, which also calls into question the appropriateness of possessing high levels of opacity for inherently governmental entities. The ﬁrst counterpoint considers the overall agency level and its relationship to its external stakeholders. Because DARPA classiﬁes as an “internally autonomized organization,” it is neither truly independent nor private; therefore, political inﬂuence can still erode efﬁciency, at least over time. In attempting to incorporate TCE into the public sector domain, Ter Bogt (2003) proffers a political transaction cost framework to account for the lack of emphasis placed on economic efﬁciency in government organizations. This frame- work analyzes each of the primary characteristics of TCE as promulgated by Williamson (1981)—asset speciﬁcity, frequency and scale, and uncertainty—in order to assess the political willingness to increase or decrease an organization’s autonomy. According to Ter Bogt’s analysis, the willingness to “autonomize” will increase for basic government functions such as the provision of student loans or road maintenance. DARPA’s case is the opposite of basic functionality. Its product, innovation, involves high asset speciﬁcity in terms of uniqueness and importance and high uncertainty in terms of the frequency with which it can be produced and the ability to measure success. Furthermore, Ter Bogt’s (2003) framework considers additional political trans- action costs associated with maximizing electoral support, the inﬂuence of special interest groups, and political opportunism with a focus on increasing political efﬁciency for inherently governmental organizations. Applying these consider- ations, DARPA’s independence as an organization could be jeopardized by two key sources. The ﬁrst source consists of special interest groups working through the A Case Study on DARPA: An Exemplar for Government Strategic Structuring. . . 113 larger DoD and military services, who might desire to control the shape and direction of DARPA-related technology development efforts. This source includes large public-private partnership companies that perform a huge proportion of defense- related R&D. Factor 1: Trust and Autonomy The second source are the taxpayers, who typically demand the very checks and balances that have been removed through “autonomization” to ensure their money is being spent wisely and competently. The higher the level of opacity within an inherently governmental organization, the more difﬁcult the challenge to safeguard against abuses. Given that DARPA explicitly regards each program manager as ﬁlling the role of a technical subject matter expert, this high level of opacity can result in what Koppl (2018, pp. 189–200) refers to as a “rule of experts” scenario, where a monopoly of experts increases the likelihood of unreliability, which can lead to bad decision-making. Another critical counterpoint involves DARPA’s autonomy internal to the orga- nization residing with the individual program managers. Miller (1992, pp. 86–89) highlights the downside of distributed control governance as explained through the Sen paradox: “. . .any organization that delegates decision-making authority to more than one subset of individuals must suffer from either incoherent behavior or inefﬁciency for some combinations of individual preferences.” The tradeoffs given the Sen paradox involve the individual self-interest of each DARPA program manager and the agency’s best interest. Thus, distributed control can evolve into a threatening construct to both the dictator and external stakeholders. However, the DARPA model exhibits additional strengths purported to combat inefﬁciency in outcomes and intransitivity in preferences. The second and third key factors of my case study analysis elaborates further on these strengths. Factor 2: Small Size and Externalization of Research To avoid the Sen paradox, Miller (1992, pp. 94–95) contends that the hierarchy must “shape and mold individual preferences into patterns that are mutually consistent.” One way DARPA mitigates the threat of incoherent behavior and inefﬁcient coor- dination is through its small manpower footprint. DARPA’s core staff size gravitates toward Dunbar’s number (~150), which is the suggested limit at which social relationships ﬂourish as each member can get to know every other person in the organization. Knowing everyone creates peer pressure through scrutiny, which pro- vides a check against abusing opacity and fosters an adherence to a common set of goals (Dunbar 1992; Reinhardt 2020). Remaining small in size may also help counter external threats to DARPA’s independence from special interest groups and the taxpayer. By staying below the radar, DARPA might avoid targeting for predation and regulation despite the higher political transactions costs associated with extremely uncertain and disruptive innovation efforts. DARPA maintains its small footprint by externalizing research, which is pro- moted as another strength of its governance model. The agency avoids the high transaction costs involved in obtaining the unique knowledge and equipment 114 R. H. Yerger required in pursuing groundbreaking research. DARPA does not establish its own labs or the bureaucracy involved in managing them (Cummings 2018; Reinhardt 2020). Instead, it outsources these assets through discrete project funding that yields a lower overhead and streamlines accountability by ensuring each project is respon- sible to one person, the program manager (Reinhardt 2020). Despite the perceived advantages of DARPA’s small size and externalization of research, there may also exist associated drawbacks. Overcoming the Sen paradox by internally streamlining preferences might restrict a sense of competition among independent program managers and instead promote expert failure by enhancing synecological bias through motives that Koppl (2018) argues are inherent in max- imizing expert utility. These motives include identiﬁcation that is tied to a common mission as well as a sympathy for and a desire to please fellow experts. Moreover, even though the organization’s small footprint might help to ward off threats to predation, it increases the detrimental effects of politicization should the willingness to decrease autonomy dominate as predicted by Ter Bogt’s political transaction framework. Factor 3: Limited Tenure and Urgency Congress grants DARPA special privileges in hiring and contracting authority. Speciﬁcally, DARPA can directly and expeditiously hire science and engineering experts from industry and academia for term appointments, typically 3 to 5 years. DARPA’s special contracting authority lowers the transaction costs of the govern- ment acquisition process in not only bypassing burdensome procurement regulations to develop ﬂexible agreements with R&D performers but also by empowering the program manager to reprioritize and reallocate funds based on performance (Gallo 2021; DARPA 2016). These authorities give DARPA distinct advantages through the motivation of active program management and ideas generation as well as in providing a counter to the Sen paradox. Limited tenure encourages program managers to take risks in funding ideas for short-term durations but with a long-term view in mind, where both the need and value proposition are uncertain (Bonvillian et al. 2019; Gallo 2021). The hiring process sets expectations upfront that the program manager position is not career oriented. Excelling in the position will not result in a promotion within the organi- zation, and funding unsuccessful long shot ideas will likely not adversely impact one’s career (Reinhardt 2020). To achieve long-term impact, program managers seek ambitious project ideas and tolerate associated failures as “the cost of supporting potentially transformative or revolutionary R&D” (Gallo 2021, p. 6). However, checks are inherent in the DARPA process that attenuate the effects of failure via the short-term funding of seedling projects, which allows the program manager to track progress and terminate and redeploy funding for those projects that underperform (Van Atta and Windham 2019a). In this manner, while DARPA externalizes research, it bears the risk for the performer, which advocates insist is a major advantage over private sector venture capitalism. Furthermore, DARPA can also bear the risks for other funding mecha- nisms by signaling technology validation, which encourages larger industry per- formers to front their own money or other government entities like the National Science Foundation to provide grants to continue development (Reinhardt 2020). In addition to incentivizing risk taking via active program management, limited tenure creates constant turnover of personnel (~25 percent per year) that should ideally result in a continued infusion of ideas. Not only does this turnover model help with new idea generation but also allows a revisiting of old ideas that might have been tried previously and failed. Factor 2: Small Size and Externalization of Research If all program managers are aligned tightly with DARPA’s director, absent bureaucracy, politicization of the director could lead to a prioritiza- tion of goals and efforts entirely dictated by external forces rather than the organi- zation’s stated mission (Reinhardt 2020). An intentional restriction in size also shapes broader ramiﬁcations for Mazzucato’s vision of strategic structuring that calls for a replication of the DARPA model to induce the entrepreneurial state. Breznitz and Ornston (2013, p. 4) argue that bastions of successful public sector entrepreneurship will more likely “occur at the periphery of the public sector, in low-proﬁle agencies with relatively few hard resources and limited political prestige.” They cite DARPA as a peripheral organization that does not suffer from the political interference found with a larger and “centrally positioned” agency. These strengths pose a signiﬁcant challenge in attempting to scale the DARPA model in order to achieve a vision of transforma- tional value creation by the public sector. Finally, there are disadvantages in externalizing research that involve tradeoffs in transaction costs. While DARPA avoids the high overhead costs associated with providing its own labs and equipment, it incurs the costs of ﬁnding and establishing relationships with appropriate and competent performers and ensuring that these performers produce value on time and on budget. These costs involve large under- takings, which typically require hierarchical control to monitor and prevent shirking (Reinhardt 2020). Koppl (2018) argues that synecological redundancy is a key tenant in mitigating expert failure. Instead, the DARPA model relies on a lone program manager tasked with multiple ventures, which exacerbates the risk of unreliability due to expert error to include making unintentional or “honest” errors given the limited cognition of an expert’s bounded rationality. Therefore, by outsourcing its potentially transformative research efforts, DARPA might ﬁnd it tempting or even necessary to outsource the centralized control mechanisms required to produce such results. Such requirements can limit research partnerships to larger, more mature companies and increase the likelihood of rent-seeking behavior. Nev- ertheless, the DARPA model provides a check against these alleged disadvantages A Case Study on DARPA: An Exemplar for Government Strategic Structuring. . . 115 by motivating active program management, which involves the third key factor of my case study analysis. Factor 3: Limited Tenure and Urgency Subtle tweaks to an old idea or simply the timing and environment in which the idea reemerges may result in improved outcomes that would not have otherwise materialized had the organization preserved the memory of past naysayers (Gallo 2021). 116 R. H. Yerger A ﬁnal advantage of limited tenure is that along with the aforementioned small manpower footprint, DARPA’s hiring ﬂexibility provides a counter to the Sen paradox associated with distributed control governance mechanisms. The DARPA director can shape coherent behavior by hiring similarly minded and motivated subordinates with preferences that align to the DARPA mission of creating and preventing technological surprise. As with the other key factors, there exist theoretical counterpoints to the pur- ported beneﬁts of DARPA’s limited tenure and ﬂexible hiring policies. An obvious drawback to excessive risk taking is that associated failures are a cost to the taxpayer and moreover, could result in destructive entrepreneurial outcomes. While logic supports the need to tolerate failure when pioneering disruptive technology advance- ment, understanding the returns to such efforts via cost-beneﬁt analysis remains an appropriate consideration. This includes taking into account the costs in revisiting or duplicating old ideas that simply will not work despite the fact that program manager turnover reinvigorates their appeal (Gallo 2021). Furthermore, while limited tenure may motivate risk-taking, it cannot completely displace familiarity bias, which inﬂuences agents to invest in and with those they trust (Reinhardt 2020). In the case of the DARPA program manager, this bias might result in allocating funding to those researchers with sound and stable reputations over less mature, smaller enterprises, which runs counter to Schumpeterian entrepreneurship. a With regard to ﬂexible hiring practices, the methods DARPA uses to streamline preferences and foster coherent behavior do not fully embrace the theoretical underpinnings required in overcoming the Sen paradox. As government employees, neither DARPA program managers nor the director are residual claimants, which is a striking difference between public sector entrepreneurs and venture capitalists. The standard solution to address the agency problem caused by decision managers not being residual claimants is via compensation that accurately reﬂects performance in the overall market for management (Fama 1980). Miller (1992, pp. 100–101) stresses that the streamlining of preferences via socialization is insufﬁcient because adverse selection causes measurement error in determining the potential ﬁt of candidate for hire. Instead, the most effective means of “reconciling transitivity, efﬁciency, and delegation” is through the compensation system. Empirical Analysis The next step of my case study analysis explores quantitative and qualitative evidence that bolsters either the points or counterpoints described above regarding the three key factors of the DARPA model. First, regarding independence, ample evidence exists that DARPA has become less autonomous over time, which is an indication that political transaction costs have inﬂuenced the willingness of political actors to tolerate a high level of opacity. Starting with the transition of ARPA to DARPA in 1972, increased oversight has inﬂuenced how DARPA spends its money. Lump sum authorization of funding by Congress has shifted to demanding annual budgets for each program that include a description of the work to be performed. Despite DARPA’s streamlined processes over other government institutions, grants for seedling projects must still go through an open and involved solicitation process. As a result of orienting DARPA’s work more to the needs of the military to counter existing threats, DoD has shaped and dictated shorter-term areas of R&D efforts to support active conﬂicts such as the Vietnam War in the 1970s and Global War on Terror in the 2000s. Finally, and perhaps the biggest example of increased politici- zation, the appointment of DARPA directors is now aligned with presidential administrations (Reinhardt 2020). Regarding the pros and cons of organizational size, DARPA has maintained a relatively small manpower footprint over time. In remaining small and ﬂat, DARPA has successfully resisted Parkinson’s Law, a crucial contributor to bureaucratic inefﬁciency where success is measured by the growth in the number of subordinates under a director’s control (Tullock 2005 [1965]). However, evidence exists that DARPA’s externalization of research suffers from the high transaction costs involved in searching for competent researchers and monitoring performance. In 2001, DARPA started awarding prime contracts almost exclusively to “established vendors,” which relegated universities and start-up ﬁrms into a teaming concept that reports through the prime contractor (Fuchs 2010, p. 1138). Sound reasons exist for the shift in awarding prime awards to established vendors. Fuchs (2010) cites the decline of corporate R&D labs over time as responsible for raising the transactions costs. A Case Study on DARPA: An Exemplar for Government Strategic Structuring. . . A Case Study on DARPA: An Exemplar for Government Strategic Structuring. . . Factor 3: Limited Tenure and Urgency While DARPA’s unique status allows for the authorization of higher salaries than compared to other government agencies, a pay gap certainly exists between similarly skilled private sector counterparts in the science and engineering communities. Consequently, DARPA must depend on the aforementioned personal gain incentives. A ﬁnal concern exists with the overall concept of active program management, which has sparked debate over the beneﬁts of DARPA’s changes to process over time. In the days of ARPA (1958–1972), program managers exercised less control over the efforts of performers, while maintaining responsibility of overall vision and funding (Worrydream 2017; Kleinrock 2014). Tracking progress and performance via standard program management techniques can focus too much priority on near- term results and derail long-term vision (Cummings 2018). This focus is bureau- cratic in nature, which ironically is what DARPA is chartered to avoid. 117 Empirical Analysis An established vendor can better perform the systems management necessary to see technology advancement through to production and thereby avoid “the Valley of Death.” Conversely, the relegation of start-ups to a supporting role in the DARPA process is concerning considering the view that newer entrepreneurial ﬁrms are the linchpin for breeding successful innovation because of ownership incentives and information advantages (Karlson et al. 2021). Further- more, the dependence on larger, more mature companies to provide the hierarchal control mechanisms for the externalization of research increases DARPA’s vulner- ability to rent seeking by special interests, which directly stunts productive entre- preneurial opportunities. In a sense, DARPA’s arrangement with established vendors might represent a transfer of expert power from the program managers to the large industry R&D performers. Koppl (2018) proffers an information choice theory model of an R. H. Yerger 118 epistemic system utilizing a sender-receiver game construct. As applied to DARPA following the shift in awarding prime contracts to established vendors, the program manager now represents the receiver (or nonexpert) beholden to a monopoly of senders (or experts) as represented by the large defense contractors. The receiver grows more powerless as rivalry among senders is reduced. Not only does this lack of rivalry increase synecological bias, but the intentional relegation of start-up companies also restricts free entry, which Koppl cites as a key contributor to expert failure: “‘Potential competition’ is more important than the number of incumbent competitors” (Koppl 2018, p. 205; cf. Baumol 1982). DARPA’s adherence to active program management might offset the increased likelihood of expert failure and vulnerability to rent seeking caused by the shift in contracting strategy. Anecdotal evidence supports the view that DARPA program managers have a healthy tolerance for failure. Over DARPA’s history, project losers ranging from research into paranormal activity to developing mechanical elephant transports to more recently, testing rapid space launch capabilities have showcased a willingness to try out challenging and quirky ideas (Gallo 2021). Of a more quantitative nature, Goldstein and Kearney (2017, 2020) conducted studies measur- ing past project selection and performance for ARPA-E, the Department of Energy’s transformational R&D organization, which can serve as a proxy for DARPA. Goldstein and Kearney (2017) ﬁnd that ARPA-E program managers exercise auton- omy via their tendency to select projects for funding that receive less consensus from external peer reviews. Empirical Analysis Furthermore, Goldstein and Kearney (2020) ﬁnd that program managers do not shirk from playing an active role in the management of their portfolio by frequently redeploying money to increase funding for stronger performing projects and decreas- ing or terminating funds for those that perform weakly. In this manner, they are exercising real options similar to the way venture capitalists monitor their invest- ments and unlike the hands-off approach that other public sector entrepreneurial mechanisms such as the Small Business Innovation Research (SBIR) program take via the provision of grants. In terms of the effectiveness of DARPA’s ﬂexible hiring practices, compensation gaps between program managers’ salaries and their private sector counterparts loom as a signiﬁcant concern. Reinhardt (2020) estimates that experienced scientists and engineers at large tech companies receive at least twice as much compensation, whereas this gap was much less severe (~20 percent) in the 1960s during the days of ARPA. The commercial high-tech sector promises to be even more competitive going forward, which may not bode well for attracting top talent to a position that entails no promotion and requires relocation to Washington, DC. In analyzing possible frictions between DARPA’s dual roles in executing trans- formative R&D and responding to threat-based time-sensitive challenges for the military, a review of DARPA’s history tells a tale of two different organizations. The ﬁrst tale involves the ARPA years from 1958–1972, when Congress and DoD exercised much less oversight over the agency and the program managers exercised much less oversight over research performers. One of the earliest DARPA directors, Jack Ruina, “valued scientiﬁc and technical merit above immediate relevance to the A Case Study on DARPA: An Exemplar for Government Strategic Structuring. . . 119 military” and delegated a high level of autonomy to his program managers (Fuchs 2010, p. 1137). The best example of this delegation involves one of the organiza- tion’s greatest successes, the R&D that led to the advent of the Internet and personal computing. J. C. R. Licklider, the program manager for these efforts, advanced an ambitious vision that foresaw computers serving as “interactive intellectual ampli- ﬁers for all humans, pervasively networked worldwide” (Worrydream 2017, para 14; Kleinrock 2014). This vision was only loosely connected to solving command and control challenges for national defense, and it did not entail a speciﬁc set of goals nor a roadmap. Empirical Analysis Instead, Licklider leveraged the power of his vision to ﬁnd and organize an impressive network of researchers and sustain investments in the underlying technologies to achieve success (Van Atta and Windham 2019b, pp. 39–40; Bonvillian 2019, pp. 94–98). It is important to note that ARPA’s considerable level of independence did not always result in productive entrepreneurial outcomes. Project AGILE supported combat operations in Vietnam and involved mismanaged efforts to improve weap- onry, which included chemical agents. The project was an unmitigated disaster, which led to the conviction of the program manager, William Godel, for embezzle- ment. Yet, because of its covert nature, the project avoided scrutiny allowing it to survive for over a decade (Van Atta and Windham 2019a; Reinhardt 2020). This example of a destructive entrepreneurial outcome calls into question the sustainabil- ity of unfettered independence for inherently governmental organizations, which provides a convenient segue to the second tale of DARPA. The shift from ARPA to DARPA in 1972 increased oversight and focused the organization’s efforts more directly on military application. By 1975, DARPA’s new director, George Heilmeier, instituted what became known as the “Heilmeier Cate- chism,” which was the genesis of active program management. Heilmeier inﬂuenced more of a top-down and mission-oriented approach for the management of projects that involved setting intermediate and long-term goals, tracking progress, and estimating the costs and beneﬁts of each research effort as it pertained to the customer (Van Atta and Windham 2019a, pp. 14–15; Fong 2019, pp. 193–194; Cheney and Van Atta 2019, pp. 233–234). Although active program management mitigates the risks of longer-term, highly uncertain technology advancement efforts and increases the success rate of technology transition, it also entails greater costs to autonomy and disincentives toward risk-taking over ARPA’s more vision-oriented approach. The ultimate empirical evidence in evaluating the effectiveness of DARPA over time would be to accurately measure return on investment in terms of innovative output. Attempts at measuring patents per award and funding per patent illustrate that DARPA performs considerably well compared to other government agencies; however, these cannot be considered apples-to-apples comparisons given the varied charters and missions of these agencies, nor do these assessments address the more important question as to how well DARPA performs compared to the private sector (Piore et al. 2019, pp. 49–52). Empirical Analysis pp Reinhardt (2020) reviews the agency’s own advertised accomplishment timeline and bins what he refers to as “outlier successes” into two categories: pre-1972 R. H. Yerger 120 (ARPA) and post-1972 (DARPA). An outlier success can be considered synony- mous with architectural innovation, which disrupts and creates markets while also outmoding existing competencies (Abernathy and Clark 1985). The results of Reinhardt’s binning excursion reveal that the vast majority (over 70 percent) of DARPA’s architectural innovation occurred during the ARPA years. The ramiﬁca- tions of this revelation do not detract from the value DARPA has provided and continues to provide to its single customer, the military; albeit this value is harder to appreciate given its speciﬁc military utility and narrow applicability. Conclusion This chapter analyzed the institutional mechanisms of DARPA as a model for strategic structuring that fosters Schumpeterian public sector entrepreneurship. In reviewing three key factors that expound the DARPA model, I explored theoretical points and counterpoints that make for a complex and inconclusive assessment as to the potentiality of DARPA’s distinctive form of organizational governance in ful- ﬁlling the vision of an entrepreneurial state. Through a unique combination of centralized and distributed control mecha- nisms, DARPA possesses a higher level of autonomy, at least compared to other government organizations; however, I ﬁnd this autonomy to be unstable. Political transaction costs associated with state-guided innovation efforts decrease the will- ingness to autonomize, which erodes independence via three discrete sources. First, concerns from the taxpayer over abuses to opacity and expert failure have led to more congressional oversight over time. Second, vulnerability to rent seeking by special interests has increased, which is evidenced by a transfer of expert power to and a growing dependence on established vendors to provide the hierarchal control mechanisms for the externalization of research. Third, pressures from external stakeholders such as the military have inﬂuenced a greater focus on shorter-term military or administration priorities, which can incentivize technology transition over risk-taking. While DARPA is better equipped than others to ward off threats to its autonomy through such advantages as ﬂexible hiring practices and special contract authorities, its model depends on employing highly competent and moti- vated program managers, and yet, subsequently cannot depend on compensation to overcome the residual claimant agency problem. My research reveals that the vast majority of DARPA’s architectural innovation occurred prior to the critical shift from ARPA to DARPA in 1972, which was a time characterized by much less external oversight and a much lower pay gap between government and private sector high-tech labor. It is important to note, however, that this correlation between ARPA’s greater autonomy and innovation success should not imply causation. Another factor at play could be the characteristics of the post- World War II era, or perhaps more speciﬁcally, the height of the Cold War, which involved a level of crisis that dictated a demand for rapid and novel change and raised alertness to entrepreneurial opportunities. Indeed, DARPA’s founding is A Case Study on DARPA: An Exemplar for Government Strategic Structuring. . . Conclusion 121 steeped in a collective mobilization across the public sector domain to counter the crisis of technological surprise. Since that time as the Cold War diminished, prepar- ing for “system-level war” shifted toward a focus on responding to “shorter-term tactical missions.” Ruttan (2006, pp. 183–184) contends that the absence of a major war, or at least the threat of one, diminishes the probability that our political system could generate the willpower and resources “required to initiate and sustain the development of major military and defense-related general-purpose commercial technologies of the past.” Another crucial concern in assessing DARPA as a model for Mazzucato’s strategic structuring vision is its scalability. Even if DARPA can effectively sustain a resistance to political interference, this would be attributed to its small footprint and its existence as a peripheral organization. The fact that DARPA’s disruptive technology efforts can threaten status quo defense acquisition processes, which can drive opposition within the military, does not support the claim that the high-risk, high reward approach inherent in Schumpeterian entrepreneurship could expand to transform large areas of the government. Even attempts at cloning DARPA for the sake of establishing other peripheral organizations dedicated to long-term revolu- tionary R&D have met with resistance and limited success. For example, despite consultation on adopting the strengths and processes of the DARPA model, ARPA- E suffers from greater hierarchical control both internally and externally. Within the organization, the program managers are outnumbered by support staff, which entails a higher level of process-driven activity. External to the organization, ARPA-E is directly funded by the Department of Energy instead of Congress, which threatens independence of basic functions such as program selection and idea generation (Fuchs 2009; Reinhardt 2020). In conclusion, DARPA undoubtedly provides value to the defense of the United States and has generated productive public sector entrepreneurial outcomes. How- ever, the agency falls short in representing a sustainable and scalable source of strategic structuring that would beﬁt the entrepreneurial state. Bonvillian, W. B. (2019). The connected science model for innovation—The DARPA Model. In W. B. Bonvillian, R. Van Atta, & P. Windham (Eds.), The DARPA Model for Transformative Technologies: Perspectives on the U.S. Defense Advanced Research Projects Agency (pp. 77–118). Cambridge, UK: Open Book Publishers. Abernathy, W. J., & Clark, K. B. (1985). Innovation: Mapping the winds of creative destruction. Research Policy, 14(1), 3–22. y Baumol, W. J. (1982). 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Some excerpts from recent Alan Kay emails. http://worrydream.com/2017-12- 30-alan/ 30-alan/ Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. g The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
https://openalex.org/W4388775844	https://journals.plos.org/ploscompbiol/article/file?id=10.1371/journal.pcbi.1010845&type=printable	English	null	AimSeg: A machine-learning-aided tool for axon, inner tongue and myelin segmentation	PLOS computational biology/PLoS computational biology	2,023	cc-by	12,334	PLOS COMPUTATIONAL BIOLOGY PLOS COMPUTATIONAL BIOLOGY Pau Carrillo-BarberàID1,2,3,4☯, Ana Maria Rondelli5,6☯, Jose Manuel Morante-Redolat1,2,3, Bertrand VernayID5,7, Anna Williams5,6, Peter BankheadID4,8* Pau Carrillo-BarberàID1,2,3,4☯, Ana Maria Rondelli5,6☯, Jose Manuel Morante-Redolat1,2, Bertrand VernayID5,7, Anna Williams5,6, Peter BankheadID4,8* 1 Centro de Investigacio´n Biome´dica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Universitat de València, Valencia, Spain, 2 Departamento de Biologı´a Celular, Biologı´a Funcional y Antropologı´a Fı´sica, Universitat de València, Valencia, Spain, 3 Instituto de Biotecnologı´a y Biomedicina (BioTecMed), Universitat de València, Valencia, Spain, 4 Centre for Genomic & Experimental Medicine, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, United Kingdom, 5 Centre for Regenerative Medicine, Institute for Regeneration and Repair, University of Edinburgh, Edinburgh BioQuarter, Edinburgh, United Kingdom, 6 MS Society Edinburgh Centre for MS Research, Edinburgh BioQuarter, Edinburgh, United Kingdom, 7 Centre d’imagerie, Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire CNRS UMR 7104—Inserm U 1258, Illkirch, France, 8 Edinburgh Pathology and CRUK Scotland Centre, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, United Kingdom ☯These authors contributed equally to this work. * pau.carrillo@ed.ac.uk (PC-B); anna.williams@ed.ac.uk (AW); p.bankhead@ed.ac.uk (PB) ☯These authors contributed equally to this work. * pau.carrillo@ed.ac.uk (PC-B); anna.williams@ed.ac.uk (AW); p.bankhead@ed.ac.uk (PB) ☯These authors contributed equally to this work. ☯These authors contributed equally to this work. * pau.carrillo@ed.ac.uk (PC-B); anna.williams@ed.ac.uk (AW); p.bankhead@ed.ac.uk (PB) Abstract Citation: Carrillo-Barberà P, Rondelli AM, Morante- Redolat JM, Vernay B, Williams A, Bankhead P (2023) AimSeg: A machine-learning-aided tool for axon, inner tongue and myelin segmentation. PLoS Comput Biol 19(11): e1010845. https://doi.org/ 10.1371/journal.pcbi.1010845 Electron microscopy (EM) images of axons and their ensheathing myelin from both the cen- tral and peripheral nervous system are used for assessing myelin formation, degeneration (demyelination) and regeneration (remyelination). The g-ratio is the gold standard measure of assessing myelin thickness and quality, and traditionally is determined from measure- ments made manually from EM images–a time-consuming endeavour with limited reproduc- ibility. These measurements have also historically neglected the innermost uncompacted myelin sheath, known as the inner tongue. Nonetheless, the inner tongue has been shown to be important for myelin growth and some studies have reported that certain conditions can elicit its enlargement. Ignoring this fact may bias the standard g-ratio analysis, whereas quantifying the uncompacted myelin has the potential to provide novel insights in the myelin field. In this regard, we have developed AimSeg, a bioimage analysis tool for axon, inner tongue and myelin segmentation. Aided by machine learning classifiers trained on transmis- sion EM (TEM) images of tissue undergoing remyelination, AimSeg can be used either as an automated workflow or as a user-assisted segmentation tool. Validation results on TEM data from both healthy and remyelinating samples show good performance in segmenting all three fibre components, with the assisted segmentation showing the potential for further improvement with minimal user intervention. This results in a considerable reduction in time for analysis compared with manual annotation. AimSeg could also be used to build larger, high quality ground truth datasets to train novel deep learning models. Implemented in Fiji, AimSeg can use machine learning classifiers trained in ilastik. This, combined with a user- friendly interface and the ability to quantify uncompacted myelin, makes AimSeg a unique tool to assess myelin growth. Editor: Drew Linsley, Brown University, UNITED STATES PLOS COMPUTATIONAL BIOLOGY PLOS COMPUTATIONAL BIOLOGY AimSeg: A new tool for axon, inner tongue and myelin segmentation Funding: P.C.B. was awarded a Formacio´n de Personal Investigador (FPI) predoctoral contract funded by the Ministerio de Ciencia e Innovacio´n (Gobierno de España). Currently, P.C.B. holds a Margarita Salas postdoctoral contract (MS21-057), which is funded by the European Union- NextGenerationEU through a call from the Ministerio de Universidades (Gobierno de España) and the Universitat de València (Valencia, Spain) for the requalification of the Spanish university system (Plan de Recuperacio´n, Transformacio´n y Resiliencia). A.M.R. was awarded a UK Medical Research Council Tissue Repair PhD fellowship. A. W. is funded by the Multiple sclerosis Society UK, Medical Research Council (MRC), and the UK Dementia Research Institute as UK DRI which was funded by the MRC, Alzheimer’s Society and Alzheimer’s Research UK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. For the purpose of open access, the author has applied a Creative Commons Attribution (CC BY) licence to any Author Accepted Manuscript version arising from this submission. Author summary Myelin is formed by specialised cells that wrap themselves around axons and has a major role in the function, protection, and maintenance of nerves. These functions are disturbed by demyelinating diseases, such as multiple sclerosis. In this work we present AimSeg, a new tool based on artificial intelligence algorithms (machine learning) to assess myelin growth from electron microscopy images. Whereas standard metrics and previous computational methods focus on quantifying compact myelin, AimSeg also quantifies the inner myelin tongue (uncompacted myelin). This structure has been largely overlooked despite the fact that it has an important role in the process of myelin growth (both during development and in the adult brain) and recent studies have reported morphological changes associated with some diseases. We report the performance of AimSeg, both as a fully automated approach and in an assisted segmentation workflow that enables the user to curate the results “on-the-fly” while reducing human intervention to the minimum. Therefore, AimSeg stands as a novel bioimage analysis tool that meets the challenges of assessing myelin growth by supporting both standard metrics for myelin evaluation and the quantification of the uncompacted myelin in different conditions. Editor: Drew Linsley, Brown University, UNITED STATES Received: January 2, 2023 Accepted: November 5, 2023 Published: November 17, 2023 Copyright: © 2023 Carrillo-Barberà 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: Fiji pipelines can be easily installed by means of a dedicated Fiji update site. Requirements, installation, usage documentation and scripts are provided in GitHub (https://github.com/paucabar/AimSeg). Additionally, the pre-trained ilastik classifiers for both pixel and object classification and the validation dataset, including both TEM images and their corresponding fibre instance labels and semantic masks, are accessible in Zenodo (doi: 10. 5281/zenodo.8351731). 1 / 21 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1010845 November 17, 2023 Introduction Competing interests: The authors have declared that no competing interests exist. The myelin sheath allows faster, saltatory conduction of nerve impulses along the underlying axon without the need to increase axon diameter [1,2]. Moreover, this lipid-rich insulating layer also provides structural protection and metabolic support to the underlying axons [3]. The myelin sheath consists of plasma membrane from either oligodendrocytes (in the central nervous system [CNS]) or Schwann cells (in the peripheral nervous system [PNS]) wrapped around axons, and is discontinuous around their length, separated by nodes of Ranvier [4]. These cells extend cytoplasmic-filled membrane processes that are guided to reach and ensheath the axon. Myelin growth occurs by the wrapping of the leading edge of the myelin membrane process (henceforth the inner tongue) around the axon, progressing underneath the previously deposited membrane in concert with the lateral extension of the individual myelin layers along the axons [5]. Myelin compaction is initiated after a few wraps, occurring first in the outermost myelin layer and progressively spreading inwards, lagging behind the inner tongue to avoid its premature compaction. During developmental myelination, the inner tongue is enlarged but it narrows as myelin matures [4,6]. Once active myelination is completed, a smaller inner tongue remains in adult myelinated fibres [5,7] (see Fig 1A), except as recently discovered in the context of some diseases [8]. Myelination is a process that takes place during development [4]. Nevertheless, in the con- text of demyelinating conditions like multiple sclerosis (MS), there is potential for damaged myelin sheaths to be replaced through remyelination, a process carried out in the CNS by oli- godendrocytes [9]. However, when remyelination does not occur, it becomes impossible to restore energy-efficient conduction, and the supportive function of myelin is forfeited. This results in energy deficiency, disruptions in axonal transport, and ultimately the degeneration of axons [9] (see Fig 1B). Generally, most myelinated fibres have a ratio of axon to fibre diameters (g-ratio; see Fig 1C) close to the optimal value for conduction velocity of neural electrical impulses, estimated from theoretical models in the PNS and the CNS [10,11]. Additionally, larger diameter axons have more myelin wraps (thicker myelin sheath) and a lower g-ratio [9,12,13]. The g-ratio is 2 / 21 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1010845 November 17, 2023 PLOS COMPUTATIONAL BIOLOGY AimSeg: A new tool for axon, inner tongue and myelin segmentation Fig 1. Introduction The myelin sheath plays a crucial role in the nervous system, making imaging essential for studying myelin formation degeneration and regeneration (A) Schematic representation of the process of myelin formation in the central Fig 1. The myelin sheath plays a crucial role in the nervous system, making imaging essential for studying myelin formation, degeneration, and regeneration. (A) Schematic representation of the process of myelin formation in the central nervous system. (B) Myelin formation is an ongoing process, starting during development (myelination) and continuing throughout lifespan. Myelin regeneration (remyelination) can occur in response to demyelination. Failure in remyelination contributes to axonal degeneration. (C) Conventionally for myelin g-ratio analysis, the axon area (green) has been annotated at Fig 1. The myelin sheath plays a crucial role in the nervous system, making imaging essential for studying myelin Fig 1. The myelin sheath plays a crucial role in the nervous system, making imaging essential for studying myelin formation, degeneration, and regeneration. (A) Schematic representation of the process of myelin formation in the central nervous system. (B) Myelin formation is an ongoing process, starting during development (myelination) and continuing throughout lifespan. Myelin regeneration (remyelination) can occur in response to demyelination. Failure in remyelination contributes to axonal degeneration. (C) Conventionally for myelin g-ratio analysis, the axon area (green) has been annotated at Fig 1. The myelin sheath plays a crucial role in the nervous system, making imaging essential for studying myelin formation, degeneration, and regeneration. (A) Schematic representation of the process of myelin formation in the central nervous system. (B) Myelin formation is an ongoing process, starting during development (myelination) and continuing throughout lifespan. Myelin regeneration (remyelination) can occur in response to demyelination. Failure in remyelination contributes to axonal degeneration. (C) Conventionally for myelin g-ratio analysis, the axon area (green) has been annotated at PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1010845 November 17, 2023 3 / 21 PLOS COMPUTATIONAL BIOLOGY AimSeg: A new tool for axon, inner tongue and myelin segmentation the inner edge of the compact myelin, thus ignoring the contribution of the area occupied by the inner tongue (diagonal orange stripes). The myelin g-ratio is determined by assimilating the axon and fibre areas to circles to estimate their respective diameters. This conventionally disregards any area contribution from the inner tongue. https://doi.org/10.1371/journal.pcbi.1010845.g001 https://doi.org/10.1371/journal.pcbi.1010845.g001 widely utilised by the scientific community as a functional and structural index of optimal axo- nal myelination, and for assessing remyelination following myelin loss. Introduction Defects in myelination in the CNS can be assessed in this way in neurodevelopmental disorders [14–17], demyelinat- ing diseases (e.g., MS) [18,19], neurodegenerative diseases [20–22], as well as in rodent models of myelin abnormalities [8,23–26]. Moreover, the remyelination process in MS is characterised by thinner myelin sheaths for the diameter of the axon, giving higher g-ratios [27,28], an extensively utilised trait for discriminating between areas of remyelination and developmental myelination (see Fig 1B). G-ratios are commonly calculated on electron microscopy (EM) images of chemically fixed samples, though progress has been made to try and measure these in vivo on MR brain scans in humans [19,29]. Despite the wide applicability and functional relevance, the g-ratio neglects the inner tongue (see Fig 1C). This is because, for its calculation, the “axon” is usually defined as the inner edge of the compact myelin, which is more readily identifiable by both researchers and computational techniques. Nonetheless, an enlarged inner tongue will bias the standard g- ratio analysis by overestimating the diameter of the axon. Consequently, researchers are adopt- ing alternative ways to perform the g-ratio analysis to assess myelination/remyelination. For example, a “corrected g-ratio” accounting for the enlarged inner tongue has been recently pro- posed [26]. Moreover, recent studies have reported an enlarged or abnormal inner tongue in transgenic mice (e.g. 20,30-cyclic nucleotide 30-phosphodiesterase (CNP)-deficient mice [23,30], in conditional knock-out of activin co-receptor Acvr1b [31] and of Pten [5] in oligo- dendrocytes), and in animal models of autoantibody-mediated- and cuprizone-induced- demyelinating disease [8,25], suggested to be secondary to stressed axons with a compensatory increase in need for metabolic support from the oligodendrocyte via the inner tongue. Several bioimage analysis approaches have been developed to analyse myelin thickness [32– 38]. Many of these approaches are implemented in semi-automated workflows that frequently require several post-processing steps. Deep learning approaches have also been applied [39,40] to segment the individual fibres and their corresponding compacted myelin. Additionally, there are methods available for the analysis of 3D EM images [41]. However, their wide appli- cation by researchers has been limited, as still only few are publicly available, well documented, and/or the code made accessible through open-source licensing. Therefore, analysis of myelin thickness from EM images is still largely performed manually by investigators, which is time- consuming and prone to selection bias, thus contributing to limited reproducibility. A bioimage analysis workflow for the analysis of myelinated axons In contrast to previous image analysis methods that were developed to calculate conventional g-ratios based upon segmenting fibres and their compact myelin alone [32–40], our goal was to develop a method to separate and analyse each of the fibre components (the axon, the com- pact myelin and the inner tongue) from TEM images (see Figs 1 and 2). To this end, it is neces- sary to outline the borders of the axon, the innermost and the outermost compact myelin. AimSeg achieves this through the segmentation of three objects with a hierarchical relation- ship: the fibre cross-section, the region enclosed by the innermost compact myelin border (henceforth inner region), and the axon. The combination of these masks allows the calcula- tion of both the standard g-ratio and other metrics for the quantification of the inner tongue area. Our strategy relies on supervised ML methods based on random forests, which have been demonstrated to be useful to analyse complex images such as those acquired through EM [44,45]. AimSeg can be applied as a fully automated image processing workflow or enable an assisted segmentation approach that includes interactive user-editing. Our workflow makes use of open-source bioimage analysis software (ilastik [42] and Fiji [43]). The AimSeg core pipeline is a Fiji script that takes as an input a series of files previously generated using ML classifiers trained using ilastik (see Fig 3). The workflow starts with two classifiers (pixel and object classification; see Fig 3A–3D) trained using supervised ML methods implemented within ilastik–although AimSeg has the potential to use classifiers trained by means of any other ML toolkit. For this work, we trained our classifiers on TEM images acquired on remyelinating tissue; specifically, the dataset includes five images of the corpus callosum from four adult mice after a demyelinating lesion was induced. Users can directly apply the ready-to-use classifiers pre-trained for this work, improve them by adding their own raw data and annotations, or train new classifiers from scratch by following the guidelines provided within the AimSeg documentation. The AimSeg pipeline is divided in three sequential stages aimed to segment each of the three fibre components from each of the fibre cross-sections in the image. In Stage 1, the mye- lin probability map is processed to segment the inner region (see Fig 3E–3H), which is used as a seed to get the fibres in Stage 2 (see Fig 3I–3L). Introduction Notably, all the above-mentioned methods ignore the inner tongue and do not support its quantification. This has motivated us to develop an open-access tool, named AimSeg, for the segmentation of the axon, the inner tongue, and the compact myelin from EM data. Our goal has been to enable a more thorough assessment of the myelin sheath thickness, while decreasing the need for manual annotation, and saving time. AimSeg uses supervised machine learning (ML) methods implemented in ilastik [42] to improve the segmentation of the fibre components, and combines automated image processing with interactive user-editing stages in Fiji [43]. AimSeg automatically stores all the generated regions of interest (ROIs) in different subsets of axonal components interrelated between them and the results table by the axon IDs. The work- flow code for AimSeg–implemented as a Groovy script–is open-source and the pre-trained ilastik classifiers are publicly available together with user documentation. 4 / 21 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1010845 November 17, 2023 PLOS COMPUTATIONAL BIOLOGY AimSeg: A new tool for axon, inner tongue and myelin segmentation AimSeg training was conducted on transmission EM (TEM) images of corpus callosum tis- sue samples obtained from mice that were undergoing remyelination following a unilateral toxin-induced demyelination lesion in the corpus callosum (see Fig 1B). We have tested Aim- Seg on both i) a validation dataset that includes TEM images of remyelinating mice and ii) a control dataset composed of images from a healthy specimen, obtaining similar results. The corpus callosum was chosen as it is a highly myelinated white matter region within the CNS, commonly affected in CNS diseases and therefore used often in preclinical studies. Further- more, segmentation of the compacted and uncompacted myelin is more technically challeng- ing after remyelination compared with the relatively straightforward task of segmenting normal myelinated fibres. As a result, our training and validation data takes into account a variety of features, including myelinated and unmyelinated axons, and a wide range of fibres with different myelin and inner tongue thickness, thereby extending the utility of AimSeg for the segmentation of myelinated fibres cross-sections from TEM images. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1010845 November 17, 2023 A bioimage analysis workflow for the analysis of myelinated axons Finally, axons are segmented from the object classifier predictions in Stage 3 (see Fig 3M–3P). AimSeg includes optional post-processing that can be applied to automatically correct the axon segmentation using different methods. Additionally, after the automated steps, each stage includes an optional user-editing step that allows manual amendment of any segmentation inaccuracies before proceeding to the Computational Biology \| https://doi.org/10.1371/journal.pcbi.1010845 November 17, 2023 5 / 21 PLOS COMPUTATIONAL BIOLOGY AimSeg: A new tool for axon, inner tongue and myelin segmentation Fig 2. Validation ground truth for the segmentation of fibre cross-sections on electron microscopy images separating the myelin sheath components (compact myelin and inner tongue) from each other and from the axon. (A, top) Examples of transmission electron microscopy (TEM) images of the corpus callosum from adult mice undergoing remyelination after inducing a demyelinating lesion. Technical artefacts of no interest, degraded myelin debris and degenerated dark axons (red asterisk) were not included. Scale bar (red line) = 1 μm. (A, bottom) Manual segmentation of the compacted myelin (blue), the inner tongue (orange), and the axon (green). (B-D) Diversity of axon/fibre size, shape or myelin thickness. (B) Histograms representing different metrics determined from the manual annotations. (C) The fibres are colour-coded based on the histogram bins to represent the distribution of fibre eccentricity, describing how much a fibre section diverges from a circle, with 0.0 representing a perfect circle. (D) The fibres are colour-coded based on the histogram bins that illustrate their myelin g-ratio distribution (ratio of diameter of the area enclosed by the innermost compact myelin border and the diameter of the whole fibre). Higher g-ratios correspond to thinner myelin, with 1.0 representing the complete absence of myelin sheath. It is worth noting that this metric does not account for the presence of the inner tongue. As a result, two fibres, one with a shrunken inner tongue and another with an enlarged inner tongue, can exhibit similar myelin g-ratios (white asterisks). https://doi org/10 1371/journal pcbi 1010845 g002 Fig 2. Validation ground truth for the segmentation of fibre cross-sections on electron microscopy images separating the myelin sheath components (compact myelin and inner tongue) from each other and from the axon. (A, top) Examples of transmission electron microscopy (TEM) images of the corpus callosum from adult mice undergoing remyelination after inducing a demyelinating lesion. Technical artefacts of no interest, degraded myelin debris and degenerated dark axons (red asterisk) were not included. https://doi.org/10.1371/journal.pcbi.1010845.g002 A bioimage analysis workflow for the analysis of myelinated axons Scale bar (red line) = 1 μm. (A, bottom) Manual segmentation of the compacted myelin (blue), the inner tongue (orange), and the axon (green). (B-D) Diversity of axon/fibre size, shape or myelin thickness. (B) Histograms representing different metrics determined from the manual annotations. (C) The fibres are colour-coded based on the histogram bins to represent the distribution of fibre eccentricity, describing how much a fibre section diverges from a circle, with 0.0 representing a perfect circle. (D) The fibres are colour-coded based on the histogram bins that illustrate their myelin g-ratio distribution (ratio of diameter of the area enclosed by the innermost compact myelin border and the diameter of the whole fibre). Higher g-ratios correspond to thinner myelin, with 1.0 representing the complete absence of myelin sheath. It is worth noting that this metric does not account for the presence of the inner tongue. As a result, two fibres, one with a shrunken inner tongue and another with an enlarged inner tongue, can exhibit similar myelin g-ratios (white asterisks). https://doi.org/10.1371/journal.pcbi.1010845.g002 6 / 21 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1010845 November 17, 2023 PLOS COMPUTATIONAL BIOLOGY AimSeg: A new tool for axon, inner tongue and myelin segmentation Fig 3. Main steps of the AimSeg bioimage analysis workflow. (A-D) AimSeg combines two machine learning (ML) classifiers for pixel and object classification. First, the pixel classifier uses (A) the electron microscopy (EM) data to generate a (B) probability map. Then, (C) potential axon instances are segmented from the axoplasm probabilities. (D) The object classifier scores each instance as an axon or inner tongue. Objects outside myelinated fibre cross-sections (marked with an asterisk) will be eliminated in the next steps. (E-H) AimSeg Stage 1 uses (E) the myelin probability channel to get an (F) inverted mask. (G) This mask is then analysed to identify the elements within the innermost compact myelin Fig 3. Main steps of the AimSeg bioimage analysis workflow. (A-D) AimSeg combines two machine learning (ML) classifiers for pixel and object classification. First, the pixel classifier uses (A) the electron microscopy (EM) data to generate a (B) probability map. Then, (C) potential axon instances are segmented from the axoplasm probabilities. (D) The object classifier scores each instance as an axon or inner tongue. Objects outside myelinated fibre cross-sections (marked with an asterisk) will be eliminated in the next steps. A bioimage analysis workflow for the analysis of myelinated axons (E-H) AimSeg Stage 1 uses (E) the myelin probability channel to get an (F) inverted mask. (G) This mask is then analysed to identify the elements within the innermost compact myelin Fig 3. Main steps of the AimSeg bioimage analysis workflow. (A-D) AimSeg combines two machine learning (ML) classifiers for pixel and object classification. First, the pixel classifier uses (A) the electron microscopy (EM) data to generate a (B) probability map. Then, (C) potential axon instances are segmented from the axoplasm probabilities. (D) The object classifier scores each instance as an axon or inner tongue. Objects outside myelinated fibre cross-sections (marked with an asterisk) will be eliminated in the next steps. (E-H) AimSeg Stage 1 uses (E) the myelin probability channel to get an (F) inverted mask. (G) This mask is then analysed to identify the elements within the innermost compact myelin PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1010845 November 17, 2023 7 / 21 PLOS COMPUTATIONAL BIOLOGY AimSeg: A new tool for axon, inner tongue and myelin segmentation border, which we call the ‘inner region’, and to exclude those representing the background. These identified elements are categorised as either selected or rejected ROIs, respectively. Running the supervised mode (optional), the user can easily toggle the ROI selection group (selected/ rejected) or use the ImageJ’s selection tools to add/edit ROIs. (H) Semantic segmentation at the end of Stage 1. (I-L) AimSeg Stage 2 (I) uses the inner region labels as seeds that expand to fill myelin regions generating (J) a label mask for the fibres, which is processed to get (K) the fibre ROIs. (L) Semantic segmentation at the end of Stage 2. (M-P) AimSeg Stage 3 combines (M) the prediction of axon and inner tongue instances with (N) the fibre binary mask. (O) This ensures that only myelinated axons are selected. Instances classified as inner tongue are marked as rejected ROIs in the supervised mode. (P) Semantic segmentation at stage 3. (Q-T) AimSeg combines the gathered sets of ROIs to conduct a thorough analysis of myelinated axons. In this process, AimSeg assigns labels to the instances of (Q) the fibre, (R) the inner region and (S) the axon establishing a hierarchical relationship among instances within the same myelinated axon. (T) Additionally, AimSeg generates a semantic mask, where each pixel is categorised as background, axon, inner tongue, or compact myelin. Scale bar (red line) = 1 μm. https://doi.org/10.1371/journal.pcbi.1010845.g003 https://doi.org/10.1371/journal.pcbi.1010845.g003 next stage. A bioimage analysis workflow for the analysis of myelinated axons At these points, the user can delete ROIs, use the Fiji selection tools to edit or add new ROIs or use a series of shortcuts provided in AimSeg to interact with the ROIs while reducing the user intervention. Additionally, those ROIs filtered out during the automated processing are also shown as rejected-mode ROIs at Stage 1 and 3, so the user can toggle them to the selected-mode without drawing them from scratch (see Fig 3G and 3O). Once the three ROI sets have been generated, an automated pipeline within the Fiji-imple- mented workflow is set to both post-process the three ROI sets and to extract the quantitative features. The post-processing step aims to: i) remove any residual pixels that may have been left by the user–during the manual editing step–by keeping only the biggest region on compos- ite ROIs (stages 1 and 2; axons are allowed to be composite ROIs); ii) ensure that, as expected, the ROIs of the innermost elements do not overflow into the outer ones (e.g., the axon ROI should never break through the inner region ROI; stages 1 and 3); iii) optionally duplicate the inner region ROI in case an axon ROI has not been selected for a fibre due to a shrunken inner tongue; and iv) constructing a hierarchical relationship between the three ROI sets corre- sponding to different fibre components (see Fig 3Q–3S). The latter step is important for the performance of a meaningful quantification, and enables the user to trace results from the final measurements table back to the image dataset. Finally, the area of each ROI in the hierar- chy is extracted and summarised in a results table. AimSeg includes additional commands to visualise the final semantic segmentation as an overlay on top of the original image (see Fig 3P), and to export the definitive ROI sets as instance (see Fig 3Q–3S) and semantic (see Fig 3T) segmentation masks. https://doi.org/10.1371/journal.pcbi.1010845.g003 Assessment of the automated and supervised segmentation First we evaluated the learning efficiency of the ML methods used to generate the pixel and object classifiers. The results obtained comparing ilastik classifiers trained on different num- bers of samples (1–5 images) showed a limited effect on the final AimSeg output. Small and sparsely annotated datasets generated results close to classifiers trained with more data. This suggests that the classifiers reach their maximum precision with few annotations; further train- ing does not seem to improve the results, and may even be counterproductive (S1 Fig). To quantitatively assess the performance of our strategy, we compared the segmentation output obtained using our workflow to analyse the images of the validation ground truth (five images from four mice), manually annotated by an expert (see Fig 2). As discussed above, one important aspect of AimSeg’s design is the user-supervised step that makes it possible to cor- rect or edit the automatic segmentation. Therefore, to evaluate the extent to which such man- ual steps are necessary or beneficial, we compared the expert’s ground truth with two different workflow outputs: a fully automated workflow without any user intervention (see Fig 4A and 4B), and a supervised (assisted segmentation) workflow that included a limited version of all the user-editing steps. For the evaluation of the supervised mode, the user was allowed to edit the ROI sets by including or discarding ROIs automatically suggested by our tool, but not PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1010845 November 17, 2023 8 / 21 PLOS COMPUTATIONAL BIOLOGY AimSeg: A new tool for axon, inner tongue and myelin segmentation Fig 4. AimSeg segmentation performance, assessed independently for the three detections performed sequentially by AimSeg: the inner region (i.e., the axon plus the inner tongue), the fibre and the axon. Evaluation of the instance segmentation performed either in (A, B) automated or (C) supervised modes. At first, since no user intervention was allowed, results included both (A) accurate and (B) loose segmentations. Note that skipping an inner region at Stage 1 caused the myelin mask of the surrounding fibres to overflow at Stage 2. (C) The supervised mode allows the user to curate the AimSeg selection during the user-edited stages. Note that toggling the rejected inner region at Stage 1 solves the overflowing issue at Stage 2 and facilitates the automated detection of the corresponding fibre and axon. Assessment of the automated and supervised segmentation (D) Quantitation of the segmentation performance is based on the F1 score, an object-based metric, plotted for increasing intersection over union (IoU) thresholds for estimating the shape matching accuracy in both the automated and the human-supervised results. Scale bar (white line) = 0.5 μm. https://doi.org/10.1371/journal.pcbi.1010845.g004 Fig 4. AimSeg segmentation performance, assessed independently for the three detections performed sequentially by AimSeg: the inner region (i.e., the axon plus the inner tongue), the fibre and the axon. Evaluation of the instance segmentation performed either in (A, B) automated or (C) supervised modes. At first, since no user intervention was allowed, results included both (A) accurate and (B) loose segmentations. Note that skipping an inner region at Stage 1 caused the myelin mask of the surrounding fibres to overflow at Stage 2. (C) The supervised mode allows the user to curate the AimSeg selection during the user-edited stages. Note that toggling the rejected inner region at Stage 1 solves the overflowing issue at Stage 2 and facilitates the automated detection of the corresponding fibre and axon. (D) Quantitation of the segmentation performance is based on the F1 score, an object-based metric, plotted for increasing intersection over union (IoU) thresholds for estimating the shape matching accuracy in both the automated and the human-supervised results. Scale bar (white line) = 0.5 μm. https://doi.org/10.1371/journal.pcbi.1010845.g004 9 / 21 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1010845 November 17, 2023 PLOS COMPUTATIONAL BIOLOGY AimSeg: A new tool for axon, inner tongue and myelin segmentation manually drawing them (see Fig 4C). This allowed us to assess the potential of AimSeg to facili- tate the user-assisted segmentation. We used the “F1 score” to assess the performance of AimSeg during the automated and supervised modes. The average F1 score of all the annotated images was calculated across a range of intersections over union (IoU) thresholds, from 0.5 to 0.9 (in increments of 0.05). The representation of the F1 score along an IoU threshold range allows one to simultaneously look at the correctly identified objects and the pixel-wise closeness of their corresponding ROIs. A higher F1 score denotes a good detection of the object while a lower score corresponds to a poor object detection. F1 scores were independently computed for the fibre, the inner region, and the axon (see Fig 4D; precision and recall in S2 Fig). Assessment of the automated and supervised segmentation The automated approach demonstrated a considerable capability to predict the fibre constituents, with the F1 score being consistently high across the IoU thresholds. We also demonstrate that these results can be substantially improved by allow- ing the user to review and amend the segmented objects for all three components, even when not allowed to use the selection tools in Fiji to upgrade the ROI selection or to create new ones. AimSeg shortcuts lead to an improved performance at the three stages, being especially relevant in the segmentation of axons. Comparing the average F1 score obtained using either the automated or the supervised AimSeg analysis, we observed that the score increased from 0.83 to 0.87 for the inner region, from 0.85 to 0.88 for the fibre, and from 0.71 to 0.83 for the axon. Quantitative validation of myelin analysis After assessing the capability of AimSeg to segment the different elements of the fibre, we wanted to investigate its adequacy to generate accurate measurements for the analysis of the myelin properties. AimSeg enables the extraction of standard fibre features such as axon, inner tongue or myelin areas, which can be combined to calculate different myelin properties. In order to evaluate the agreement between the measurements obtained from the manual annota- tion and the segmentation obtained with AimSeg, the computed fibre areas (see Fig 5A) were compared using the Lin’s concordance correlation coefficient (CCC). Since the CCC requires a list of matched samples, fibres with an IoU lower than 0.5 were rejected from the analysis (15% false negatives, 5.8% false positives). Fibre areas agreed with a CCC of 0.9987 (95% confi- dence interval (CI) 0.9984–0.9989). Fibre areas detected using AimSeg were, on average, 0.01 μm2 smaller than those manually annotated and the limits of agreement were between -0.06 and 0.03 μm2 (see Fig 5B). Following the segmentation of the inner tongue, we distinguish between two types of “g- ratio”: 1) the classical g-ratio, here called the “myelin g-ratio”, the ratio of inner region to the fibre diameter, describing the thickness of the compact myelin relative to the axon and the inner tongue (see Fig 5C) and 2) the “axon g-ratio”, the ratio of the axonal diameter (excluding the inner tongue) to the fibre diameter, describing the thickness of both the compact and the non compact myelin relative to the axon diameter (see Fig 5D). Therefore, the difference of the myelin and the axon g-ratios can be used as a relative measurement to estimate the inner tongue enlargement, where 0 is equivalent to an absent structure. Myelin g-ratios agreed with a CCC of 0.83 (95% CI 0.8–0.86). On average, AimSeg analysis returned myelin g-ratios 0.01 smaller than manual segmentation, while the limits of agreement were between -0.07 and 0.06 (see Fig 5E). The CCC for the axon g-ratio was 0.75 (95% CI 0.7– 0.79). The mean difference of the axon g-ratios calculated by AimSeg and the validation ground truth is close to zero, even if the limits of agreement were between -0.13 and 0.12 (see Fig 5F). 10 / 21 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1010845 November 17, 2023 PLOS COMPUTATIONAL BIOLOGY AimSeg: A new tool for axon, inner tongue and myelin segmentation Fig 5. Quantitative validation of myelin analysis Agreement between the validation ground truth and the AimSeg measurements for the analysis of myelin properties. (A) Example image for the semantic segmentation of the fibres. Scale bar (white line) = 1 μm. (B) Comparison of the fibre areas obtained by manually segmenting the images or using AimSeg. (B, left) The measurement agreement is calculated as the Lin’s concordance correlation coefficient (CCC), (B, right) while the measurement bias is assessed by means of a Bland-Altman analysis. Diagonal line in the CCC plot represents perfect agreement (y = x). (C) Illustration of the myelin g-ratio measurement, calculated as the ratio of the inner region diameter to the fibre diameter. (D) Illustration of the axon g-ratio measurement, calculated as the ratio of the axon diameter to the fibre diameter. CCC and Bland-Altman plot for (E) the myelin and (F) the axon g-ratios. https://doi.org/10.1371/journal.pcbi.1010845.g005 Fig 5. Agreement between the validation ground truth and the AimSeg measurements for the analysis of myelin properties. (A) Example image for the semantic segmentation of the fibres. Scale bar (white line) = 1 μm. (B) Comparison of the fibre areas obtained by manually segmenting the images or using AimSeg. (B, left) The measurement agreement is calculated as the Lin’s concordance correlation coefficient (CCC), (B, right) while the measurement bias is assessed by means of a Bland-Altman analysis. Diagonal line in the CCC plot represents perfect agreement (y = x). (C) Illustration of the myelin g-ratio measurement, calculated as the ratio of the inner region diameter to the fibre diameter. (D) Illustration of the axon g-ratio measurement, calculated as the ratio f h d h f b d CCC d l d Al l f ( ) h l d ( ) h Fig 5. Agreement between the validation ground truth and the AimSeg measurements for the analysis of myelin properties. (A) Example image for the semantic segmentation of the fibres. Scale bar (white line) = 1 μm. (B) Comparison of the fibre areas obtained by manually segmenting the images or using AimSeg. (B, left) The measurement agreement is calculated as the Lin’s concordance correlation coefficient (CCC), (B, right) while the measurement bias is assessed by means of a Bland-Altman analysis. Diagonal line in the CCC plot represents perfect agreement (y = x). (C) Illustration of the myelin g-ratio measurement, calculated as the ratio of the inner region diameter to the fibre diameter. Quantitative validation of myelin analysis (D) Illustration of the axon g-ratio measurement, calculated as the ratio of the axon diameter to the fibre diameter. CCC and Bland-Altman plot for (E) the myelin and (F) the axon g-ratios. Fig 5. Agreement between the validation ground truth and the AimSeg measurements for the analysis of myelin properties. (A) Example image for the semantic segmentation of the fibres. Scale bar (white line) = 1 μm. (B) Comparison of the fibre areas obtained by manually segmenting the images or using AimSeg. (B, left) The measurement agreement is calculated as the Lin’s concordance correlation coefficient (CCC), (B, right) while the measurement bias is assessed by means of a Bland-Altman analysis. Diagonal line in the CCC plot represents perfect agreement (y = x). (C) Illustration of the myelin g-ratio measurement, calculated as the ratio of the inner region diameter to the fibre diameter. (D) Illustration of the axon g-ratio measurement, calculated as the ratio of the axon diameter to the fibre diameter. CCC and Bland-Altman plot for (E) the myelin and (F) the axon g-ratios. https://doi.org/10.1371/journal.pcbi.1010845.g005 https://doi.org/10.1371/journal.pcbi.1010845.g005 We aimed to determine if the enhanced segmentation quality achieved through the utilisa- tion of AimSeg shortcuts results in a more precise analysis of myelin properties. As expected, we observed a decrease in the number of fibres rejected by the 0.5 IoU filter when employing the supervised mode (14% false negatives, 0.56% false positives). The CCC of the fibre area remained close to 1, while the myelin g-ratio experienced a slight increase to 0.84 CCC. The axon g-ratio benefited most from the supervised workflow, reaching a CCC of 0.87. The limits of agreement for all the measurements investigated remained largely unchanged (see S3 Fig). PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1010845 November 17, 2023 11 / 21 PLOS COMPUTATIONAL BIOLOGY AimSeg: A new tool for axon, inner tongue and myelin segmentation Computation time The time required to manually annotate the validation ground truth per image was approxi- mately one hour compared to an average of 6.34 seconds with automatic processing with Aim- Seg. We further assessed the computational time required for each automated step of the AimSeg core workflow. The time required for the automated processing steps (i.e., excluding parameterisation, data import and user supervision) of each stage per image was: 0.74 s (Stage 1), 1.97 s (Stage 2), and 2.01 s (Stage 3) and 1.62 s for post-processing and quantification. Therefore, the computational time for automated processing across the three stages was negli- gible when compared with the highly time-consuming endeavour of annotating the images manually. Performance on non-remyelinating samples Tissue undergoing remyelination contains axons whose myelin sheath may present a wide variety of states and, additionally, a high proportion of unmyelinated axons. Therefore, it seemed suitable data for training and validating AimSeg. We also tested its performance on control, healthy, more uniform tissue (without a demyelinating lesion) (see S4 Fig). Overall, the segmentation metrics and the biological baseline proved to be very similar to the validation dataset (see Fig 6A). The statistical analysis performed on the identified objects within the con- trol dataset (with a 5.9% false negative rate and a 6.6% false positive rate) revealed a CCC of 0.9953 (95% CI 0.9943–0.9962) for fibre areas. Fibre areas detected using AimSeg were, on average, 0.01 μm2 smaller than those manually annotated and the limits of agreement were between -0.05 and 0.03 μm2 (see Fig 6B). Myelin g-ratios on control samples agreed with a CCC of 0.86 (95% CI 0.83–0.89). Myelin g-ratios, on average, were 0.01 smaller than manual segmentation and the limits of agreement were between -0.05 and 0.04 (see Fig 6C). The CCC for the axon g-ratio of the healthy mouse was 0.75 (95% CI 0.7–0.79). On average, the axon g- ratios calculated by AimSeg were 0.04 smaller than those manually annotated. In this case, the limits of agreement were between -0.11 and 0.03 (see Fig 6D). Discussion The g-ratio is the gold standard for the assessment of the optimal myelination of axons. How- ever, the calculation of this highly used metric neglects the existence of the uncompacted mye- lin of the inner tongue; a fibre component with relevance during myelination and remyelination, and whose thickness variation may contribute to the identification of both physiological and pathological processes. In fact, our dataset on remyelinating white matter clearly shows that fibres of similar cross-section diameter, but differing in the presence of inner tongue would often render almost identical classic myelin g-ratio values evidencing the risk of overlooking relevant biological conditions. The lack of bioimage analysis tools account- ing for the inner tongue makes its quantification a tedious task, requiring the manual annota- tion of EM images by experts: this is a common bottleneck that often hinders the quantification of larger datasets. Here we present AimSeg, a bioimage analysis tool for axon, inner tongue, and myelin segmentation of fibre-cross sections from EM images. The AimSeg workflow was built using open-source bioimage analysis software to combine supervised ML with an image processing pipeline to facilitate the annotation of the fibre com- partments. To this end, it takes advantage of the user-friendly and interactive ML tools pro- vided by ilastik (which are readily accessible for users without coding experience), the versatility of Fiji, and the interoperability of both toolkits. A post-processing pipeline corrects some common annotation errors and establishes a hierarchical relationship between different ROI sets before quantifying key myelin metrics. In this context, we propose differentiating PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1010845 November 17, 2023 12 / 21 PLOS COMPUTATIONAL BIOLOGY AimSeg: A new tool for axon, inner tongue and myelin segmentation Fig 6. Segmentation metrics and agreement between the control ground truth and the AimSeg measurements for the analysis of myelin properties. (A) Segmentation performance for the instances detected at each AimSeg stage. (A, left) F1 score plotted for increasing intersection over union (IoU) thresholds. (A, right) Average F1 score, precision, recall, and Jaccard index. (B) Comparison of the fibre areas obtained by manually segmenting the images or using AimSeg. (B, left) The measurement agreement is calculated as the Lin’s concordance correlation coefficient (CCC), (B, right) while the measurement bias is assessed by means of a Bland-Altman analysis. Diagonal line in the CCC plot represents perfect agreement (y = x). Discussion CCC and Bland-Altman plot for (C) the myelin and (D) the axon g-ratios. https://doi.org/10.1371/journal.pcbi.1010845.g006 Fig 6. Segmentation metrics and agreement between the control ground truth and the AimSeg measurements Fig 6. Segmentation metrics and agreement between the control ground truth and the AimSeg measurements for the analysis of myelin properties. (A) Segmentation performance for the instances detected at each AimSeg stage. (A, left) F1 score plotted for increasing intersection over union (IoU) thresholds. (A, right) Average F1 score, precision, recall, and Jaccard index. (B) Comparison of the fibre areas obtained by manually segmenting the images or using AimSeg. (B, left) The measurement agreement is calculated as the Lin’s concordance correlation coefficient (CCC), (B, right) while the measurement bias is assessed by means of a Bland-Altman analysis. Diagonal line in the CCC plot represents perfect agreement (y = x). CCC and Bland-Altman plot for (C) the myelin and (D) the axon g-ratios. https://doi.org/10.1371/journal.pcbi.1010845.g006 Fig 6. Segmentation metrics and agreement between the control ground truth and the AimSeg measurements for the analysis of myelin properties. (A) Segmentation performance for the instances detected at each AimSeg stage. (A, left) F1 score plotted for increasing intersection over union (IoU) thresholds. (A, right) Average F1 score, precision, recall, and Jaccard index. (B) Comparison of the fibre areas obtained by manually segmenting the images or using AimSeg. (B, left) The measurement agreement is calculated as the Lin’s concordance correlation coefficient (CCC), (B, right) while the measurement bias is assessed by means of a Bland-Altman analysis. Diagonal line in the CCC plot represents perfect agreement (y = x). CCC and Bland-Altman plot for (C) the myelin and (D) the axon g-ratios. https://doi.org/10.1371/journal.pcbi.1010845.g006 13 / 21 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1010845 November 17, 2023 PLOS COMPUTATIONAL BIOLOGY AimSeg: A new tool for axon, inner tongue and myelin segmentation between the myelin g-ratio, which corresponds to the classic g-ratio, and the axon g-ratio, which takes into account the inner tongue area in addition to the myelin sheath. By combining both metrics, we can identify enlarged inner tongues that may bias myelin analysis, and explore variations in the inner tongue as an independent subject of investigation. Functioning as a fully automated tool, AimSeg has demonstrated both significant segmen- tation accuracy and the capacity to produce its measurements for analysing myelin properties that closely align with those obtained directly from the ground truth. Discussion However, we have observed occasional underestimation of the axon area likely due to the presence of electron- dense bodies such as mitochondria or neurofilaments within the axoplasm. Additionally, we have noted variations in segmentation performance between well-preserved fibres and those affected by tissue-processing artefacts, emphasising the significance of sample preparation, a common challenge in bioimage analysis. It is important to clarify that AimSeg is designed to segment all myelinated axons independently of their quality. To address these or any other potential segmentation issues, AimSeg has been designed as a flexible tool that includes several selectable automatic operations to correct the predicted axon ROIs, such as automated convex hull estimation. On the other hand, throughout the three segmentation stages, users can make use of an interactive, shortcut-assisted, supervised ROI edition workflow. Our validation results demonstrate that the application of these correction/edition tools clearly improves the segmentation output and the accuracy of the obtained data with minimum impact on the pro- cessing time when compared to fully manual annotation. The end goal of AimSeg is to provide tools that enable more sophisticated bioimage analysis in the field of myelin biology. We have demonstrated AimSeg in combination with conven- tional ML using random forests, because this provides a flexible workflow that can be readily integrated into different laboratories with minimal effort (i.e. sparsely annotating only a few images is sufficient to train models adapted to a new image type). Although beyond the scope of the current work, this flexibility may be useful for the analysis of images obtained using other microscopy modalities, either EM (scanning electron microscopy, scanning transmis- sion electron microscopy) or optical microscopy (e.g., brightfield, confocal). While the limita- tion on the spatial resolution achieved by some of these modalities may prevent the analysis of the inner tongue, AimSeg is capable of determining classic myelin g-ratios, for which the higher resolution of TEM is not essential. Moreover, the AimSeg pipeline can readily be adapted to use other pixel and object classifier outputs, for example generated using standard supervised deep learning-based approaches. Future work will explore training more generalised deep learning models for fibre compart- ment segmentation and classification, based on training data gathered from different laborato- ries. This has the potential to make deployment even easier, by removing the need for classifier training on a per-lab, per-modality basis. Data preprocessing AimSeg includes a preprocessing command that can be used to resize and/or normalise the image dataset. If the normalisation option is checked, images are converted to 32-bit and their pixel values are normalised to be floats in a 0 to 1 range. Data normalisation is conducted using ImageJ’s ContrastEnhancer class. Bit conversion uses ImageJ’s ImageConverter class. The TEM dataset used to train the ilastik classifiers and validate AimSeg was normalised enabling a 1% of saturated pixels and resized using a downsampling factor of 4, generating new data with image dimensions 8.62 μm x 8.62 μm (1024 x 1024 pixels; pixel size 8.4182 x 10−3 x 8.4182 x 10−3 μm). The TEM dataset used to train the ilastik classifiers and validate AimSeg was normalised enabling a 1% of saturated pixels and resized using a downsampling factor of 4, generating new data with image dimensions 8.62 μm x 8.62 μm (1024 x 1024 pixels; pixel size 8.4182 x 10−3 x 8.4182 x 10−3 μm). Validation and control ground truth Evaluation was performed on corpus callosum tissue samples obtained from independent mice undergoing remyelination different from those selected for training the classifiers. Addi- tional tests were conducted on control samples to assess AimSeg performance on samples that had not undergone demyelination. Manual annotations of the three ROIs (axon, inner region, and fibre) were done by a single expert in QuPath [47], thus generating the ground truth for the respective ROI set of entire images. The validation ground truth consists of five TEM images from four mice. The control ground truth consists of three TEM images from a healthy specimen. Annotations were exported using QuPath’s scripting language to generate three independent instance masks (axon, inner region, and fibre images) and one semantic mask (axon, inner tongue, and compact myelin pixels) per image applying a downsampling factor of 4. TEM dataset Experimental protocols involving mice were performed under UK Home Office project licence PADF15B79 (A.W.) issued under the Animals (Scientific Procedures) Act. Adult mouse cor- pus callosum tissue from remyelinating and healthy specimens was obtained and processed for EM as described in [18,46]. TEM images used in this study were collected on a JEOL JEM- 1400 Plus TEM with GATAN OneView camera at 7.1 K magnification with image dimensions 8.62 μm x 8.62 μm (4096 x 4096 pixels; pixel size 2.1046 x 10−3 x 2.1046 x 10−3 μm). Discussion The efficient, computer-assisted annotation features currently within AimSeg will help in generating the ground truth for such models at scale, while the full AimSeg pipeline will remain important to translate the deep learning outputs into biologically meaningful quantitative results. Any future work, whether conducted by us or the scientific community, can benefit from AimSeg’s ground truth data along with the QuPath scripts for the generation of compatible datasets, all openly shared. Collectively, these comple- mentary assets hold the potential to serve as a foundational resource for projects aiming to achieve a comprehensive segmentation of myelinated axons. With this work, we contribute to filling the gap between myelin biology and bioimage anal- ysis. We believe that AimSeg may facilitate the study of the long-neglected inner tongue by providing a user-friendly, open-source platform for its quantification. Moreover, our assisted segmentation approach enhances the throughput capability of the analysis while enabling manual annotation. Overall, AimSeg’s features and novel metrics have the potential to support PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1010845 November 17, 2023 14 / 21 PLOS COMPUTATIONAL BIOLOGY AimSeg: A new tool for axon, inner tongue and myelin segmentation more sensitive and high-throughput approaches to analyse myelin ultrastructure beyond the standard g-ratio. Pixel and object classification training and learning efficiency ML tools implemented within ilastik 1.3.3post3 [42] were used to perform a pixel classification and an object classification. Pixel classification was conducted as an ilastik autocontext workflow [42], which performs two sequential pixel classifications using the predictions of the first classifier as additional channels for the input of the second classifier. Four different classes were defined for the first pixel classification: i) compacted myelin, ii) axoplasm iii) membrane–such as the axolemma or the inner tongue membrane–and, iv) mitochondria. The second pixel classifier uses the same classes but merges mitochondria within the axoplasm class to prevent holes on the final axon instances. The pixel classifier uses all the intensity, edge and texture features implemented within ilastik with different σ (0.3, 0.7, 1.0, 1.6, 3.5, 5.0, 10.0, 15.0, 30.0, 50.0). 15 / 21 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1010845 November 17, 2023 PLOS COMPUTATIONAL BIOLOGY AimSeg: A new tool for axon, inner tongue and myelin segmentation The object classification pipeline starts performing an instance segmentation, taking as input the probability map generated by the pixel classifier. The axoplasm probability channel is smoothed (σ = 2.0 sigma), thresholded (0.6 threshold) and size-filtered (with objects smaller than 10 pixels rejected) to compute potential axon instances. However, there are electron- lucent structures that can be segmented along the axons, such as cells or inner tongue sections. Therefore, we defined three different classes: two representing axon cross-sections (larger or smaller), and the other representing inner tongue cross-sections. The rest of the objects obtained through the instance segmentation, including cells or unmyelinated axons, are not annotated during the training process, and thus their predictions are ignored. The classifier uses all the shape and intensity distribution features implemented within ilastik for the object classification; conversely, the location features were ignored. Both pixel and object classifiers were trained interactively using a subset of five images ran- domly selected as the training set from one mouse. To assess the learning efficiency, five pairs of pixel-object classifiers were trained using a different number of images (from 1 to 5), with an increment of 1 image per step. Image processing methods implemented in AimSeg AimSeg is implemented as a Fiji workflow and maintained through an update site. AimSeg han- dles the segmentation output as three different types of data, including binary masks, ROIs and label masks. Basic binary operations (erode, dilate, open, close, fill) are implemented as modifi- cations of ImageJ 1.x source code [48]. Logical operators use ImageJ’s ImageCalculator class. Binary reconstruction is part of the morphological operations provided at Fiji’s Morphology update site [49]. Connected components from binary masks are detected, filtered and converted into ROIs with the ImageJ ParticleAnalyzer plugin. AimSeg handles different ROI sets by means of independent ImageJ RoiManager instances. The RoiManager is also used to transform ROIs into binary or label masks. Operations with individual ROIs use ImageJ Roi, PolygonRoi and ShapeRoi classes. These include calculating the convex hull, filling of ShapeRois, filtering PolygonRois contained in ShapeRois by specified criteria, and calculating the intersection of two ROIs. Additionally, ROI erosion and dilation is performed with ImageJ RoiEnlarger plugin. Operations with label masks are implemented using the MorphoLibJ library [50] by accessing the MarkerControlledWatershedTransform2D and ReplaceLabelValues classes. Label masks are transformed into ROIs by means of the ImageJ’s ThresholdToSelection plugin. Metrics for the assessment of instance segmentation To evaluate the performance of the segmentation we used precision, recall and the F1 score. We computed object-based metrics rather than pixel-based metrics because the goal of our pipeline is to perform the instance segmentation of each individual fibre and its components to extract mor- phometric features. Briefly, the metrics used are based on computing the overlapping degree between the target (T), i.e., the validation ground truth annotated by an expert, and the prediction (P) masks, automatically generated by AimSeg. First, the overlap between T and P is calculated for each object as the intersection over union (IoU) metric (also known as Jaccard index). IoU T; P ð Þ ¼ T \ P T [ P ð1Þ ð1Þ Where the intersection (T\P) is the count of the pixels shared by both T and P, whereas the union (T[P) is the count of the pixels that are part of either T, P or both. Therefore, the IoU has a value of 1.0 for identical objects, while a value of 0 indicates that there is no overlap between T and P. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1010845 November 17, 2023 16 / 21 PLOS COMPUTATIONAL BIOLOGY AimSeg: A new tool for axon, inner tongue and myelin segmentation Then, an IoU threshold is set to label each object as a true positive (TP), a false negative (FN) or a false positive (FP). Precision is determined by the proportion of predicted objects that had a match with the annotated ground truth and is defined as: Precision ¼ TP TP þ FP ð2Þ ð2Þ Recall is determined by the proportion of target objects that had a match on the prediction mask, calculated as: Recall is determined by the proportion of target objects that had a match on the prediction mask, calculated as: Recall ¼ TP TP þ FN ð3Þ ð3Þ The F1 score is defined as the harmonic mean of precision and recall: F1 ¼ 2 precision recall precision þ recall ð4Þ ð4Þ Therefore, the F1 score can be calculated as: F1 ¼ 2TP 2TP þ FN þ FP ð5Þ ð5Þ We computed the three metrics along a range of IoU values, since the selection of a single IoU threshold may be considered an arbitrary measure. We excluded IoU values below 0.5 to avoid the conflict of pairing a T object with two P objects, or vice versa. Metrics for the assessment of instance segmentation Notably, a perfect overlap is practically unattainable, even when comparing the annotations of two human operators. Therefore, we used a range from 0.5 to 0.9 with increments of 0.05. The average precision, recall and F1 score is calculated as the mean of all the scores obtained at all the IoU thresholds previously described. Additionally, the Jaccard index average is calculated as the IoU mean of all T individual objects. The assessment of AimSeg segmentation performance (IoU, precision, recall, and F1 score) has been computed using a customised script based on the evaluation pipeline implemented by Caicedo et al [51]. Correlation and bias analysis for AimSeg myelin quantification The CCC was used to determine the level of agreement in measurements between myelin metrics obtained from AimSeg segmentation masks and the manual segmentation performed by an expert (validation and control ground truth). Unlike other methods, CCC relies on concordance, not just linearity. Therefore, all the spots in the scatter plot comparing two samples with a perfect CCC are expected to fall on the x = y line. The expected bias was computed by means of a Bland-Altman analysis. The validation of myelin analysis was conducted using a customised script based on the val- idation pipeline implemented by Matthews et al [52]. Image processing and myelin analysis (fibre area, myelin g-ratio, and axon g-ratio) for correlation and bias analysis were carried out using Python’s skimage [53], numpy [54] and pandas [55] libraries. Since it is not possible to extract reli- able myelin metrics from incomplete fibres, CCC and Bland-Altman analyses were performed after eliminating the image borders from both the validation ground truth and the AimSeg prediction. Supporting information S1 Fig. Learning efficiency of the classifiers. Evaluation of the number of training images required to train efficient classifiers for AimSeg. Different classifiers were used to run AimSeg and compute the F1 score obtained for the segmentation of (A) the inner region (axon plus inner tongue), (B) the fibre and (C) the axon. (TIF) S2 Fig. AimSeg segmentation precision and recall, assessed independently for the three exported ROI sets: the inner region (i.e., the axon plus the inner tongue), the fibre and the axon. AimSeg was run in automated and supervised modes to compute precision and recall for the segmentation of (A) the inner region, (B) the fibre, and (C) the axon. (TIF) S2 Fig. AimSeg segmentation precision and recall, assessed independently for the three exported ROI sets: the inner region (i.e., the axon plus the inner tongue), the fibre and the axon. AimSeg was run in automated and supervised modes to compute precision and recall for the segmentation of (A) the inner region, (B) the fibre, and (C) the axon. (TIF) S3 Fig. Agreement between the control ground truth and the supervised AimSeg measure- ments for the analysis of myelin properties. (A) Comparison of the fibre areas obtained by manually segmenting the images or using AimSeg. (A, left) The measurement agreement is calculated as the Lin’s concordance correlation coefficient (CCC), (A, right) while the mea- surement bias is assessed by means of a Bland-Altman analysis. Diagonal line in the CCC plot represents perfect agreement (y = x). CCC and Bland-Altman plot for (B) the myelin and (C) the axon g-ratios. (TIF) S4 Fig. Control ground truth for the instance segmentation of fibres and the semantic seg- mentation of myelin, inner tongue, and axon. (A, top) Examples of transmission electron microscopy (TEM) images of the corpus callosum from a healthy, adult mouse. Scale bar (red line) = 1 μm. (A, bottom) Manual segmentation of the compacted myelin (blue), the inner tongue, (orange) and the axon (green). (B-D) Diversity of axon/fibre size, shape or myelin thickness. (B) Histograms representing different metrics determined from the manual annota- tions. (C) The fibres are colour-coded based on the histogram bins to represent the distribu- tion of fibre eccentricity, describing how much a fibre section diverges from a circle, with 0.0 representing a perfect circle. Acknowledgments The authors would like to thank Stephen Mitchell for helping with the acquisition of TEM data, and Ana Domingo-Muelas and Chiara Sgattoni for help with designing the graphics. Supporting information (D) The fibres are colour-coded based on the histogram bins that illustrate their g-ratio distribution (ratio of diameter of the area enclosed by the innermost compact myelin border and the diameter of the whole fibre). Higher g-ratios correspond to thinner myelin, with 1.0 representing the complete absence of myelin sheath. (TIF) Hardware Computation time quantification was performed on a HP OMEN 15-DC0000NS laptop with an Intel Core i7-8750H processor, 16 GB of RAM and an NVIDIA GeForce GTX 1060 graphic card. 17 / 21 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1010845 November 17, 2023 PLOS COMPUTATIONAL BIOLOGY AimSeg: A new tool for axon, inner tongue and myelin segmentation Author Contributions Conceptualization: Pau Carrillo-Barberà, Ana Maria Rondelli, Bertrand Vernay, Anna Williams, Peter Bankhead. Data curation: Pau Carrillo-Barberà, Ana Maria Rondelli. Data curation: Pau Carrillo-Barberà, Ana Maria Rondelli. Formal analysis: Pau Carrillo-Barberà, Peter Bankhead. Formal analysis: Pau Carrillo-Barberà, Peter Bankhead. Funding acquisition: Pau Carrillo-Barberà, Anna Williams. Funding acquisition: Pau Carrillo-Barberà, Anna Williams. Investigation: Ana Maria Rondelli. 18 / 21 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1010845 November 17, 2023 PLOS COMPUTATIONAL BIOLOGY AimSeg: A new tool for axon, inner tongue and myelin segmentation Methodology: Pau Carrillo-Barberà, Jose Manuel Morante-Redolat, Peter Bankhead. Project administration: Pau Carrillo-Barberà, Jose Manuel Morante-Redolat, Peter Bankhead. Resources: Bertrand Vernay, Anna Williams, Peter Bankhead. Software: Pau Carrillo-Barberà, Peter Bankhead. Supervision: Jose Manuel Morante-Redolat, Bertrand Vernay, Anna Williams, Peter Bankhead. Validation: Pau Carrillo-Barberà. Visualization: Pau Carrillo-Barberà. Writing – original draft: Pau Carrillo-Barberà, Ana Maria Rondelli, Jose Manuel Morante- Redolat. Writing – review & editing: Pau Carrillo-Barberà, Ana Maria Rondelli, Jose Manuel Mor- ante-Redolat, Bertrand Vernay, Anna Williams, Peter Bankhead. References Owen JP, Marco EJ, Desai S, Fourie E, Harris J, Hill SS, et al. 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https://openalex.org/W2950105613	https://europepmc.org/articles/pmc5677151?pdf=render	English	null	Comparison of Single Genome and Allele Frequency Data Reveals Discordant Demographic Histories	G3	2,017	cc-by	15,689	Copyright © 2017 Beichman et al. doi: https://doi.org/10.1534/g3.117.300259 Manuscript received July 12, 2017; accepted for publication September 1, 2017; published Early Online September 11, 2017. This is an open-access article 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 the original work is properly cited. Supplemental material is available online at www.g3journal.org/lookup/suppl/ doi:10.1534/g3.117.300259/-/DC1. 1Corresponding author: Department of Ecology and Evolutionary Biology, University of California, Los Angeles, 621 Charles E. Young Dr. South, Los Angeles, CA 90095- 1606. E-mail: klohmueller@ucla.edu Comparison of Single Genome and Allele Frequency Data Reveals Discordant Demographic Histories Annabel C. Beichman,* Tanya N. Phung,† and Kirk E. Lohmueller,†,‡,1 Department of Ecology and Evolutionary Biology, †Interdepartmental Program in Bioinformatics, and ‡Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, California 90095 ORCID IDs: 0000-0002-6991-587X (A.C.B.); 0000-0003-2685-6977 (T.N.P.); 0000-0002-3874-369X (K.E.L.) ABSTRACT Inference of demographic history from genetic data is a primary goal of population genetics of model and nonmodel organisms. Whole genome-based approaches such as the pairwise/multiple sequentially Markovian coalescent methods use genomic data from one to four individuals to infer the demographic history of an entire population, while site frequency spectrum (SFS)-based methods use the distribution of allele frequencies in a sample to reconstruct the same historical events. Although both methods are extensively used in empirical studies and perform well on data simulated under simple models, there have been only limited comparisons of them in more complex and realistic settings. Here we use published demographic models based on data from three human populations (Yoruba, descendants of northwest-Europeans, and Han Chinese) as an empirical test case to study the behavior of both inference procedures. We ﬁnd that several of the demographic histories inferred by the whole genome-based methods do not predict the genome-wide distribution of heterozygosity, nor do they predict the empirical SFS. However, using simulated data, we also ﬁnd that the whole genome methods can reconstruct the complex demographic models inferred by SFS-based methods, suggesting that the discordant patterns of genetic variation are not attributable to a lack of statistical power, but may reﬂect unmodeled complexities in the underlying demography. More generally, our ﬁndings indicate that demographic inference from a small number of genomes, routine in genomic studies of nonmodel organisms, should be interpreted cautiously, as these models cannot recapitulate other summaries of the data. KEYWORDS pairwise sequentially Markovian coalescent site frequency spectrum population genetics demographic inference nonmodel organisms The pairwise sequentially Markovian coalescent (PSMC) and related methods have become a popular tool to estimate the history of a population from genetic variation data (McVean and Cardin 2005; Li and Durbin 2011; Schiffels and Durbin 2014). These methods use whole genome sequences from one to four individuals to infer the demographic history of an entire population. Speciﬁcally, they estimate the local time to the most recent common ancestor (TMRCA) for small regions in the genome and then use the distribution of these coalescent times to infer an overarching demographic history. INVESTIGATION KEYWORDS pairwise sequentially Markovian coalescent site frequency spectrum population genetics demographic inference nonmodel organisms Comparison of Single Genome and Allele Frequency Data Reveals Discordant Demographic Histories They therefore often give information about events occurring millions of years ago, but the reliability of those results remains uncertain (Li and Durbin 2011). Further, demographic models inferred from human populations using the SFS were unable to recapitulate the empirical distribution of identity-by-state (IBS) tracts across the genome, while PSMC-derived models and an IBS-derived model were better able to match the IBS tract distribution (Harris and Nielsen 2013). However, the IBS-derived model did not predict the empirical SFS. Despite their widespread prominence, there is concern over the validityofdemographicmodelsobtainedfromthissetofwholegenome- based methods. Particularly, Mazet et al. (2015b) found that PSMC captures the inverse instantaneous coalescent rate (IICR) rather than an absolute measure of population size. The IICR corresponds to the effective population size if the population is panmictic, but it can differ from the population size due to gene ﬂow and population structure, which affect the time to coalescence between subgroups. Thus, popu- lation structure can give a false signal of population growth or contrac- tion, a notorious problem in demographic inference (Ptak and Przeworski 2002; Chikhi et al. 2010; Peter et al. 2010; Gattepaille et al. 2013; Heller et al. 2013; Mazet et al. 2015a,b; Orozco-terWengel 2016). Given these possible confounders, the degree to which whole genome-based plots derived from PSMC and its successors correspond to actual population size changes, rather than other demographic phe- nomena, remains unclear. Due to these different strengths and weaknesses of approaches using singletypesofdata, new methodshavebeendeveloped whichattempt to combine linkage disequilibrium (LD) information and the SFS (Bunnefeld et al. 2015; Boitard et al. 2016; Terhorst et al. 2017; Weissman and Hallatschek 2017). One of the most recent methods is Terhorst et al.’s (2017) SMC++, which combines a PSMC-like ap- proach with the SFS to condition an SFS calculated from many indi- viduals on the distribution of TMRCA from a single unphased genome. This approach is fast and potentially very powerful, but has the same barrier to entry for those studying nonmodel organisms as the other SFS methods, as it requires sequence data from many individuals. An alternative approach to infer population demography from genetic data uses the site frequency spectrum (SFS). The SFS represents the distribution of alleles at different frequencies in a sample of indi- viduals from a population (Nielsen 2000; Wakeley 2009). Comparison of Single Genome and Allele Frequency Data Reveals Discordant Demographic Histories For instance, if many regions of the genome coalesce at a speciﬁc time, it may be evidence for a population contraction, which would reduce the number of genetic lineages. The great appeal of these methods is that they do not rely on deep sequencing of multiple individuals in a population; in- stead, a single genome can be used to infer the demographic history of an entire population. PSMC and its successors have been used to infer the demographic histories and split times of many human populations (Li and Durbin 2011; Kidd et al. 2012; Schiffels and Durbin 2014; 1000 Genomes Project Consortium 2015; Henn et al. 2016), and were re- cently featured in three prominent articles that reconstructed human history using whole genome sequencing data from over 20 populations (Malaspinas et al. 2016; Mallick et al. 2016; Pagani et al. 2016). PSMC plots have also become a cornerstone of many studies of nonmodel organisms lacking resources for the sequencing of numerous individuals, including archaic hominins (Meyer et al. 2012; Prufer et al. 2014), great apes (Prado-Martinez et al. 2013), wild boars and domestic pigs (Groenen et al. 2012; Bosse et al. 2014), canids (Freedman et al. Volume 7 \| November 2017 \| 3605 2014; Wang et al. 2016), horses (Orlando et al. 2013), over 38 bird species (Nadachowska-Brzyska et al. 2013, 2015, 2016; Hung et al. 2014; Murray et al. 2017), pandas (Zhao et al. 2012), dromedaries (Fitak et al. 2016), ﬂowering plants (Albert et al. 2013; Ibarra-Laclette et al. 2013; Holliday et al. 2016), and even woolly mammoths (Palkopoulou et al. 2015). an extension to PSMC that uses the SMC9 algorithm (Marjoram and Wall 2006) and can infer demography from two, four, or eight haplo- types (also known as PSMC9 when inferring from two haplotypes). The incorporation of multiple genomes in MSMC is speciﬁcally meant to improve estimates of recent growth (Schiffels and Durbin 2014). The SFS may be limited in the degree to which it can detect ancient bottlenecks .2Ne (effective population size) generations ago and in its ability to detect population declines (Bunnefeld et al. 2015; Terhorst and Song 2015; Boitard et al. 2016). Whole genome-based approaches are not constrained a priori by the number of population size changes as is common in the SFS-based approaches (but see the “stairway plot” approach of Liu and Fu 2015). Comparison of Single Genome and Allele Frequency Data Reveals Discordant Demographic Histories The distri- bution of single nucleotide polymorphisms (SNPs), ranging from rare “singletons” which appear only once in the sample, to high-frequency variants that may appear in the majority of individuals, is directly affected by the demographic history of the population (Nielsen 2000; Wakeley 2009). Population contractions (“bottlenecks”) can lead to a dearth of rare variants (Nei et al. 1975), whereas a rapid population expansion can lead to an overabundance (Tajima 1989; Slatkin and Hudson 1991; Keinan and Clark 2012). The SFS is a sufﬁcient statistic for unlinked SNPs and has been used extensively in population genetic inference of demography (Nielsen 2000; Polanski and Kimmel 2003; Adams and Hudson 2004; Marth et al. 2004; Keinan et al. 2007; Gutenkunst et al. 2009; Gravel et al. 2011; Excofﬁer et al. 2013). SFS- based demographic inference has been implemented in programs such as @a@i (Gutenkunst et al. 2009), moments (Jouganous et al. 2017), fastsimcoal2 (Excofﬁer et al. 2013), stairway plot (Liu and Fu 2015), fastNeutrino (Bhaskar et al. 2015), and others (Schraiber and Akey 2015). The SFS requires less sequence data per individual than the whole genome methods, but requires a greater number of individuals to be studied, with a minimum of 10 per population typically used (Gutenkunst et al. 2009; Excofﬁer et al. 2013). While the SFS is imprac- tical if one can only sequence one or two individuals per population, population genomic studies based on many short loci scattered throughout the genome are beginning to be carried out on nonmodel organisms. RAD-seq data or gene transcript data from RNA-seq can readily be used for SFS-based demographic inference (McCoy et al. 2014; Trucchi et al. 2014; Sovic et al. 2016). Due to anthropological and biomedical interest, humans have been extensively studied using numerous demographic inference methods and provide a means to quantitatively compare these demographic inferenceapproachesusingthesameempiricalpopulations.Gutenkunst et al. (2009) and Gravel et al. (2011) carried out SFS-based inference of human demography using the diffusion approximation in @a@i, while Li and Durbin (2011) and Schiffels and Durbin (2014) estimated human demography from the same populations using PSMC and MSMC, respectively. Although the results are in some ways generally similar, the demographic models inferred for three human populations using MSMC (Schiffels and Durbin 2014) differ from demographic models for the same populations derived from SFS-based methods (Gutenkunst et al. 2009). 3606 \| A. C. Beichman, T. N. Phung, and K. E. Lohmueller Comparison of Single Genome and Allele Frequency Data Reveals Discordant Demographic Histories MSMC infers ancient ancestral sizes and periods of growth and decline (the characteristic “humps” in MSMC trajectories) that were not detected in the SFS-derived models as well as inferring greater recent growth (Figure 1). The models inferred using MSMC also vary depending on the number of genomes used for the inference (Figure 1). Terhorst et al. (2017) analyzed the same populations with the com- bined whole genome and SFS method, SMC++, ﬁnding an ancestral size more in line with Gutenkunst et al.’s (2009) model, but with greater recent growth and ancestral bottlenecks more resembling the MSMC models (Figure 1). The reasons why these approaches to demographic inference yield different estimates remain poorly understood. SFS-based and whole genome-based methods may have different strengths and weaknesses for demographic inference (Schraiber and Akey 2015). Theoretical and empirical data show that SFS-based ap- proaches using large numbers of individuals can accurately estimate recent population growth (Nelson et al. 2012; Tennessen et al. 2012; Gazave et al. 2014; Bhaskar et al. 2015; Gao and Keinan 2016). In contrast, whole genome-based methods are less able to do so (Li and Durbin 2011). Recently, however, Schiffels and Durbin (2014) de- veloped the multiple sequentially Markovian coalescent (MSMC), Here weleverage humans as a model system toperform an empirical comparison of the performance of whole genome, SFS, and combined methods of demographic inference. Speciﬁcally, we determine which published models of human demography described above (Figure 1) best ﬁt the empirical distributions of genome-wide heterozygosity, LD decay, and the observed SFS. 3606 \| A. C. Beichman, T. N. Phung, and K. E. Lohmueller We ﬁnd that the models inferred using the SFS or the combined method, SMC++, accurately recapitulate heterozygosity and the ob- served SFS. Among the MSMC models inferred by Schiffels and Durbin (2014), only the MSMC models based on a single genome were able to accurately recapitulate heterozygosity, and none of the MSMC models predicted an SFS that matched the empirical SFS. None of the demo- graphic histories accurately predicted LD decay, but the histories de- rived from MSMC using four genomes (eight haplotypes), the SFS, and SMC++-based models ﬁt better than the MSMC models based on one or two genomes. Our results provide a cautionary tale against the literal interpretation of demographic models inferred using one type of data, instead arguing for considering multiple summaries of the data when making detailed demographic inferences in nonmodel species. Comparison of Single Genome and Allele Frequency Data Reveals Discordant Demographic Histories the genome-wide SFS. The distribution of heterozygosity across windows of the genome retains some spatial information and is more similar to what is used by the MSMC inference approach. Empirical heterozygosity: 1000 Genomes data from the CEU, CHB, and YRI populations were downloaded. 10 unrelated individuals per population (see Supplementary Note 3 in File S1 for sequence IDs) were randomly chosen so that comparisons could be made with Gutenkunst et al.’s (2009) empirical SFS, described below. For all our empirical analyses, only sites that passed the 1000 Genomes “Strict Mask” ﬁlter were considered (1000 Genomes Project Consortium 2015). Expected heterozygosity per site (p) was calculated in nonoverlap- ping 100-kb windows from the whole genome data (Supplementary Note 3 in File S1) as: Published demographic models used in this study Their model parameters were made available both in @a@i and Hudson’s ms (Hudson 2002) format, and include gene ﬂow between the three populations (here referred to as the “Gutenkunst” model). Simulated heterozygosity: For each demographic model, whole ge- nome data for 10 individuals were simulated in MaCS (Chen et al. 2009) over 20,000 · 100-kb independent blocks, each with a different recom- bination rate drawn from the distribution of recombination rates cal- culated by Phung et al. (2016) from the pedigree-based genetic map assembled by the deCODE project (Kong et al. 2010). Additionally, 6300 · 10-kb independent blocks per 10 individuals were simulated for comparison to the neutral regions from the 1000 Genomes data set (1000 Genomes Project Consortium 2015). Each 10-kb block was sim- ulated using a recombination rate matched to that of one of the empir- ically neutral 10-kb windows, linearly interpolated from the deCODE project (Kong et al. 2010). For both sets of simulations, the expected heterozygosity across the 10 individuals was calculated using the equation above in msstats (Hudson 2002). The next nine models were inferred by Schiffels and Durbin (2014) using whole genome Complete Genomics (Drmanac et al. 2010) se- quence data of two, four, and eight statistically phased genomic hap- lotypes (one, two, and three individual genomes) per population to infer demographic histories using MSMC (here referred to as the “MSMC 2-haplotype,” “MSMC 4-haplotype,” and “MSMC 8-haplotype” models; Supplemental Material, Supplementary Note 1 in File S1). To analyze their models with @a@i, we converted these nine de- mographic models (CEU, CHB, and YRI populations, each based on two, four, and eight haplotypes) into stepwise models of population size changes over small time intervals (Figure S1 and Supplementary Note 2 in File S1). TheﬁnalsetofmodelswasinferredbyTerhorstetal.(2017) inSMC++, a combined SFS plus whole genome approach. For the whole genome portion of the analysis, they used high coverage sequence data from Complete Genomics, and generated an SFS based on a combination of 1000 Genomes and Complete Genomics whole genome data for each population (Drmanac et al. 2010; 1000 Genomes Project Consortium 2015; Terhorst et al. 2017). We converted these SMC++ models to @a@i and ms format in the same manner as the MSMC models (here referred to as the “SMC++” models; Supplementary Note 2 in File S1). Published demographic models used in this study Published demographic models used in this study We determined which, if any, of the published models of human demography (Figure 1) described below could accurately predict multiple summaries of the genetic variation data. Demographic models that ﬁt the data well should produce patterns of genetic variation that match the empirical patterns in the data. We focused on three human populations: Utah residents with Northern and Western European ancestry from the Centre d’Etude du Polymorphism Humain collection (CEU); Han Chinese in Beijing, China (CHB); and Yoruba in Ibadan, Nigeria (YRI). The ﬁrst set of demographic models was jointly inferred for the three populations in @a@i by Gutenkunst et al. (2009) using a three- population joint SFS based on data from intronic regions. Their model parameters were made available both in @a@i and Hudson’s ms (Hudson 2002) format, and include gene ﬂow between the three populations (here referred to as the “Gutenkunst” model). We determined which, if any, of the published models of human demography (Figure 1) described below could accurately predict multiple summaries of the genetic variation data. Demographic models that ﬁt the data well should produce patterns of genetic variation that match the empirical patterns in the data. We focused on three human populations: Utah residents with Northern and Western European ancestry from the Centre d’Etude du Polymorphism Humain collection (CEU); Han Chinese in Beijing, China (CHB); and Yoruba in Ibadan, Nigeria (YRI). where p is the frequency of one allele, L is the total number of callable sites in the window, and n is number of sampled chromosomes (n = 20 for 10 diploid individuals). Because genetic variation can be affected by linked natural selection (Gazave et al. 2014; Schrider et al. 2016), we also calculated expected heterozygosity for a set of 6333 · 10-kb neutral windows that were selected using the Neutral Region Explorer (NRE) (Arbiza et al. 2012) (Figure S2 and Supplementary Note 3 in File S1). The NRE is a useful tool that allows for the quick identiﬁcation of putatively neutral regions that have high recombination rates and high B-values (indicating less linked selection). For the full set of parameters used in selection of putatively neutral regions, see Supplementary Note 3 in File S1. The ﬁrst set of demographic models was jointly inferred for the three populations in @a@i by Gutenkunst et al. (2009) using a three- population joint SFS based on data from intronic regions. LD decay predicted by demographic models We calculated LD between pairs of SNPs using genotype data from 10 individuals from each of the four populations in the 1000 Genomes Project data. We removed singletons and sites where all 10 individuals werehomozygousforthereferencealleleandthencalculatedgenotyper2 using vcftools (Danecek et al. 2011). All pairs of SNPs were then placed into bins based on their physical distance (bp) between each other, from 0–1000 bp (bin 1) to 50,000–51,000 bp (bin 51). Within each bin, the average r2 was calculated by dividing the sum of r2 values of each pair of SNPs in the bin by the total number of SNP pairs in that bin. METHODS p ¼ n n 2 1 PL i¼12pi 1 2 pi L Volume 7 November 2017 \| Disparate Demographic Histories \| Heterozygosity predicted by demographic models We experimented with down-sampling the results and found no change in the LD decay curve due to the smaller amount of data. average r2 was calculated as described above. The MSMC 8-haplotype YRI and MSMC 4-haplotype CEU, CHB, and YRI models have extremely large ancestral sizes, and so their simulations involve so many SNPs that the LD calculations become highly computationally intensive. Therefore, for these models only 5000 · 100-kb blocks were used for LD decay calculations, with 20,000 · 100-kb blocks used for the other models. We experimented with down-sampling the results and found no change in the LD decay curve due to the smaller amount of data. LD, we simulated data in the manner described above under a simple model of extreme population decline (from 100,000 ancestral individ- uals to 1000) using both MaCS and MSMS (Ewing and Hermisson 2010) (which does not use the SMC9 approximation) and ran it through the same LD decay pipeline used for our other simulated data (Figure S3 in File S1). LD, we simulated data in the manner described above under a simple model of extreme population decline (from 100,000 ancestral individ- uals to 1000) using both MaCS and MSMS (Ewing and Hermisson 2010) (which does not use the SMC9 approximation) and ran it through the same LD decay pipeline used for our other simulated data (Figure S3 in File S1). Heterozygosity predicted by demographic models 2009) simulator was not biasing our estimates of LD, we simulated data in the manner described above under a simple model of extreme population decline (from 100,000 ancestral individ- uals to 1000) using both MaCS and MSMS (Ewing and Hermisson 2010) (which does not use the SMC9 approximation) and ran it through the same LD decay pipeline used for our other simulated data (Figure S3 in File S1). SFS predicted by demographic models We used the diffusion approximation in @a@i (Gutenkunst et al. 2009) to calculate the expected SFSs under the Gutenkunst, MSMC Figure 1 Demographic histories for the (A) CEU, (B) CHB, and (C) YRI populations. Trajectories are log scaled and in terms of physical units (diploid individuals and years). Models were either inferred using SFS-based methods (Gutenkunst) by Gutenkunst et al. (2009); from a sequentially Markovian coalescent-based approach (MSMC) from two, four, and eight haplotypes by Schiffels and Durbin (2014); or using a combined SFS and whole genome approach (SMC++) by Terhorst et al. (2017). The Gutenkunst models also include migration between all three populations, not depicted here. Models are scaled by the gener- ation times used in each study [Gutenkunst et al. (2009): 25 yr/generation; Schiffels and Durbin (2014): 30 yr/generation; Terhorst et al. (2017): 29 yr/generation]. Figure 1 Demographic histories for the (A) CEU, (B) CHB, and (C) YRI populations. Trajectories are log scaled and in terms of physical units (diploid individuals and years). Models were either inferred using SFS-based methods (Gutenkunst) by Gutenkunst et al. (2009); from a sequentially Markovian coalescent-based approach (MSMC) from two, four, and eight haplotypes by Schiffels and Durbin (2014); or using a combined SFS and whole genome approach (SMC++) by Terhorst et al. (2017). The Gutenkunst models also include migration between all three populations, not depicted here. Models are scaled by the gener- ation times used in each study [Gutenkunst et al. (2009): 25 yr/generation; Schiffels and Durbin (2014): 30 yr/generation; Terhorst et al. (2017): 29 yr/generation]. 2 average r2 was calculated as described above. The MSMC 8-haplotype YRI and MSMC 4-haplotype CEU, CHB, and YRI models have extremely large ancestral sizes, and so their simulations involve so many SNPs that the LD calculations become highly computationally intensive. Therefore, for these models only 5000 · 100-kb blocks were used for LD decay calculations, with 20,000 · 100-kb blocks used for the other models. Heterozygosity predicted by demographic models We compared the distribution of expected heterozygosity from data simulated under each demographic model to empirical 1000 Genomes data from the same populations to determine which models most accurately predict this broad summary of the data (Figure 2 and Table S1 in File S1). While heterozygosity is a summary of the SFS, we considered it valuable to examine both statistics since information re- garding the spatial correlation among SNPs along the genome is lost in The same procedure was carried out for the data simulated in MaCS (Chen et al. 2009) that were used for the calculations of heterozygosity above. The MaCS output was converted to vcf format using a custom bash script. Genotype r2 was calculated in vcftools (Danecek et al. 2011) for each 100-kb simulated window, the SNP pairs were binned by distance, and Volume 7 November 2017 \| Disparate Demographic Histories 3607 Figure 1 Demographic histories for the (A) CEU, (B) CHB, and (C) YRI populations. Trajectories are log scaled and in terms of physical units (diploid individuals and years). Models were either inferred using SFS-based methods (Gutenkunst) by Gutenkunst et al. (2009); from a sequentially Markovian coalescent-based approach (MSMC) from two, four, and eight haplotypes by Schiffels and Durbin (2014); or using a combined SFS and whole genome approach (SMC++) by Terhorst et al. (2017). The Gutenkunst models also include migration between all three populations, not depicted here. Models are scaled by the gener- ation times used in each study [Gutenkunst et al. (2009): 25 yr/generation; Schiffels and Durbin (2014): 30 yr/generation; Terhorst et al. (2017): 29 yr/generation]. average r2 was calculated as described above. The MSMC 8-haplotype YRI and MSMC 4-haplotype CEU, CHB, and YRI models have extremely large ancestral sizes, and so their simulations involve so many SNPs that the LD calculations become highly computationally intensive. Therefore, for these models only 5000 · 100-kb blocks were used for LD decay calculations, with 20,000 · 100-kb blocks used for the other models. We experimented with down-sampling the results and found no change in the LD decay curve due to the smaller amount of data. To demonstrate that the use of the SMC9 approximation in the MaCS (Chen et al. 3608 \| A. C. Beichman, T. N. Phung, and K. E. Lohmueller SFS predicted by demographic models To demonstrate that the use of the SMC9 approximation in the MaCS (Chen et al. 2009) simulator was not biasing our estimates of We used the diffusion approximation in @a@i (Gutenkunst et al. 2009) to calculate the expected SFSs under the Gutenkunst, MSMC 3608 \| A. C. Beichman, T. N. Phung, and K. E. Lohmueller Figure 2 Kernel density distribution of expected heterozygosity (p per site). Heterozygosity was calculated across 100-kb windows from whole genome 1000 Genomes Project data for (A) CEU, (B) CHB, and (C) YRI, and from 20,000 · 100-kb blocks for data simulated under each demographic model. The black dot and bars indicate the mean 6 2 SD for each distribution. Note the log-10 scaling on the y-axis. Figure 2 Kernel density distribution of expected heterozygosity (p per site). Heterozygosity was calculated across 100-kb windows from whole genome 1000 Genomes Project data for (A) CEU, (B) CHB, and (C) YRI, and from 20,000 · 100-kb blocks for data simulated under each demographic model. The black dot and bars indicate the mean 6 2 SD for each distribution. Note the log-10 scaling on the y-axis. Figure 2 Kernel density distribution of expected heterozygosity (p per site). Heterozygosity was calculated across 100-kb windows from whole genome 1000 Genomes Project data for (A) CEU, (B) CHB, and (C) YRI, and from 20,000 · 100-kb blocks for data simulated under each demographic model. The black dot and bars indicate the mean 6 2 SD for each distribution. Note the log-10 scaling on the y-axis. 2-haplotype, MSMC 4-haplotype, MSMC 8-haplotype, and SMC++ models for the CEU, CHB, and YRI populations. We compared the SFSs expected under each of these models both to the empirical SFS used by Gutenkunst et al. (2009) to infer the demographic histories of these three populations [“observed (Gutenkunst),” Figure 4 and Fig- ure 5], as well as to the SFSs based on low-coverage 1000 Genomes whole genome sequencing data [“1000 Genomes (whole genome),” Figure 6] and SFSs based on putatively neutral regions in the 1000 Ge- nomes data set [“1000 Genomes (neutral)”, Figure 6]. We assessed the ﬁt of different models to the observed SFS by comparing their log- likelihoods (see below, Table 1, Supplementary Note 4 in File S1, and Tables S2–S4 in File S1). 20 chromosomes for a total of 17,446 segregating SNPs polarized against chimp, with a correction for ancestral misidentiﬁcation applied. Effect of uncertainty in ancestral population size To investigate whether changing the ancestral population size (NA) in the MSMC trajectories would result in SFSs that better ﬁt the observed SFS, we adjusted the CEU MSMC 2-haplotype model to have a variety of NA values. We also trimmed the model to remove ancient events (older than 225.5 KYA) to better match the time period (in years) encompassed by the Gutenkunst et al.’s (2009) model. These adjusted stepwise models were then used to calculate the expected SFS in @a@i, as above. Supplementary Note 7 in File S1 describes the values of NA used when testing the trimmed and untrimmed models (Figures S10–S13 in File S1). MSMC population size trajectories for demographic models inferred from the SFS u ¼ 4NAimL; where NAi is the oldest ancestral size inferred in each model and L is the sequence length (4.04 Mbp) in Gutenkunst et al. (2009). u for the Gutenkunst model used the authors’ preferred mutation rate of m = 2.35 · 1028 mutations per base per generation, and u for the MSMC and SMC++ models used the authors’ preferred mutation rate of m = 1.25 · 1028 mutations per base per generation (see Supple- mentary Note 5 in File S1 for scaling using alternate mutation rates). Extreme recent growth and Neanderthal admixture: We simulated data under more complex demographic histories, ﬁrst to explore the relative abilities of the MSMC 2-haplotype and 8-haplotype models to inferextremerecentgrowth,andthentodeterminewhethertheaddition of Neanderthal admixture may lead to MSMC trajectories resembling those inferred from real data by Schiffels and Durbin (2014) (Supple- mentary Note 6 and Figures S8 and S9 in File S1). Assessing SFS ﬁt: Log-likelihoods were calculated for eachproportional SFS relative to each of the three observed SFSs [observed (Gutenkunst), 1000 Genomes (whole genome), and 1000 Genomes (neutral)] using a multinomial log-likelihood (Table 1, Supplementary Note 4 in File S1, and Tables S2 and S4 in File S1). The ﬁt of different models was compared by examining their decrease in log-likelihood compared to that of each of the observed SFSs to itself (Table 1, Supplementary Note 4 in File S1, and Tables S2 and S4 in File S1). Due to the un- certainty of singleton SNP calls using high-throughput sequencing data, log-likelihoods were calculated both with singletons and with the SFS renormalized without the singletons category when comparing to the 1000 Genomes SFSs (Figure S7 and Table S4 in File S1). MSMC population size trajectories for demographic models inferred from the SFS To determine whether MSMC is capable of inferring a demography as complexastheoneinferredintheGutenkunstmodel,weusedcoalescent simulations to generate long chromosomal sequence data for each population under the Gutenkunst et al. (2009) inferred demographic model [see Gutenkunst et al.’s (2009) ﬁgure 2B and table 1 for full model], and then ran MSMC on these simulated data sets to assess whether the program is capable of recovering the underlying demo- graphic model. Data to data denotes the best log-likelihood possible when replacing the proportions predicted by the model with the observed proportions from the SFS used in Gutenkunst et al.’s (2009) study (see Supplementary Note 4 in File S1). Gutenkunst denotes the model inferred by Gutenkunst et al. (2009) ﬁt to the observed SFS. SMC++ denotes the model inferred by Terhorst et al. (2017) using a combined whole genome and SFS approach. MSMC 2-, 4-, and 8-haplotype denote the demographic models inferred by Schiffels and Durbin (2014) using MSMC on two, four, and eight haplotypes, respectively. Simulations were carried out using MaCS (Chen et al. 2009). For each population, we simulated 50 replicate “genomes,” made up of 80 independent 30-Mb “chromosomes,” each made up of 300 linked 100-kb recombination blocks, with per-block recombination rates cal- culated by Phung et al. (2016) from the pedigree-based genetic map assembled by the deCODE project (Kong et al. 2010). We generated both the proportional (Figure 4 and Figure S5 in File S1) and absolute (i.e., SFS based on SNP counts) SFSs (Figure 5 and Figure S6 in File). The proportional SFS was calculated by dividing each bin of the SFS output by @a@i by the sum of the bins. The absolute SFS was calculated by scaling the SFS output by @a@i (which is relative to u ¼ 1) by Each simulated genome was then used for a separate MSMC inference, using the default parameters (Schiffels and Durbin 2014) (Figure 7A). To determine whether these inferred MSMC trajecto- ries would lead to SFSs matching those predicted by Gutenkunst et al.’s (2009) model, the MSMC trajectories were averaged and the average was converted into a stepwise @a@i model. This model was then used to calculate the expected SFS under the averaged model based on simulated data (Figure 7, B and C). The multinomial and Poisson log-likelihoods for the proportional and SNP count SFSs were calculated as described in Supplementary Note 4 in File S1 (Tables S2 and S3 in File S1). SFS predicted by demographic models We marginalized the SFS using @a@i (Gutenkunst et al. 2009) to have one SFS per population (Figure 4 and Figure 5). To make sure our results were consistent with SFSs derived from other sequencing methodologies and different genomic regions, we also generated folded proportional genome-wide and neutral SFSs from the 1000 Genomes data described above [1000 Genomes (whole genome) and 1000 Genomes (neutral)] (1000 Genomes Project Consortium 2015; Figure 6, Figure S7 in File S1, and Supplementary Note 3 in File S1). Expected SFSs under published demographic models: Expected SFSs for a sample size of 10 diploid individuals were calculated in @a@i (Gutenkunst et al. 2009) for each of the published demographic models extrapolating calculations across three grid points (40, 50, and 60) (Figure 4 and Figure 5). To test whether the effect of differences in mutation rate between the studies may be responsible for discrepancies, we also considered an alternative scaling of the MSMC models using a higher mutation rate (Supplementary Note 5 in File S1). Empirical SFSs: The primary empirical SFSs used in our comparisons were produced by Gutenkunst et al. (2009) and used to infer the joint demographic histories of CEU, CHB, and YRI populations in their study [observed (Gutenkunst)]. As described in their supplementary material, the joint SFS represents 4.04 Mb of Sanger sequencing data from 12 YRI, 12 CHB, and 22 CEU individuals projected down to Volume 7 November 2017 \| Disparate Demographic Histories \| 3609 3609 n Table 1 Multinomial log-likelihoods comparing the ﬁt of various models to the observed SFS derived from Sanger sequencing data and used by Gutenkunst et al. (2009) for their inference (SFSs in Figure 4) Model Multinomial LL ΔLL (Model 2 Data) CEU Data to data 221,546 0 Gutenkunst 221,555 29 SMC++ 221,599 253 MSMC 2-haplotype 221,698 2152 MSMC 8-haplotype 221,816 2270 MSMC 4-haplotype 222,760 21214 CHB Data to data 220,154 0 Gutenkunst 220,202 248 SMC++ 220,277 2123 MSMC 8-haplotype 220,343 2188 MSMC 2-haplotype 220,370 2216 MSMC 4-haplotype 221,411 21257 YRI Data to data 229,630 0 Gutenkunst 229,647 217 SMC++ 229,779 2150 MSMC 2-haplotype 230,003 2373 MSMC 8-haplotype 231,282 21652 MSMC 4-haplotype 232,976 23346 Log-likelihoods were calculated for each absolute SFS (in terms of SNP counts) using a Poisson likelihood relative to the observed (Gutenkunst) SFS (Supplementary Note 4 and Table S3 in File S1). Data availability All code to simulate data under each demographic model, calculate hetero- zygosity,andgeneratetheSFSfromsimulatedandempiricaldataareavailable on GitHub: github.com/LohmuellerLab/Compare_Demographic_Models. RESULTS We ﬁnd that the Gutenkunst demographic model inferred from the SFS, the MSMC 2-haplotype model, and the SMC++ model all yielded distributions of heterozygosity that resemble the empirical whole ge- nome distribution of heterozygosity, with MSMC 2-haplotype ﬁtting the mean most closely (Figure 2). However, we found that the higher haplotype MSMC models (MSMC 4-haplotype and 8-haplotype) yielded distributions of heterozygosity that were highly divergent from the empirical distribution (Figure 2 and Table S1 in File S1). The MSMC 4-haplotype modelsﬁt worst duetotheir extremely high inferredancestralsizeacrossallthreepopulations(Figure1andTableS2 in File S1) (CEU, 187,514; CHB, 191,238; YRI, 205,845 individuals; compared to 4000–40,000 individuals in the other models), with mean whole genome heterozygosity distributions nearly seven times larger than that of the empirical whole genome distribution (Figure 2 and RESULTS We compared published models of demography for three human populations (CEU, CHB, and YRI) inferred using different methods 3610 \| A. C. Beichman, T. N. Phung, and K. E. Lohmueller 3610 Figure 3 LD decay patterns. LD decay was calculated across 100-kb windows from 1000 Ge- nomes data and simulated data under each de- mographic model for (A) CEU, (B) CHB, and (C) YRI. Pairs of SNPs are binned based on physical distance (bp) between them, up to 51 kb. Aver- age genotype r2 is calculated within each dis- tance bin. Figure 3 LD decay patterns. LD decay was calculated across 100-kb windows from 1000 Ge- nomes data and simulated data under each de- mographic model for (A) CEU, (B) CHB, and (C) YRI. Pairs of SNPs are binned based on physical distance (bp) between them, up to 51 kb. Aver- age genotype r2 is calculated within each dis- tance bin. Figure 3 LD decay patterns. LD decay calculated across 100-kb windows from 100 nomes data and simulated data under eac mographic model for (A) CEU, (B) CHB, an YRI. Pairs of SNPs are binned based on ph distance (bp) between them, up to 51 kb. age genotype r2 is calculated within eac tance bin. Figure 3 LD decay patterns. LD decay was calculated across 100-kb windows from 1000 Ge- nomes data and simulated data under each de- mographic model for (A) CEU, (B) CHB, and (C) YRI. Pairs of SNPs are binned based on physical distance (bp) between them, up to 51 kb. Aver- age genotype r2 is calculated within each dis- tance bin. distributions of heterozygosity based on whole genome and putatively neutral sequence data from the 1000 Genomes Project. for demographic inference: (1) using the SFS in @a@i (Gutenkunst) (Gutenkunst et al. 2009), (2) using whole genomes in MSMC (MSMC 2-, 4-, and 8-haplotype) (Schiffels and Durbin 2014), and (3) using a combined SFS plus whole genome approach in SMC++ (SMC++) (Terhorst et al. 2017). The evaluation of the MSMC models involves three models per population because Schiffels and Durbin’s (2014) inference was carried out using two, three, or eight chromosomal hap- lotypes (from one, two, and four individuals), sometimes resulting in fundamentally different demographic parameter estimates. We evalu- ated whether the method’s performance was improved using certain numbers of haplotypes. Volume 7 November 2017 \| Disparate Demographic Histories \| 3611 Heterozygosity predicted by demographic models Thedistributionofexpectedheterozygosityacross100-and10-kbblocks was calculated from data simulated under each published demographic model for each of the three populations and compared to empirical Volume 7 November 2017 \| Disparate Demographic Histories \| 3611 3611 Figure 4 Unfolded proportional site fre- quency spectra for (A) CEU, (B) CHB (B), and (C) YRI populations. The “observed” SFS is from noncoding sequence used by Gutenkunst et al. (2009) to infer demo- graphic histories for these three popula- tions. See Figure S5 in File S1 for scaling using alternative mutation rates. Figure 4 Unfolded proportional site fre- quency spectra for (A) CEU, (B) CHB (B), and (C) YRI populations. The “observed” SFS is from noncoding sequence used by Gutenkunst et al. (2009) to infer demo- graphic histories for these three popula- tions. See Figure S5 in File S1 for scaling using alternative mutation rates. Table S1 in File S1). The MSMC 8-haplotye model for YRI infers a similarly large ancestral size and has a similarly high mean heterozy- gosity as the 4-haplotype YRI model. The MSMC 8-haplotype models for CEU and CHB, however, infer much lower ancestral sizes (CEU, 2147; CHB, 5666) (Figure 1). Due to the low ancestral size, these models also do not ﬁt the empirical distribution well, yielding distributions of heterozygosity with means that are two to four times lower than the empirical distributions. 4-haplotype models. For YRI SNP pairs ,10 kb apart, SMC++ and MSMC 8-haplotype predict similar LD decay curves and are close to the empirical distribution, with Gutenkunst still ﬁtting better than MSMC 2-haplotype and 4-haplotype (Figure 3C). At distances .10 kb apart, all demographic models predict there to be more LD than seen in the empirical data (Figure 3). We found that the lack of ﬁt is not due to the use of the SMC9 approximation in the simulator MaCS (Chen et al. 2009), as both MaCS and MSMS (Ewing and Hermisson 2010), a coalescent simulator which does not use the SMC9 approximation, yielded highly similar LD decay curves when simulating data under the same simple population con- traction model (Figure S3 in File S1). When examining the 1000 Genomes data, we found that heterozy- gosity in the neutral regions was higher than that seen for the genome- wide distribution of heterozygosity calculated in 10-kb windows (Table S1 in File S1; e.g., CEU mean heterozygosity per site for whole genome: 7.8 · 1024 vs. Heterozygosity predicted by demographic models neutral: 9.4 · 1024), suggesting that natural selection has directly and/or indirectly affected genome-wide patterns of heterozy- gosity. When the published demographic models were compared to the neutral heterozygosity distributions, we found similar trends to those seen for the whole genome data (Figure S2 in File S1). SFS predicted by demographic models Lastly, we examined which of the demographic models could match the SFSofthe empirical data. Toaccount for the possibility of overﬁtting the SFS-based Gutenkunst model to the SFS it was inferred from, we also compared all models to empirical SFSs based on low-coverage, high-throughput 1000 Genomes sequence data from the same three populations. LD predicted by demographic models SFSs are scaled using the ancestral population size given by each model, the mutation rate used to scale each model by the authors, and the sequence length of the empirical data set (4.04 Mb). See Figure S6 in File S1 for scaling using al- ternative mutation rates. and (C) YRI populations SFS is from noncoding s Gutenkunst et al. (2009 graphic histories for the tions. SFSs are scaled u population size given by mutation rate used to s by the authors, and the of the empirical data se Figure S6 in File S1 for ternative mutation rates et al. (2009) demographic history matches the observed SFS closely, being only 9 log-likelihood units worse than the best possible ﬁt (com- paring the empirical SFS to itself) for CEU, 48 units worse for CHB, and 17 units worse for YRI (Table 1). In comparison, the best-ﬁtting MSMC models for each population are 152, 188, and 373 log-likelihood units below the best possible ﬁt (Table 1). The combined whole genome plus SFS method SMC++ has an intermediate ﬁt, with a log-likelihood well below the Gutenkunst model, but consistently better than any of the MSMC models (Table 1). ﬁt; followed by MSMC 2-haplotype which fell 278 (CEU), 378 (CHB), and 455 (YRI) log-likelihood units below the optimal ﬁt (Table S3 in File S1). In all three populations, the MSMC 4-haplotype and 8-haplotype models are thousands of log-likelihood units worse than the best possible ﬁt, showing no improvement based on using a larger number of individuals in the inference (Table S3 in File S1). The over- estimation of SNPs in the 4-haplotype model is due to the model’s extremely high predicted ancestral size (200,000 individuals for each population) (Table S3 in File S1). Interestingly, there is not a consistent improvement in ﬁt to the observed SFS when increasing the number of individuals used for the MSMC inference. For each population, the 4-haplotype model has the worst ﬁt (Figure 4 and Table 1). For CEU and YRI, the MSMC 2-haplotype models ﬁt best out of the MSMC models, but both are .100 log-likelihood units worse than the Gutenkunst model. For CHB, the 8-haplotype model ﬁts best, but is still 140 units worse than the Gutenkunst model (Table 1). For both the proportional and absoluteSFSs, we found that rescaling themodelsusingahighermutationratedidnotproducelargequalitative differencesinhow the MSMC modelsﬁt the observed(Gutenkunst)SFS (Supplementary Note 5 and Figures S4–S6 in File S1). LD predicted by demographic models None of the published demographic models could perfectly recapitulate the empirical LD decay curve (Figure 3). For SNP pairs ,10 kb apart, the MSMC-8 haplotype model comes closest to the empirical curve for the CEU and CHB populations (Figure 3, A and B), but underes- timates the amount of LD, while all other models predict too much LD. The Gutenkunst and SMC++ models predict similar LD curves and are closer to the empirical curve than the MSMC 2-haplotype and Comparing to the observed Gutenkunst SFS: For each population, the SFSs predicted by the three MSMC models do not match the empirical proportional SFS from Gutenkunst et al. (2009), regardless of the mu- tation rate or number of genomes used (Figure 4, Table 1, and Figure S5 and Table S2 in File S1). The expected SFS based on the Gutenkunst 3612 \| A. C. Beichman, T. N. Phung, and K. E. Lohmueller Figure 5 SNP count SFSs using the counts of SNPs for the (A) CEU, (B) CHB, and (C) YRI populations. The “observed” SFS is from noncoding sequence used by Gutenkunst et al. (2009) to infer demo- graphic histories for these three popula- tions. SFSs are scaled using the ancestral population size given by each model, the mutation rate used to scale each model by the authors, and the sequence length of the empirical data set (4.04 Mb). See Figure S6 in File S1 for scaling using al- ternative mutation rates. Figure 5 SNP count SFSs using the counts of SNPs for the (A) CEU, (B) CHB, and (C) YRI populations. The “observed” SFS is from noncoding sequence used by Gutenkunst et al. (2009) to infer demo- graphic histories for these three popula- tions. SFSs are scaled using the ancestral population size given by each model, the mutation rate used to scale each model by the authors, and the sequence length of the empirical data set (4.04 Mb). See Figure S6 in File S1 for scaling using al- ternative mutation rates. Figure 5 SNP count SFSs using the counts of SNPs for the (A) CEU, (B) CHB, and (C) YRI populations. The “observed” SFS is from noncoding sequence used by Gutenkunst et al. (2009) to infer demo- graphic histories for these three popula- tions. LD predicted by demographic models The “1000 Genomes (WG)” SFS is from low-coverage whole genome 1000 Genomes data, and the “1000 Genomes (Neut)” SFS is from 6333 · 10-kb putatively neutral regions in the 1000 Genomes data. ForYRI,theGutenkunstmodelisthebest-ﬁttingmodelforthewhole genome and neutral 1000 Genomes SFSs, both with and without singletons, with all other models having a much worse ﬁt (the next best model, SMC++, is hundreds to thousands of log-likelihood units below the ﬁt of the Gutenkunst model) (Figure 6C and Table S4 in File S1). For CEU and CHB, if singletons are included, SMC++ ﬁts the whole genome and neutral 1000 Genomes SFSs best. For CEU, the Gutenkunst model then ﬁts second best, with the MSMC models far behind (Figure 6A and Table S4 in File S1). For CHB, the MSMC 2-haplotype ﬁts second best after SMC++, with the Gutenkunst model coming third, but both are .10,000 log-likelihood units below SMC++ (Figure 6B and Table S4 in File S1). If singletons are excluded for CEU and CHB, then the Gutenkunst model ﬁts best, with SMC++ coming in second, and the MSMC models all ranking far below (Table S4 in File S1). inferred using other methods, could be artifacts that are causing the SFS predicted by these models to deviate from the true SFS. To testthis hypothesis, we took the best ﬁtting of the MSMC models, the CEU 2-haplotype model, and carried out a series of adjustment experimentstodeterminewhetherchangestothemodelcouldprovide a better ﬁt to the observed SFS. Without adjusting the time period encompassed by the model, we altered the ancestral population size toavarietyofvaluesincludingthoseinferredbyGutenkunstetal.(2009) (Supplementary Note 7 and Figures S10 and S11 in File S1). We also truncated the MSMC trajectory to remove ancient events and better match the time period (in years) encompassed by the Gutenkunst et al. (2009) model. We again adjusted the ancestral population size to a variety of plausible values (Supplementary Note 7 and Figures S12 and S13 in File S1). Wefoundtrimmingawaytheancient(olderthan225KYA)partof the demographic trajectory and lowering the ancestral population size to 10,000–12,300 (compared to 41,261 inferred initially) dramatically improved the ﬁt of the proportional SFSs predicted under these ad- justed models to the observed (Gutenkunst) SFS (Figure S12 and Table S5 in File S1). The best-ﬁt model with ancestral size (NA) equal to 12,300 was brought to within 38 log-likelihood units of the best possible likelihood (Figure S12D and Table S5 in File S1), only 29 units below the Gutenkunst model. LD predicted by demographic models When repeating this procedure using the SFS based on counts, the SFSs under these adjusted models showed a dif- ferent pattern of improvement. Here the untrimmed models that did not have ancient events .225 KYA trimmed away, but had a lowered LD predicted by demographic models Comparing to the folded low-coverage 1000 Genomes SFS: To avoid givingtheGutenkunstmodelanunfairadvantagebyﬁttingallmodelsto the SFS used to infer that particular model, we also compared all models to proportional folded SFSs based on whole genome and neutral data from the 1000 Genomes Project (Figure 6 and Figure S7 in File S1). The ﬁt to the empirical singletons bin was poor for all models, except for SMC++, which was, in part, ﬁt to an SFS based on 1000 Genomes data. Calling singletons is notoriously difﬁcult in low-coverage data, making that bin the least reliable in the 1000 Genomes data (Kim et al. 2011; Nielsen et al. 2011; Han et al. 2014, 2015). We therefore calculated likelihoods for all models relative to the data both with singletons in- cluded and again with the SFSs renormalized without the singletons category (Figure S7 and Table S4 in File S1). The above comparisons considered the proportions of SNPs at speciﬁc frequencies in the sample. We also performed a comparison of the number of SNPs in each bin of the SFS, the absolute SFS, to the observed absolute SFS used in Gutenkunst et al.’s (2009) inference using a Poisson likelihood. The absolute SFS expected under the Gutenkunst et al. (2009) model ﬁts the observed SFS best (Figure 5 and Table S3 in File S1), and is only 9, 49, and 17 log-likelihood units below the best possible ﬁts for CEU, CHB, and YRI models, respectively. The SMC++ models have the next-best ﬁt to the absolute SFS, but come 86 (CEU), 176 (CHB), and 193 (YRI) log-likelihood units below the best possible Volume 7 November 2017 \| Disparate Demographic Histories \| 36 3613 Figure 6 Folded proportional SFSs for (A) CEU, (B) CHB, and (C) YRI populations. The “1000 Genomes (WG)” SFS is from low-coverage whole genome 1000 Genomes data, and the “1000 Genomes (Neut)” SFS is from 6333 · 10-kb putatively neutral regions in the 1000 Genomes data. Figure 6 Folded p for (A) CEU, (B) C Figure 6 Folded proportional SFSs for (A) CEU, (B) CHB, and (C) YRI populations. The “1000 Genomes (WG)” SFS is from low-coverage whole genome 1000 Genomes data, and the “1000 Genomes (Neut)” SFS is from 6333 · 10-kb putatively neutral regions in the 1000 Genomes data. Figure 6 Folded proportional SFSs for (A) CEU, (B) CHB, and (C) YRI populations. 3614 \| A. C. Beichman, T. N. Phung, and K. E. Lohmueller Effect of uncertain ancestral population size The pink bars denote the expected SFS under the average of the 50 MSMC 2-haplotype demographic model trajectories for each population. Note that these three SFSs agree. (Figure 7A). Both of these phenomena were also noted by Bunnefeld et al. (2015). ancestral population size of 7300–12,300, showed the most improve- ment (Figures S11 and S12 in File S1). However, their ﬁt was still .100 log-likelihood units worse than the Gutenkunst model (Figure S12 and Table S6 in File S1). The SFSs predicted by the demographic models inferred using MSMC on the simulated dataﬁt the SFSexpectedunder the Gutenkunst model and the observed Gutenkunst SFSs better than the MSMC demographic models inferred by Schiffels and Durbin (2014) (Figure 7, B and C). The proportional MSMC simulated data SFSs were only 40, 74, and 10 log-likelihood units below the Gutenkunst model SFS (Table S2 in File S1), with the SFSs based on SNP counts showing a similar pattern (Table S3 in File S1). Therefore, if the Gutenkunst model is the true demographic model for human history, MSMC accurately cap- tures the population size changes and produces an appropriate SFS. Effect of uncertain ancestral population size The accuracy of ancient ancestral population sizes, particularly .3 MYA (.100,000 generations), using the whole genome-based methods remains unclear (Li and Durbin 2011). As discussed above, the MSMC 2-haplotype and 4-haplotype models infer large ancestral sizes for each population that are not supported by previous inferences of human demographic history (Adams and Hudson 2004; Keinan et al. 2007; Boyko et al. 2008; Gutenkunst et al. 2009; Nielsen et al. 2009; Gravel et al. 2011). We hypothesized that these extreme ancestral sizes, as well as ancient bottlenecks and population growth (the signature humps of MSMC trajectories), which do not appear in demographic models 3614 \| A. C. Beichman, T. N. Phung, and K. E. Lohmueller 3614 Figure 7 MSMC 2-haplotype can accurately infer the demographic model predicted by Gutenkunst et al. (2009). (A) The results of running MSMC 2-haplotype on 50 independent two-haplotype data sets simulated under the Gutenkunst et al. (2009) model of human demographic history (Gutenkunst, heavy purple line). The resulting MSMC 2-haplotype trajectories (“MSMC Sim. Gut. Data,” ﬁne pink lines) show the MSMC trajectories inferred from these 50 data sets. Note that these trajectories accurately track the demographic model used to simulate the data. (B) and (C) show proportional and SNP count SFSs for each population, respectively. The gray bars (observed) denote the empirical SFS used by Gutenkunst et al. (2009). The purple bars denote the expected SFS under the inferred Gutenkunst demographic models. The pink bars denote the expected SFS under the average of the 50 MSMC 2-haplotype demographic model trajectories for each population. Note that these three SFSs agree. Figure 7 MSMC 2-haplotype can accurately infer the demographic model predicted by Gutenkunst et al. (2009). (A) The results of running MSMC 2-haplotype on 50 independent two-haplotype data sets simulated under the Gutenkunst et al. (2009) model of human demographic history (Gutenkunst, heavy purple line). The resulting MSMC 2-haplotype trajectories (“MSMC Sim. Gut. Data,” ﬁne pink lines) show the MSMC trajectories inferred from these 50 data sets. Note that these trajectories accurately track the demographic model used to simulate the data. (B) and (C) show proportional and SNP count SFSs for each population, respectively. The gray bars (observed) denote the empirical SFS used by Gutenkunst et al. (2009). The purple bars denote the expected SFS under the inferred Gutenkunst demographic models. Volume 7 November 2017 \| Disparate Demographic Histories \| 3615 DISCUSSION g p g p We found that the models based on MSMC inference from four oreighthaplotypesdidnotimprovetheﬁtoftheexpectedSFScompared to that based on two haplotypes; in fact, in most cases the 4- and 8-haplotype models ﬁt much worse than the 2-haplotype models. The 4-haplotype modelsforCEU,CHB, andYRI and the 8-haplotype model for YRI appear to ﬁt poorly due to their extremely high ancestral sizes and ancient humps of growth and decline (Figure 1). The expected SFSs under the 8-haplotype models for CEU and CHB show a skew toward low-frequency variants that may be due to their low ancestral size followed by extreme recent growth (Figure 1). We ﬁnd that MSMC 8-haplotype vastly overestimates recent growth in simulated data, which may be contributing to the lack of ﬁt to the SFS (Figure S8 in File S1). This result is at odds with the ﬁndings of Schiffels and Durbin (2014), who suggested that using eight haplotypes instead of two should increase accuracy of population size inference in the recent past, though they also noted a bias toward smaller ancient population sizes when using an increased number of haplotypes. Changing the scaling of the mutation rate did not generally help the MSMC models to ﬁt the expected SFS better (Figures S4–S6 in File S1). It is worth noting that the model inferred in SMC++ used the same mutation rate as MSMC, yet ﬁt the empirical SFSs much better (Figure 4, Figure 5, Figure 6, Table 1, and Tables S2–S4 in File S1), indicating that mutation rate differences between the whole genome- and SFS-based studies is not the source of the discrepancies. Alternatively, if the Gutenkunst et al. (2009) demographic model is largely accurate, biases or other factors that exist in real data but not in simulated data may be affecting MSMC inference, resulting in the method failing to recover an underlying demography that matches Gutenkunst et al.’s (2009) model. For example, Song et al. (2016) found that statistical phasing could affect MSMC estimates of population split times; and Nadachowska-Brzyska et al. (2016) found that per-site se- quencing depth, mean genome coverage, and the amount of missing data led to differences in PSMC curve amplitudes, expansions and contractions, and the timing and values of Ne. They therefore recom- mended only using samples with a mean genome coverage of $18· and ,25% missing data, and employing a per-site sequencing depth ﬁlter of $10 (Nadachowska-Brzyska et al. 2016). DISCUSSION It is worth noting that the model inferred in SMC++ used the same mutation rate as MSMC, yet ﬁt the empirical SFSs much better (Figure 4, Figure 5, Figure 6, Table 1, and Tables S2–S4 in File S1), indicating that mutation rate differences between the whole genome- and SFS-based studies is not the source of the discrepancies. f d h dd ﬁ h lS S h S C We tested which published models of human demographic history, inferred using either whole genome sequence data, the SFS, or a combined approach, can recapitulate multiple summaries of human genetic variation data. We found that no model was able to recapitulate all summaries of the data, but some models still performed better than others. In particular, none of the models was able recapitulate LD decay, but the GutenkunstSFS-basedmodels andthe combined wholegenome and SFS-based SMC++ models were able to recapitulate empirical heterozygosity and the SFS. MSMC 2-haplotype was able to recapitulate heterozygosity, but not the SFS, and MSMC 4-haplotype and 8-haplotype could ﬁt neither heterozygosity nor the SFS, though MSMC 8-haplotype did ﬁt LD decay slightly better than the other models. These results highlight the uncertainties of demographic inference from one, or even two, types of data and the need to assess the ﬁt of demographic models using multiple summaries of the data. y While our results indicate that features of MSMC trajectories, particularly ancient events, should be regarded with caution, we also found that MSMC 2-haplotype is able to accurately recapitulate a complex demography (with the exception of steep drops in population size, extreme recent growth, and some false periods of growth) from simulated data, supporting the validity of the method, at least for use on simulated data (Figure 7). Migration between populations did not ap- pear to cause deviations in MSMC trajectories from the underlying model (Figure 7), nor did a small degree of Neanderthal admixture (Figure S9 in File S1), indicating that MSMC is robust to small amounts of gene ﬂow. The fact that the 2-haplotype model based on real data did not ﬁt the observed SFS very well (Figure 4, Figure 5, Figure 6, Table 1, and Tables S2–S4 in File S1) suggests that the true underlying pattern of human demography is more complex than either type of inference (@a@i or MSMC) is capturing, potentially revealing weaknesses in both methods. MSMC population size trajectories for demographic models inferred from the SFS Given that the SFSs predicted by the demographic models inferred using MSMC do not ﬁt the observed SFS, we examined whether MSMC is capable of recovering a complex demography such as the one inferred by Gutenkunst et al. (2009) from a single simulated genome. We ﬁnd that MSMC performs relatively well at inferring the underlying demography from the simulated data. Figure 7A shows the underlying Gutenkunst demographic model for each population (purple) (as in the other Gutenkunst model simulations, migration is included in the model, but is not depicted in our dia- grams), with the results of 50 independent MSMC inferences on each 2-haplotype simulated data set coming close to the underlying demography. However, sharp bottlenecks are inferred as long pop- ulation declines (as noted by Li and Durbin 2011 and Schiffels and Durbin 2014). Additionally, we found evidence of MSMC detecting a false spurt of growth in the YRI population 1350 generations ago p p g p pp p It is well established that two-haplotype, whole genome-based in- ference (PSMC and MSMC 2-haplotype, also known as PSMC9) is not able to detect recent demographic events (Li and Durbin 2011; Schiffels and Durbin 2014). However, the ability to detect recent growth by using more than two haplotypes in the inference is cited as a feature of MSMC (Schiffels and Durbin 2014). We ran MSMC 2-haplotype and 8-haplotype on data sets simulated under the Gutenkunst model and a Gutenkunst model plus extreme recent growth (Figure S8 and Supple- mentary Note 6 in File S1). Unsurprisingly, MSMC 2-haplotype was not able to detect extreme recent growth. Its estimates of current population size were fairly accurate for the original Gutenkunst model Volume 7 November 2017 \| Disparate Demographic Histories \| 3615 3615 (Figure 7A), but the method dramatically underestimated the growth for data simulated under the Gutenkunst plus growth model (Figure S8 in File S1). The results from 8-haplotype MSMC inference were most surprising. We found that for both models, MSMC 8-haplotype inferred extreme recent growth as many as four orders of magnitude beyond that in the underlying model, with a high degree of variance between replicates (Figure S8 in File S1). Despite the high degree of variance, the average of the MSMC trajectories all showed a strong upward bias in estimates of the recent past (Figure S8 in File S1). MSMC population size trajectories for demographic models inferred from the SFS While the ability to detect recent growth is meant to be a feature of MSMC, our ﬁndings indicate that the magnitude of growth may not be esti- mated well. distribution of heterozygosity (Figure 2), which may be surprising as the genome-wide distribution of heterozygosity is a major feature of the data used by MSMC. As with the SFS, the reason for the lack of ﬁt for these models appears to be the extremely high ancestral size inferred in the 4-haplotype models for all three populations and in the 8-haplotype YRI model, and the low ancestral size inferred in the 8-haplotype models for CEU and CHB (Figure 1). Since the most ancient size in the MSMC trajectory will have a large inﬂuence on heterozygosity and the SFS and the most ancient bin of the MSMC trajectory may be unreliable (Li and Durbin 2011; Schiffels and Durbin 2014), we explored the effect of altering this ancient size and removing ancient growth events in the CEU MSMC 2-haplotype model. We found that selective trimming could improve the ﬁt to the SFS (Figures S10–S13 in File S1). However, the ﬁnal bin of the model cannot explain all of the lack of ﬁt of the MSMC models to the data as the CEU and CHB MSMC 8-haplotype trajectories do not show the extreme ancestral sizes in the last bin, yet these models also dramatically deviate from empirical heterozygosity and the SFS. In other words, simple exclusion of the ﬁnal high ancestral size is not sufﬁcient to improve model ﬁt to other summaries of the data. Our trimming experiments were only made possible by the abundance of human sequence data and demographic models previously ﬁt to the data. Since many MSMC trajectories are calculated for species for which there is no prior information about ancient demographic history, the “informed trimming” we carried out is not a practicable solution to improve the reliability of MSMC inference. We had hypothesized that Neanderthal admixture could cause deviation between the MSMC and Gutenkunst demographic models, butfoundthattheadditionofNeanderthaladmixturetoourGutenkunst model simulations did not substantively change the MSMC trajectories orexpectedSFSs(FigureS9,SupplementaryNote6,andTablesS2andS3 in File S1). 3616 \| A. C. Beichman, T. N. Phung, and K. E. Lohmueller DISCUSSION DISCUSSION We tested which published models of human demographic history, inferred using either whole genome sequence data, the SFS, or a combined approach, can recapitulate multiple summaries of human genetic variation data. We found that no model was able to recapitulate all summaries of the data, but some models still performed better than others. In particular, none of the models was able recapitulate LD decay, but the GutenkunstSFS-basedmodels andthe combined wholegenome and SFS-based SMC++ models were able to recapitulate empirical heterozygosity and the SFS. MSMC 2-haplotype was able to recapitulate heterozygosity, but not the SFS, and MSMC 4-haplotype and 8-haplotype could ﬁt neither heterozygosity nor the SFS, though MSMC 8-haplotype did ﬁt LD decay slightly better than the other models. These results highlight the uncertainties of demographic inference from one, or even two, types of data and the need to assess the ﬁt of demographic models using multiple summaries of the data. We found that the models based on MSMC inference from four oreighthaplotypesdidnotimprovetheﬁtoftheexpectedSFScompared to that based on two haplotypes; in fact, in most cases the 4- and 8-haplotype models ﬁt much worse than the 2-haplotype models. The 4-haplotype modelsforCEU,CHB, andYRI and the 8-haplotype model for YRI appear to ﬁt poorly due to their extremely high ancestral sizes and ancient humps of growth and decline (Figure 1). The expected SFSs under the 8-haplotype models for CEU and CHB show a skew toward low-frequency variants that may be due to their low ancestral size followed by extreme recent growth (Figure 1). We ﬁnd that MSMC 8-haplotype vastly overestimates recent growth in simulated data, which may be contributing to the lack of ﬁt to the SFS (Figure S8 in File S1). This result is at odds with the ﬁndings of Schiffels and Durbin (2014), who suggested that using eight haplotypes instead of two should increase accuracy of population size inference in the recent past, though they also noted a bias toward smaller ancient population sizes when using an increased number of haplotypes. Changing the scaling of the mutation rate did not generally help the MSMC models to ﬁt the expected SFS better (Figures S4–S6 in File S1). DISCUSSION (2015) found that they could not recover empirical LD patterns in Drosophila, despite matching the SFS, number of segregating sites (S), and number of pairwise differences ðpÞ: Garud et al. (2015) sug- gested the lack of ﬁt could either be due to linked positive selection or to an incompleteness of the demographic model, demonstrating how models that ﬁt some summaries of the data may not recapitulate others. It is more surprising that the MSMC 2-haplotype and 4-haplotype models do not ﬁt the data well, as the method uses LD information in its inference, though different summaries of LD may be affected by demography in distinct ways (Plagnol and Wall 2006). Other possible factors that could lead to the lack of ﬁt of all models to empirical LD decay patterns include the absence of natural selection, gene conver- sion, and ﬁne-scale recombination hotspots in our simulations (Ardlie et al. 2001; Frisse et al. 2001; Wall and Pritchard 2003). Further, if the true mutation rate is actually smaller than the relatively high value used by Gutenkunst et al. (m ¼ 2.35 · 1028 mutations/bp/generation), then the population sizes would have to be larger than those estimated by Gutenkunst et al. (2009). Larger population sizes would yield larger values of the population scaled recombination rate ðrÞ than what was used in our simulations under the Gutenkunst model. Larger values of r would then lead to a decrease in LD in the simulations, which might better match the empirical LD decay curves. Deep ancestral structure has been put forward as explanation for the humps detected by the whole genome-based methods by the developers of PSMC and others (Li and Durbin 2011; Henn et al. 2012; Mazet et al. 2015a,b; Orozco-terWengel 2016). While Blum and Jakobsson (2011) used the TMRCA to postulate an ancient bottleneck 150 KYA, they also were not able to reject a model of ancestral structure. Strikingly, Mazet et al. (2015b) were able to perfectly recapitulate the human PSMC humps without invoking a single size change in the population by simulating data from a highly structured ancestral population (10 sub- populations) and modulating the amount of gene ﬂow between these populations. Therefore, the large “population size changes” inferred in MSMC, which cause the models not to match the empirical SFS, may in fact be due to complex structure and large-scale changes in gene ﬂow. DISCUSSION The Complete Geno- mics genomes used by Li and Durbin (2011) were .40· coverage (Drmanac et al. 2010), indicating that lack of coverage is not respon- sible for their divergence from estimates based on the SFS. However, the standards suggested by Nadachowska-Brzyska et al. (2016) may not always be attainable in de novo genome projects and, thus, data quality issues may affect nonmodel organism PSMC and MSMC inferences WefoundthatinadditiontonotﬁttingtheempiricalSFS,theMSMC 4-haplotype and 8-haplotype models did not predict the genome-wide 3616 \| A. C. Beichman, T. N. Phung, and K. E. Lohmueller Figures S10–S13 in File S1). The fact that they are not seen in the observed SFS suggests that either the size changes did not occur, and the inferred size changes are artifacts, or instead, the true demography is more complex than currently modeled using either approach. Our conclusion of ﬁnding little evidence for the ancient population size changes is supported by the study of Sjödin et al. (2012). They employed an ABC approach to directly test models with ancient pop- ulation size changes in Africa and found little support for such ancient bottlenecks. Figures S10–S13 in File S1). The fact that they are not seen in the observed SFS suggests that either the size changes did not occur, and the inferred size changes are artifacts, or instead, the true demography is more complex than currently modeled using either approach. Our conclusion of ﬁnding little evidence for the ancient population size changes is supported by the study of Sjödin et al. (2012). They employed an ABC approach to directly test models with ancient pop- ulation size changes in Africa and found little support for such ancient bottlenecks. more acutely. Future work should also examine the impact of artifacts of genome assembly errors and structural variants on PSMC inference. For example, collapsing duplicate regions of the genome on top of each other could result in regions of the genome having excess heterozygos- ity, which could in turn affect inference of demography. We found that no model was able to accurately recapitulate the empirical distribution of LD decay. The lack of ﬁt of the SFS-based modelsisperhapsunsurprising,as Harris andNielsen (2013) foundthat the Gutenkunst model cannot recapitulate empirical IBS distributions (a ﬁner-scale summary of the data related to LD), and Garud et al. DISCUSSION This ancient structure may have a large effect on MSMC trajectories and LD patterns, but may not strongly inﬂuence the SFS (see ﬁgure 7 in Lohmueller et al. 2009), potentially resolving the discrepancy between the methods (Henn et al. 2012). Our work provides a cautionary tale for understanding population history in nonmodel organisms. Our results argue against a literal interpretation of humps and other jumps in MSMC plots as reﬂecting population size changes. This problem is exacerbated for putative ancient size changes. Given the ever-increasing generation of genomic data from nonmodel taxa and the application of whole genome-based approaches to such data (Groenen et al. 2012; Meyer et al. 2012; Zhao et al. 2012; Albert et al. 2013; Ibarra-Laclette et al. 2013; Nadachowska- Brzyska et al. 2013, 2015, 2016; Orlando et al. 2013; Prado-Martinez et al. 2013; Bosse et al. 2014; Freedman et al. 2014; Hung et al. 2014; Prufer et al. 2014; Palkopoulou et al. 2015; Holliday et al. 2016; Wang et al. 2016), our ﬁndings are especially concerning. We recommend employing other model-based types of demographic inference leverag- ing either SFS-based or other summary statistics in an ABC framework to test whether important demographic features suggested by PSMC or MSMC plots can be recapitulated using other features in the data. We also recommend, as done in Thornton and Andolfatto (2006), Freedman et al. (2014), Cahill et al. (2016), Hsieh et al. (2016), and Song et al. (2016), that the PSMC or MSMC plots and TMRCA esti- mates be used themselves as summary statistics for model comparison, rather than the actual population size estimates. In other words, more complex demographic models can be simulated and tested to see whether they recapitulate the observed whole genome-based trajecto- ries. Of course, this approach will not be successful if the trajectories are strongly inﬂuenced by bioinformatic artifacts or other features not captured within the simulations, such as natural selection. For both PSMC/MSMC and SFS-based inference methods, we also recommend testing whether the estimated models can predict multiple features of the data. Speciﬁcally, researchers should check whether their inferred model can recapitulate the genome-wide distribution of heterozygosity. The genome-wide distribution of heterozygosity may be the most prac- tical and useful statistic for studies of nonmodel organisms that only have a handful of genomes available to them. SMC++ and other new approaches that leverage multiple types of data (Bunnefeld et al. Volume 7 November 2017 \| Disparate Demographic Histories \| 3617 DISCUSSION 2015; Boitard et al. 2016; Weissman and Hallatschek 2017) are promising Natural selection may affect both SFS- and whole genome-based methods of demographic inference. Li and Durbin (2011) found that masking exonic sequence did not alter PSMC trajectories. However, Schrider et al. (2016) examined the impact of selective sweeps on de- mographic inference using the SFS in @a@i, approximate Bayesian computation (ABC), and PSMC and found that all three methods were inﬂuenced to varying degrees and in slightly different directions by the presence of selective sweeps, with @a@i the most robust to these effects. This is a concern for published human demographic models as Gutenkunst et al. (2009) used noncoding sequence from autosomal genes in their study, which may be subject to linked selection (Gazave et al. 2014; Schrider et al. 2016). Schiffels and Durbin (2014) used whole genome sequences that included genic and nongenic re- gions, some of which are certainly under selection. Thus, the sensitivity of these methods to selection may partially explain why both perform well on simulated data without selection, yet have such divergent results when run on empirical data. Our results have implications for understanding human demo- graphic history. First, there has been controversy concerning the pres- ence of ancient bottlenecks (.100 KYA) in human populations (Takahata et al. 1995; Harpending et al. 1998; Takahata and Satta 1998; Hawks et al. 2000; Garrigan and Hammer 2006; Fagundes et al. 2007; Scholz et al. 2007; Blum and Jakobsson 2011; Sjödin et al. 2012). 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W1868552708.txt	http://www.scielo.br/pdf/pusf/v17n2/v17n2a13.pdf	en		Evaluation of sleepiness in college students from different shifts	Psico-USF	2,012	cc-by	4,908	Psico-USF, Bragança Paulista, v. 17, n. 2, p. 295-302, mai./ago. 2012 295 Avaliação da sonolência em estudantes universitários de turnos distintos Danilo de Freitas Araújo – Universidade Federal do Rio Grande do Norte, Natal, Rio Grande do Norte, Brasil Katie Moraes de Almondes – Universidade Federal do Rio Grande do Norte, Natal, Rio Grande do Norte, Brasil Resumo Estudantes de graduação possuem altas chances de apresentar Sonolência Diurna Excessiva, devido aos horários escolares e às demandas acadêmicas. Por isso, pretendeu-se analisar níveis de sonolência de estudantes de turnos distintos. O universo foi constituído por 109 graduandos do turno matutino e 125 do noturno. Utilizou-se a Escala de Sonolência de Epworth. A amostra apresentou média total de 9,38 (DP=4,03), sendo 9,03 (DP=4,01) para o turno matutino e 9,7 (DP=3,93) para o noturno. Foram detectadas diferenças significativas nos níveis de sonolência entre turnos (η2 =0,10; p < 0,00), e entre os indivíduos do gênero masculino e feminino nos dois turnos (η2=0,45; p<0,00). Assim, deve ser considerado o papel das demandas acadêmicas em cada turno, já que elas afetam diretamente a qualidade do sono. Palavras-chave: Sono; Escala de Sonolência Epworth; Estudantes universitários. Evaluation of sleepiness in college students from different shifts Abstract Undergraduate students are a population with high chances of developing Excessive Daytime Sleepiness, due to school schedules and academic demands. Our aim was to examine the levels of sleepiness of students belonging to morning and night classes. The universe has consisted of 109 undergraduate students of the morning shift and 125 of the night shift. We have used the Epworth Sleepiness Scale. The sample had an average of 9.38 (SD=4.03), and 9.03 (SD=4.01) for the morning shift and 9.7 (SD=3.93) for the night shift. Statistical analysis showed significant differences in sleepiness between shifts (η2=0,10; p<0,00), and among male subjects and female in the two shifts (η2=0,45; p<0,00). It should therefore be considered the role of academic demands, since they are directly affecting the quality of sleep and the demands inherent to women. Keywords: Sleep; Epworth Sleepiness Scale; College students. Evaluación de la somnolencia en estudiantes universitarios de turnos distintos Resumen Estudiantes de graduación poseen altas posibilidades de presentar Somnolencia Diurna Excesiva, debido a los horarios escolares y a las demandas académicas. Por eso, se pretendió analizar niveles de somnolencia de estudiantes de turnos distintos. El universo fue constituido por 109 graduandos del turno matutino y 125 del nocturno. Se utilizó la Escala de Somnolencia de Epworth. La muestra presentó promedio total de 9,38 (DP=4,03), siendo 9,03 (DP=4,01) para el turno matutino y 9,7 (DP=3,93) para el nocturno. Fueron detectadas diferencias significativas en los niveles de somnolencia entre turnos (η2=0,10; p<0,00), y entre los individuos del género masculino y femenino en los dos turnos (η2=0,45; p<0,00). Así, debe ser considerado el papel de las demandas académicas en cada turno, ya que ellas afectan directamente la calidad del sueño. Palabras-clave: Sueño; Escala de Somnolencia Epworth; Estudiantes universitarios. Estima-se que os distúrbios do sono afetam, no Brasil, pelo menos 10 a 20 milhões de pessoas (Danda, Ferreira, Azenha, Souza & Bastos, 2005). Dentre os sintomas provocados pelos distúrbios do sono encontram-se mal-estar, fadiga, irritabilidade, prejuízo na agilidade e eficiência mental (Campos & Martino, 2004). Outro sintoma muito recorrente diz respeito à Sonolência Diurna Excessiva (SDE). Estatísticas apontam que 4% a 12% da população em geral se queixam deste sintoma (Souza, Souza, Arashiro & Schaedler, 2007), que é uma das principais causas de acidentes automobilísticos, acidentes de trabalho, erros em atividades que demandam atenção (controle de tráfego aéreo, por exemplo), dentre outras (Moreno, Fischer & Rotenberg, 2003). A sonolência diurna excessiva relaciona-se ao sono que ocorre nas situações em que seria esperado que o indivíduo estivesse alerta e desperto. Por isso, este estado é constituído por ataques do sono, cochilos involuntários e necessidade subjetiva para dormir Disponível em www.scielo.br (Bittencourt, Silva, Santos, Pires & Mello, 2007). Dessa forma, sonolência excessiva pode ser conceituada como um estado em que ocorre desordem dos processos fisiológicos e cognitivos, repercutindo durante o estado de vigília do indivíduo (García & colaboradores, 2004). Associa-se, muitas vezes, a irregularidades do padrão de sono e duração de sono encurtada ou privação de sono, o que afeta a qualidade de sono. Assim, ela afeta diversos aspectos da vida de um indivíduo, como saúde, estudo, trabalho e relações interpessoais (Bittencourt & colaboradores, 2007). Dentre os prejuízos na esfera cognitiva estão a dificuldade de dirigir e manter constante a atenção, comprometimento na capacidade de planejamento, lapsos de memória, déficits na agilidade e precisão da coordenação motora, dentre outros (Danda & colaboradores, 2005). Dados da literatura apontam para as altas chances dos estudantes (sobretudo os do ensino superior) 296 Araújo, D. F. & Almondes, K. M. Sonolência em estudantes universitários desenvolverem sonolência excessiva diurna. Justifica-se essa afirmativa ao se mencionarem os períodos extensos destinados aos horários de aulas, aos estudos e às outras atividades curriculares (Medeiros & colaboradores, 2002), típicos de quem cursa a graduação de nível superior. Um exemplo disso é o estudo de Sai (2007) com uma amostra constituída por 150 estudantes de graduação chineses, cujos dados demonstraram uma correlação significativa entre altos níveis de sonolência e a duração do sono, sugerindo que o grande número de demandas acadêmicas afetava a duração do sono desses indivíduos, influenciando diretamente seus níveis de sonolência. Já o estudo de Morales, Flores, Meneses, Figueiras e Guerrero (2005) discute não só a prevalência de sonolência em estudantes, como também as consequências desta. Nessa pesquisa (realizada com 64 graduandos de psicologia), eles mostraram que havia altos níveis de sonolência diurna excessiva na amostra, relacionados a decréscimos nos níveis de compreensão e resolução de problemas matemáticos, indicando, com isso, uma associação significativa entre sonolência excessiva e rendimento acadêmico. O turno em que os graduandos estudam também pode se constituir num importante fator que influencia os níveis de sonolência diurna excessiva. De acordo com Lima, Medeiros e Araújo (2002), numa pesquisa com alunos de graduação, aqueles que estudavam muito cedo no turno matutino, em detrimento daqueles que estudavam mais tarde, apresentavam qualidade do sono ruim e, dessa forma, sonolência diurna. Natal e colaboradores (2009) demonstram que os estudantes com o sono mais negativamente afetado eram aqueles que cursavam o turno da manhã. Em ambos os estudos, as aulas começavam às 7h, e os estudantes tinham que acordar mais cedo para chegar a tempo nas aulas. Além disso, como resultado de cumprir as demandas acadêmicas, eles sofriam de privação de sono durante a semana e, por isso, a organização de suas atividades diárias em função dos seus horários de aula estava fortemente relacionada à privação. Por outro lado, além das demandas mencionadas, muitos dos estudantes de graduação dedicam-se também ao trabalho. Numa pesquisa com estudantes que cursavam o turno noturno e, além disso, trabalhavam, Fischer, Oliveira, Teixeira, Teixeira e Amaral (2003) encontraram que 46,3% desses alunos afirmaram sentir sonolência excessiva, e que ela estava relacionada à redução da duração do sono noturno, já que deviam acordar mais cedo para trabalhar. Teixeira (2002), em seu estudo, afirma que os padrões de sonolência de estudantes não-trabalhadores são diferentes dos de estudantes trabalhadores, uma vez que estes exercem atividades de trabalho que demandam horários alternativos para estudar (depois das 22h, por exemplo). Por isso, eles dormem mais tarde e têm menor duração do sono noturno, bem como menor qualidade do sono. Dessa forma, dados apontam que tanto no turno matutino como no turno noturno ocorrem exigências que afetam os níveis de sonolência dos estudantes (Agudelo & colaboradores, 2005; Natal & colaboradores, 2009). Em primeiro lugar, os horários de estudo no turno matutino que iniciam muito cedo (por volta das 7h) podem afetar a qualidade do sono e acarretar sonolência excessiva. Em segundo lugar, no turno noturno o trabalho pode se constituir num fator que estende o período de vigília e, juntamente com as atividades acadêmicas, afeta a duração de sono. Assim, a proposta deste estudo é analisar os níveis de sonolência de estudantes universitários, pertencentes a turmas de turnos diferentes (manhã e noite) de uma faculdade privada da cidade de Natal (RN), e verificar se há diferenças nos níveis de sonolência entre elas. Método Participantes O universo foi constituído por graduandos de uma faculdade privada de Natal, Rio Grande do Norte, totalizando 234 estudantes, dos quais 109 cursavam o turno matutino e 125 o turno noturno. A média de idade da amostra geral foi de 24 anos (DP=8,09 anos). No turno matutino constatou-se uma média de idade de 23,26 (DP=7,03 anos), enquanto no turno noturno os alunos apresentaram uma média de idade de 25,1 (DP=8,85 anos). Dos graduandos que compuseram a amostra, 66,7% foram mulheres, enquanto 33,3% foram homens, sendo que na amostra do turno matutino 73,4% foram do gênero feminino e 26,6% do gênero masculino, e na amostra do turno noturno, 60,8% foram do gênero feminino e 39,2% do masculino. A pesquisa, desta forma, coletou informações dos turnos da manhã (composto pelos cursos de Enfermagem, Nutrição, Fisioterapia e Direito) e da noite (abrangendo os cursos de Administração, Psicologia, Ciências Contábeis e Direito). As aulas do turno matutino iniciavam às 7h40, com término às 12h00, e as do turno noturno iniciavam às 19h00 e terminavam às 22h35. Instrumento e procedimentos Logo após ter sido efetivada a aprovação da pesquisa pelo Comitê de Ética em Pesquisa com Seres Humanos da instituição onde se realizou o presente estudo (sob o nº de protocolo 12/2007), procedeu-se ao levantamento dos cursos e turmas existentes. Assim, Psico-USF, Bragança Paulista, v. 17, n. 2, p. 295-302, mai./ago. 2012 Araújo, D. F. & Almondes, K. M. Sonolência em estudantes universitários optou-se por realizar a pesquisa com os discentes que se encontravam no segundo semestre do primeiro ano, pois essas turmas já estavam adaptadas ao regime de horários e demandas inerentes a uma instituição de ensino superior. Por meio de ofícios, requereu-se a autorização de cada coordenador dos cursos de graduação para a realização da pesquisa. Obtida essa autorização, efetivou-se a coleta: antes do começo das aulas ou ao término (com o consentimento dos professores que ocupavam o horário), adentrava-se nas salas de aula e procedia-se à leitura do Termo de Consentimento Livre e Esclarecido, ao mesmo tempo em que os objetivos da pesquisa eram expostos. Após a leitura do Termo, os estudantes voluntários o assinavam, concordando em participar do estudo; em seguida, preenchiam uma ficha de identificação, em que constavam os dados demográficos de perfil da amostra, como nome, gênero, local de trabalho, horários de aula e horários de esquema de trabalho, presença de problemas de saúde, dentre outros. O instrumento que se prestou a cumprir os objetivos desta pesquisa foi a Escala de Sonolência de Epworth (ESE), um questionário constituído por oito situações, tanto ativas como passivas, associadas a diferentes graus de sonolência (Johns, 1991). O indivíduo deveria responder qual seria a chance de cochilar em cada uma das ocasiões apresentadas, pontuando de 0 (nenhuma chance) a 3 (alta chance). Escores obtidos acima de 10 são diagnósticos de sonolência diurna excessiva. Análise dos dados Foi realizado o teste de Kolmogorov-Smirnov para avaliar a existência de normalidade dos referidos dados. Foram realizadas, ainda, análises descritivas (frequências, médias e desvios padrão). Foi usada a ANOVA de duas vias, turno e gênero, com o objetivo de investigar a interação entre os dois fatores. Foi utilizado também o teste quiquadrado para comparar as frequências de ocorrência de sonolência excessiva entre os dois turnos. E ainda, foi calculado o Coeficiente de Correlação de Pearson entre os níveis de sonolência diurna excessiva e os dados sociodemográficos, nos dois turnos. Foi utilizado o software Statistic (versão 11.0). Resultados Os dados obtidos foram considerados normais pelo teste de Kolmogorov-Smirnov. A amostra da presente pesquisa foi constituída por um total de 234 estudantes. Desse total, 13,2% já possuíam uma Psico-USF, Bragança Paulista, v. 17, n. 2, p. 295-302, mai./ago. 2012 297 formação de nível superior (correspondendo a 12% dos estudantes do turno matutino e 15% do turno noturno); 83,8% dos estudantes eram solteiros (86,2% do turno matutino e 81,6% do noturno); e 82,9% não possuíam filhos (88,1% do turno matutino e 78,4% do turno noturno). Além disso, 70,6% do turno matutino e 32% do turno noturno afirmaram não possuir nenhuma outra ocupação além dos estudos, demonstrando que no turno noturno havia um número maior de indivíduos com vínculo empregatício (ver Tabela 1). Em relação à sonolência, verificou-se que a amostra apresentou uma média total de 9,4 (DP=4,03) na Escala de Sonolência de Epworth (conforme apresentado na Tabela 2). O turno matutino obteve uma média de 9,03 (DP=4,01) na escala, enquanto o turno noturno alcançou uma média de 9,7 (DP=3,93). Esses dados apontam que os indivíduos dos dois grupos não foram diagnosticados com sonolência excessiva, mas as médias elevadas e o desvio padrão demonstraram tendência para o diagnóstico de sonolência. A ANOVA demonstrou que há diferenças significativas entre os níveis de sonolência do turno matutino e os níveis de sonolência do turno noturno (η2=0,10; p<0,00), sugerindo que o turno noturno obteve médias de níveis de sonolência significativamente maiores em relação ao turno matutino. Considerando a tendência para o diagnóstico de sonolência excessiva nos dois turnos, verificou-se que 33,94% dos estudantes do turno matutino e 42,4% dos estudantes do turno noturno foram diagnosticados com sonolência diurna excessiva (Tabela 2). Os dados sugerem que o turno noturno não apresentou ocorrência significativamente maior de casos diagnosticados de sonolência excessiva, em relação ao turno matutino, já que a diferença não foi estatisticamente significativa, segundo o Teste Quiquadrado (χ2=0,84; p=0,36). Na análise da interação entre as variáveis gênero e turno, os homens que estudavam no turno matutino obtiveram média de 7,93 (DP=3,98), ao passo que as mulheres alcançaram 9,42 (DP=4,09). Os homens que estudavam no turno noturno obtiveram 9,31 (DP=4,29) na Escala de Sonolência e as mulheres, 9,95 (DP=3,74). A ANOVA demonstrou que há diferenças significativas entre os níveis de sonolência dos indivíduos do gênero masculino e os indivíduos do gênero feminino nos dois turnos (η2=0,45; p<0,00), revelando que as mulheres apresentaram níveis maiores de sonolência excessiva do que os homens, independentemente dos turnos. Quando foi realizado o Teste de Correlação de Pearson para analisar se havia correlação entre os níveis 298 Araújo, D. F. & Almondes, K. M. Sonolência em estudantes universitários de sonolência e os dados demográficos, os resultados indicaram apenas uma correlação entre os níveis de sonolência e a condição de ter filhos no turno noturno (r=-0,18; p<0,05), sugerindo que os filhos parecem ser uma variável influenciadora para os indivíduos que apresentaram altos níveis de sonolência excessiva no turno noturno (Tabela 3). Tabela 1. Características sociodemográficas dos estudantes de graduação, nos dois turnos de estudo (n=234) Matutino Noturno Amostra Total Variáveis (n=109) (n=125) (n=234) Tipo sociodemográficas n % n % n % Gênero Masculino 29 26,6 49 39,2 78 33,3 Feminino 80 73,4 76 60,8 156 66,7 Estado civil Solteiro 94 86,2 102 81,6 196 83,8 Casado 15 13,8 23 18,4 38 16,2 Separado 0 0 0 0 0 0 Divorciado 0 0 0 0 0 0 Graduação Sim 13 12 19 15 32 13,7 anterior Não 96 88 106 85 202 86,3 Indivíduos que Sim 13 11,9 27 21,6 40 17,1 possuem filhos Não 96 88,1 98 78,4 194 82,9 Indivíduos que Sim 32 29,4 85 68 117 50 trabalham Não 77 70,6 40 32 117 50 Atividades extraOutros 4 3,7 4 3,2 8 3,4 faculdade Atividades voluntárias 0 0 3 2,4 3 1,3 Aula em outro curso 4 3,7 3 2,4 7 3 Trabalha 7 6,4 8 6,4 15 6,4 Pratica esportes 16 14,7 24 19,2 40 17,1 Faz cursos 4 3,7 8 6,4 12 5,1 Não realiza 54 49,5 61 48,8 115 49,1 Não respondeu 20 18,3 14 11,2 34 14,5 Idade M DP M DP M DP 23,26 7,03 25,1 8,85 24,24 8,09 Tabela 2. Valores em média e percentuais dos níveis de sonolência, por turno e gênero Geral Masculino Feminino Sonolência Turnos M DP M DP M DP n % Amostra total 9,40 4,03 8,79 4,20 9,68 3,92 90 38,46 Turno matutino 9,03 4,01 7,93 3,98 9,42 4,09 37 33,94 Turno noturno 9,70 3,93 9,31 4,29 9,95 3,740 53 42,40 Sem sonolência n % 144 61,64 72 66,06 72 57,60 Tabela 3. Coeficientes de correlação de Pearson entre níveis de sonolência e variáveis sociodemográficas Variáveis sociodemográficas Turno matutino Turno Noturno Gênero 0,16 0,16* Idade -0,19 0,04* Estado civil -0,08 0,02* Ter filhos 0,17 -0,18* Ter vínculo empregatício -0,02 -0,06* Problemas de saúde 0,14 -0,06* Uso de medicação -0,09 -0,07* *p<0,05 Discussão e considerações finais O presente estudo buscou avaliar os níveis de sonolência diurna excessiva em estudantes de graduação, pertencentes a turmas de turnos diferentes (manhã e noite). Em relação à amostra geral e aos turnos matutino e noturno, embora os dados não tenham apontado para o diagnóstico de sonolência Psico-USF, Bragança Paulista, v. 17, n. 2, p. 295-302, mai./ago. 2012 Araújo, D. F. & Almondes, K. M. Sonolência em estudantes universitários excessiva, as médias encontradas se mostraram elevadas, com tendência para o diagnóstico. Tal fato indica a influência que as demandas inerentes a cada turno exercem sobre o sono dos estudantes de graduação, e demonstra que as médias de sonolência não dependem diretamente do turno de estudo. Considerando os achados, não se pode ignorar que ocorrem, em ambos os turnos, atividades acadêmicas, extensos horários direcionados aos estudos, demandas extracurriculares inerentes ao curso universitário e aulas, que podem estar relacionados à privação do sono, acarretando, com isso, níveis elevados de sonolência ao longo do dia (Laberge & colaboradores, 2010). É possível perceber, por isso, que durante os anos de graduação, ocorre uma diminuição da duração total do sono, bem como um atraso em seu início (Carskadon, Acebo & Seifer, 2001; Ohayon, Carskadon, Guilleminault & Vitiello, 2004). O estudante é obrigado a restringir o sono durante a semana e a estendê-lo nos fins de semana e feriados para compensá-lo, ocasionando o chamado “efeito sanfona” (Thorleifsdottir, Björnsson, Benediktsdottir, Gislason & Kristbjarnarson, 2002). A sonolência excessiva, dessa forma, influencia de modo significativo a vida do estudante universitário, principalmente quanto ao seu rendimento acadêmico. Segundo Gianotti e Cortesi (1997), estudantes italianos que dormiam menos durante a semana queixavam-se mais de sonolência diurna e cochilavam um número maior de vezes do que a população em geral e, em decorrência disso, havia uma forte associação entre sono e baixo desempenho acadêmico. Num outro estudo, Gibson e colaboradores (2006) procuraram avaliar a sonolência em estudantes e verificaram níveis elevados de SDE nessa amostra, comprometendo a consolidação da memória e, consequentemente, o êxito acadêmico. Os autores discutem que o atraso do início do sono é um dos responsáveis pela queda do rendimento acadêmico. Na presente investigação, os níveis de sonolência do turno noturno foram significativamente maiores em relação aos estudantes do turno matutino. Considerando que 68% dos alunos do turno noturno trabalhavam (em detrimento dos alunos do turno matutino, cuja parcela de 29,4% possuía vínculo empregatício), é possível perceber que a variável trabalho assume um papel de destaque, já que foi um tipo de demanda frequente dos alunos participantes da amostra que estudavam no turno noturno. Assim, muitos estudantes de graduação que estudam à noite também trabalham durante o dia. Em estudos anteriores, buscou-se avaliar a relação entre os horários de aula e vínculo de trabalho. Assim, conforme estudo proposto por Machado, Varella e Psico-USF, Bragança Paulista, v. 17, n. 2, p. 295-302, mai./ago. 2012 299 Andrade (1998), alunos que desempenham alguma atividade rotineira (trabalho, por exemplo) têm maior chance de relatar privação do sono. Os autores verificaram que estudantes comprometidos com atividades laborais, sejam pela manhã, sejam pela noite, demonstraram privação parcial do sono em dias da semana. Isso era mais evidente no grupo dos estudantes que trabalhavam à noite, que não atrasaram horários de dormir em fins de semana, talvez por causa da maior necessidade de sono, acumulado durante a semana. No entanto, os estudantes do turno matutino também apresentaram forte tendência para sonolência diurna excessiva. Em especial, ao contrário das demais pesquisas sobre a temática, esses alunos iniciavam suas aulas um pouco mais tarde, às 7h40min. Isso reflete que eles também estão privados de sono, isto é, o atraso de fase de sono verificado e consequente mudança no ritmo circadiano, provocado pela necessidade de acordarem mais cedo pela manhã, restringe a duração do seu sono (Gibson & colaboradores, 2006). Assim, Machado e colaboradores (1998) também buscaram avaliar no seu estudo os níveis de sonolência de estudantes de graduação do turno matutino, verificando que a dificuldade para despertar espontaneamente foi maior, por causa da obrigação de acordar mais cedo para ir às aulas (que iniciavam às 7h30). Também foi relatado no estudo da autora um aumento da duração do sono nos fins de semana, para compensar o débito acumulado de sono no decorrer da semana. Outras atividades diárias, como atividades acadêmicas e horários dedicados ao lazer, tiveram um papel importante na organização de fase e na duração do sono dos estudantes universitários. Além disso, quando foi analisado se havia diferenças nos níveis de SDE por gênero, encontrou-se que as mulheres apresentavam níveis maiores de sonolência excessiva do que os homens, independentemente dos turnos. Tal fato pode indicar que as demandas vivenciadas pelo gênero feminino podem ser muito maiores em relação ao gênero masculino. Rotenberg, Portela, Marcondes, Moreno e Nascimento (2001) discutem que sobre o gênero feminino recaem responsabilidades inerentes ao cuidado da casa, bem como tarefas ligadas ao marido e aos filhos. Assim, no estudo que realizaram com trabalhadores do gênero masculino e feminino, observaram uma tendência nas trabalhadoras sonolentas, casadas e com filhos, a dormir menos de manhã e a dormir mais vezes por dia, quando comparadas às colegas que eram solteiras e não tinham filhos. No presente estudo, níveis de sonolência e a condição de ter filhos no turno noturno estavam 300 Araújo, D. F. & Almondes, K. M. Sonolência em estudantes universitários correlacionados, apontando para o fato de que os estudantes com filhos do turno noturno, apresentaram maiores níveis de sonolência em relação aos indivíduos sem filhos. Danda e colaboradores (2005), num estudo com alunos de medicina, não encontraram diferenças significativas ente os gêneros. No entanto, reconhecem que os estudantes de graduação são considerados uma população suscetível a desenvolver privação do sono, uma vez que, além de enfrentar uma carga curricular em horário integral, em busca de uma boa qualificação profissional, complementam o curso com atividades extracurriculares como plantões, pesquisas e monitorias. Assim, submetem-se a forte pressão e estresse, sobretudo em decorrência das exigências de alto rendimento acadêmico e pelo tempo demandado em estudos. Dedicam muito do seu tempo livre para conseguir atender a tais solicitações, inclusive horários que deveriam ser dedicados ao sono. Consequentemente, privados de sono, tenderão a experimentar os sintomas mencionados. Considerando que outros estudos poderão ampliar o alcance dos achados, algumas limitações podem ser destacadas. Por exemplo, ao restringir os participantes da amostra aos alunos do segundo semestre, a avaliação do sono focou apenas nesse período de graduação. No entanto, com a participação de turmas de outros semestres, a influência da sonolência diurna excessiva seria analisada ao longo de toda a graduação, que possui grande variação no número de demandas entre os períodos (é o caso dos últimos semestres, com a presença de estágios). No presente estudo também não foram abordadas as consequências da sonolência excessiva sobre o cotidiano acadêmico dos estudantes. Poderia ser considerado o rendimento acadêmico e possíveis correlações com a sonolência diurna excessiva, como forma de avaliação dos efeitos desse sintoma. Estudos posteriores poderão verificar também até que ponto a sonolência está relacionada às demandas diárias dos estudantes de graduação, analisando outros horários de aula. Numa sociedade que funciona 24 horas por dia, 7 dias por semana, e que busca produzir cada vez mais bens e serviços (Rotenberg & colaboradores, 2001), estudantes de graduação se constituem numa população amplamente afetada. Em virtude de estarem inseridos nessa sociedade 24 horas, é importante considerar a influência do turno vespertino ou de aulas em mais de um turno, por exemplo, para só então investigar achados que beneficiem o bem-estar dos alunos de graduação. Referências Agudelo, H. A. M., Rodrígues, S. S., Vivanco, D., Aristizábal, N., Berrio, M. C., & Alpi, S. V. (2005). Factores culturales que privan de sueño y causan somnolencia excesiva en estudiantes universitários: un estúdio piloto. Psicología y Salud, 15(1), 57-68. Bittencourt, L. R. A., Silva, R. S., Santos, R. F., Pires, M. L. N., & Mello, M. T. (2007). Excessive daytime sleepiness. Revista Brasileira de Psiquiatria, 27(1), 16-21. Campos, M. L. P., & Martino, M. M. F. (2004). Aspectos cronobiológicos do ciclo vigília-sono e níveis de ansiedade dos enfermeiros nos diferentes turnos de trabalho. Revista da Escola de Enfermagem da USP, 38(4), 415-421. Carskadon, M. A., Acebo, C., & Seifer, R. (2001). Extended nights, sleep loss, and recovery sleep in adolescents. Archives of Italian Biology, 139(3), 301312. Danda, G. J. N., Ferreira, G. R., Azenha, M., Souza, K. F. R., & Bastos, O. (2005). Padrão do ciclo sonovigília e sonolência excessiva diurna em estudantes de medicina. Jornal Brasileiro de Psiquiatria, 54(2), 102-106. Fischer, F. M., Oliveira, D. C., Teixeira, L. R., Teixeira, M. C. T. V., & Amaral, M. A. (2003). Effects of work on the health of adolescents. Ciência & Saúde Coletiva, 8(4), 973-984. García, M. R., Flores, M. V., Ayala, M. V. S., Castaño, V. A., Rojas, J. M., Hernández, J. S., & cols. (2004). Somnolencia diurna excesiva: causas y medición. Revista Mexicana de Neurociencias, 5(2), 147-155. Giannoti, F., & Cortesi, F. (1997). Sleep pattern and daytime functions in italian adolescents. Marina Del Rey, California: Contemporary Perspectives on Adolescent Sleep. Gibson, E. S., Powles, A. C. P., Thabane, L., O’Brien, S., Molnar, D. S., Trajanovic, N., & cols. (2006). Sleepiness is serious in adolescence: two surveys of 3235 Canadian students. BMC Public Health, 116(6), 1-9. Johns, M. W. (1991). A new method for measuring daytime sleepiness: the Epworth Sleepiness Scale. Sleep, 14(6), 540-545. Laberge, L., Petit, D., Simard, C., Vitaro, F., Tremblay, R. E., & Montplaisir, J. (2010). Development of sleep patterns in early adolescence. Journal of Sleep Research, 10, 59-67. Psico-USF, Bragança Paulista, v. 17, n. 2, p. 295-302, mai./ago. 2012 Araújo, D. F. & Almondes, K. M. Sonolência em estudantes universitários Lima, P. F., Medeiros, A. L. D., & Araújo, J. F. (2002). Sleep-wake pattern of medical students: early versus late class starting time. Brazilian Journal of Medical and Biological Research, 35(11), 1373-1377. Machado, E. R. S., Varella, V. B. R., & Andrade, M. M. M. (1998). The influence of study schedules and work on the sleep-wake cycle of college students. Biological Rhythm Research, 29(5), 578-584. Medeiros, A. L. D., Lima, P. F., Almondes, K. M., Dias Junior, A. S., Rolim, S. A. M., & Araújo, J. F. (2002). Hábitos de sono e desempenho em estudantes de medicina. Revista Saúde do Centro de Ciências da Saúde (UFRN), 16(1), 49-54. Morales, R. M. C., Flores, M. V., Meneses, A. C., Figueiras, S. C., & Guerrero, J. M. (2005). Sleepiness, performance and mood state in a group of Mexican undergraduate students. Biological Rhythm Research, 36(1/2), 9-13. Moreno, C. R. C., Fischer, F. M., & Rotenberg, L. (2003). A saúde do trabalhador na sociedade 24 horas. São Paulo em Perspectiva, 17(1), 34-46. Natal, C. L., Lourenço, T. J., Silva, L. A., Boscolo, R. A., Silva, A., Tufik, S., & cols. (2009). Gender differences in the sleep habits of 11-13 years old. Revista Brasileira de Psiquiatria, 31(4), 358-361. Ohayon, M. M., Carskadon, M. A., Guilleminault, C., & Vitiello, M. V. (2004). Meta-analysis of quantitative sleep parameters from childhood to old age in healthy individuals: developing normative sleep values across the human lifespan. Sleep, 27(7), 1255-1273. Psico-USF, Bragança Paulista, v. 17, n. 2, p. 295-302, mai./ago. 2012 301 Rotenberg, L., Portela, L. F., Marcondes, W. B., Moreno, C., & Nascimento, C. P. (2001). Gênero e trabalho noturno: sono, cotidiano e vivências de quem troca a noite pelo dia. Cadernos de Saúde Pública, 17(3), 639-649. Sai, L. P. (2007). Sleep quality versus sleep quantity: relationship between sleep and measures of health, wellbeing and sleepiness in university students (Tese de Doutorado). Faculty of Medicine The Department of Psychiatry, Hong Kong. Souza, J. C., Souza, N., Arashiro, E. S. H., & Schaedler, R. (2007). Sonolência diurna excessiva em prévestibulandos. Jornal Brasileiro de Psiquiatria, 56(3), 184-187. Teixeira, L. R. (2002). Análise dos padrões do ciclo vigíliasono de adolescentes trabalhadores e não trabalhadores, alunos de escola pública no município de São Paulo (Tese de Doutorado). Universidade de São Paulo, São Paulo, Brasil. Thorleifsdottir, B., Björnsson, J. K., Benediktsdottir, B., Gislason, T., & Kristbjarnarson, H. (2002). Sleep and sleep habits from childhood to young adulthood over a 10-year period. Journal of Psychosomatic Research, 53, 529-537. Recebido em 10/02/2011 Reformulado em 16/04/2012 Aprovado em 08/05/2012 302 Araújo, D. F. & Almondes, K. M. Sonolência em estudantes universitários Sobre os autores: Danilo de Freitas Araújo é graduado em Psicologia pela Faculdade Natalense para o Desenvolvimento do Rio Grande do Norte (2009) finalizando mestrado em Psicologia pela Universidade Federal do Rio Grande do Norte (2012). É psicólogo clínico/saúde. Katie Moraes de Almondes possui graduação em Psicologia pela Universidade Estadual da Paraíba (1996), mestrado em Psicobiologia pela Universidade Federal do Rio Grande do Norte (2001) e doutorado em Psicobiologia pela Universidade Federal do Rio Grande do Norte (2007). É psicóloga clínica e hospitalar/saúde e professora adjunta do Departamento de Psicologia e da Pós-Graduação em Psicologia da Universidade Federal do Rio Grande do Norte. Contato com os autores: Campus Universitário – Caixa Postal 1622 – CEP 59078-970 – Lagoa Nova – Natal-RN. E-mail: kmalmondes@ufrnet.br Psico-USF, Bragança Paulista, v. 17, n. 2, p. 295-302, mai./ago. 2012
https://openalex.org/W2460033878	https://www.scielo.br/j/ni/a/k4QgZXgDLBKxK4GyVTnhrqJ/?lang=en&format=pdf	Latin	null	Cytogenetic analysis of Baryancistrus xanthellus (Siluriformes: Loricariidae: Ancistrini), an ornamental fish endemic to the Xingu River, Brazil	Neotropical ichthyology/Neotropical Ichthyology	2,016	cc-by	7,301	1Instituto Nacional de Pesquisas da Amazônia, Programa de Pós-Graduação em Genética, Conservação e Biologia Evolutiva. Av. André Araújo 2936, Petrópolis, 69.067-375 Manaus, AM, Brazil. (LAM) larissaazmedeiros@gmail.com (corresponding author), (EF) feldberg@inpa.gov.br 2Instituto Nacional de Pesquisas da Amazônia, Programa de Pós-Graduação em Entomologia. Av. André Araújo 2936, Petrópolis, 69067-375 Manaus, AM, Brazil. edu.ginani@gmail.com 3Universidade Federal do Pará, Campus de Altamira, Faculdade de Ciências Biológicas. Av. Coronel José Porfírio, 2515, Bairro São Sebastião, 68372-040 Altamira, PA, Brazil. leandro.m.sousa@gmail.com 4Instituto Nacional de Pesquisas da Amazônia, Coleções de Peixes. Av. André Araújo, 2936, Petrópolis, 69067-375 Manaus, AM, Brazil. lucia.rapp@gmail.com Neotropical Ichthyology, 14(2): e150108, 2016 DOI: 10.1590/1982-0224-20150108 Neotropical Ichthyology, 14(2): e150108, 2016 DOI: 10.1590/1982-0224-20150108 Journal homepage: www.scielo.br/ni Published online: 27 June 2016 (ISSN 1982-0224) Journal homepage: www.scielo.br/ni Published online: 27 June 2016 (ISSN 1982-0224) Larissa A. Medeiros1, Eduardo G. Ginani2, Leandro M. Sousa3, Lúcia H. Rapp Py- Daniel4 and Eliana Feldberg1 Larissa A. Medeiros1, Eduardo G. Ginani2, Leandro M. Sousa3, Lúcia H. Rapp Py- Daniel4 and Eliana Feldberg1 Baryancistrus xanthellus is a species from the Ancistrini tribe known commonly as “amarelinho” or “golden nugget pleco”. It is one of the most popular and valued ornamental fishes due to its color pattern. Also, it is an endemic species from the Xingu River occurring from Volta Grande do Xingu, region where the Belo Monte Hydropower Dam is being built, to São Félix do Xingu. The current study aimed to cytogenetically characterize B. xanthellus. Results point to the maintenance of 2n=52, which is considered the most common condition for the tribe, and a single nucleolus organizer region (NOR). Mapping of the 18S rDNA confirmed the NOR sites, and the 5S rDNA was mapped in the interstitial position of a single chromosome pair. The 18S and 5S rDNA located in different pairs constitute an apomorphy in Loricariidae. Large blocks of heterochromatin are present in pairs 1 and 10 and in the regions equivalent to NOR and the 5S rDNA. Data obtained in this study corroborated with the currently accepted phylogenetic hypothesis for the Ancistrini and demonstrate evidence that the genus Baryancistrus occupies a basal position in the tribe. Baryancistrus xanthellus é uma espécie da tribo Ancistrini conhecida popularmente como “amarelinho” ou “cascudo pepita de ouro”. É um dos peixes ornamentais mais populares e valorizados, devido aos padrões de cor. Também é uma espécie endêmica do rio Xingu, ocorrendo a partir da Volta Grande do Xingu, região onde a Usina Hidrelétrica de Belo Monte está sendo construída, até São Félix do Xingu. O presente estudo teve como objetivo caracterizar citogeneticamente B. xanthellus. Os resultados apontam para a manutenção do 2n=52, considerado a condição mais comum para a tribo, e região organizadora de nucléolo (RON) simples. O mapeamento do DNAr 18S confirmou a marcação da RON e o DNAr 5S foi localizado na posição intersticial de apenas um par cromossômico. A localização dos DNAr 18S e 5S em diferentes pares configura uma apomorfia em Loricariidae. Grandes blocos de heterocromatina estão presentes nos pares 1 e 10 e nas regiões equivalentes à RON e ao DNAr 5S. Os dados obtidos neste estudo corroboram a hipótese filogenética atualmente mais aceita para Ancistrini e demonstram evidências que o gênero Baryancistrus ocupa uma posição basal na tribo. Keywords: FISH, Habitat endangered, rDNA, Volta Grande do Xingu. Cytogenetic analysis of Baryancistrus xanthellus (Siluriformes: Loricariidae: Ancistrini), an ornamental fish endemic to the Xingu River, Brazil Xingu River, Brazil Cytogenetic analysis of Baryancistrus xanthellus Neotropical Ichthyology, 14(2): e150108, 2016 2 Neotropical Ichthyology, 14(2): e150108, 2016 2 Cytogenetic analysis of Baryancistrus xanthellus Neotropical Ichthyology, 14(2): e150108, 2016 2 Cytogenetic analysis of Baryancistrus xanthellus Cytogenetic analysis of Baryancistrus xanthellus Ancistrini belongs to the Hypostominae and has around 217 species (Fisch-Muller, 2003) distributed in 24 genera (Armbruster, 2004; Ferraris, 2007). This tribe includes several species that are taxonomically poorly known and are often misidentified (Alves et al., 2003). In addition, the majority of available studies are based only on morphological data (Isbrücker, 1980; Schaefer, 1986, 1987). them but also to design conservation strategies, since their habitats are being seriously impacted. For the Baryancistrus genus, only B. aff. niveatus has cytogenetic data published (Souza et al., 2004). Therefore, the present study investigated the conventional and molecular karyotype macrostructure of one more specie of Baryancistrus, B. xanthellus, in order to increase the information on the genetic diversity of Ancistrini on Amazon region. p g ( ) Baryancistrus Rapp Py-Daniel, 1989 is allocated into the Ancistrini and has six described species (Rapp Py-Daniel et al., 2011) that are unique due to their exuberance and diversity of coloration and are, therefore, highly demanded in the fishkeeping market. The presence of yellow spots throughout its body and yellow markings on its dorsal and caudal fins characterize this species. Due to the presence of these spots, which vary in size and intensity, this species is commonly known as “amarelinho” or “golden nugget pleco”. This species is rheophilic, and its distribution is strongly linked to the rapids of the Xingu River (Rapp Py-Daniel et al., 2011), which is target for several constructions to take advantage of its hydroelectric potential (Junk & Mello, 1990). Near the middle of its course, the Xingu River receives the Iriri River and posteriorly suffers an accentuated deflection, forming the region known as the Volta Grande do Xingu. According to Zuanon (1999), the most commonly found species in this part of the river are from the Loricariidae family. Also, according to a report developed by several specialists on Belo Monte dam environmental impacts (Painel de Especialistas - Análise Crítica do Estudo de Impacto Ambiental do Aproveitamento Hidrelétrico de Belo Monte, 2009), the situation of the rheophilic fish there is dire. Introduction reformulations (Reis et al., 2006). According to Eschmeyer & Fong (2015) and Lujan et al. (2015), this family holds around 800 valid species in six subfamilies: Delturinae, Hypoptopomatinae, Hypostominae, Lithogeninae, Loricariinae, and Neoplecostominae (Schaefer, 1987; Reis et al., 2003; Armbruster, 2004; Reis et al., 2006). Loricariidae is a widespread family of fish in the Neotropical region, from Costa Rica until Argentina (Reis et al., 2003). Subfamilies classification and propositions of correlations among the genera has been the subject of constant Material and Methods Thirteen specimens of B. xanthellus (Fig. 1) (six males, four females, and three of unidentified sex) were collected in the Xingu River in the rapids of Volta Grande do Xingu, municipality of Altamira, State of Pará (03º 36’31,5” S 51º 34’57,4” W; 03º 23’28,2” S 51º 44’29,3” W; 03º 22’29,7” S 51º 42’25,0” W; 03º 35’38,6” S 51º 49’36,0” W). Collection was performed during free dives in the rapids using a collecting permit (ICMBio SISBIO 10609-1/2007) in the name of Eliana Feldberg, and the specimens were deposited in the fish collection of INPA: INPA 43926, 43927, 43928 and 43929. The Parecer Consubstanciado Sobre Protocolos de Pesquisas no Uso de Animais, number 030/2013, was obtained for the experiments with the specimens. Mitotic induction was performed with the application of a yeast solution according to the protocol of Oliveira et al. (1988). Mitotic chromosomes were obtained from kidney cells through the air drying technique modified for fishes by Bertollo et al. (1978). For the characterization of the nucleolus organizer regions (NORs), an AgNO3 stain was done according to Howell & Black (1980). The heterochromatic regions were identified according to the protocol of Sumner (1972). Thus, all studies involving these species are extremely important not only for acquiring basic knowledge about Fig. 1. Live photograph of Baryancistrus xanthellus, LIA 1629. Fig. 1. Live photograph of Baryancistrus xanthellus, LIA 1629. L. A. Medeiros, E. G. Ginani, L. M. Sousa, L. H. Rapp Py-Daniel & E. Feldberg Neotropical Ichthyology, 14(2): e150108, 2016 3 L. A. Medeiros, E. G. Ginani, L. M. Sousa, L. H. Rapp Py-Daniel & E. Feldberg Neotropical Ichthyology, 14(2): e150108, 2016 3 L. A. Medeiros, E. G. Ginani, L. M. Sousa, L. H. Rapp Py-Daniel & E. Feldberg Neotropical Ichthyology, 14(2): e150108, 2016 3 DNA extraction followed Sambrook et al. (1989). An amplification through PCR (polymerase chain reaction) was done for the development of 18S and 5S rDNA probes using the primers 18Sf (5’-CCG CTT TGG TGA CTC TTG AT-3’) and 18Sr (5’-CCG AGG ACC TCA CTA AAC CA-3’) (Gross et al., 2010) and primers 5Sa (5’ TAC GCC CGA TCT CGT CCG ATC-3’) and 5Sb (5’-CAGGCT GGT ATG GCC GTA AGC-3’) (Martins & Galetti, 1999), respectively. The final volume of each reaction was of 25µl containing 200ng of genomic DNA, 10X buffer with 1.6mM of MgCl2, Taq DNA polimerase (5U/µl), dNTPs (1mM), primer pair (5mM) and Milli-Q water. Material and Methods The 18S rDNA probe obtained was isolated and labeled with digoxigenin-11-dUTP (Roche Applied Science) through the Nick Translation method and the signal detection was performed using anti-digoxigenin-rhodamine (Roche Applied Science). The 5S rDNA probe was labeled with biotin-16-dUTP (Roche Applied Science) using Nick translation and signal detection was performed using a conjugated avidin-fluorescein (FITC). with a mounted Olympus DP71 camera through the Image- Pro MC 6.3 software. The karyotypes were organized with the aid of the Adobe Photoshop CS6 software, measured with the ImageJ software and classified according to Levan et al. (1964). Results Karyotype of Baryancistrus xanthellus: a) C-banding; b) Mapping of rDNA 18S (red signal) and 5S (green signal) through double FISH. Fig. 3. Karyotype of Baryancistrus xanthellus: a) C-banding; b) Mapping of rDNA 18S (red signal) and 5S (green signal) through double FISH Fig. 3. Karyotype of Baryancistrus xanthellus: a) C-banding; b) Mapping of rDNA 18S (red signal) and 5S (green signal) through double FISH. ryancistrus xanthellus: a) C-banding; b) Mapping of rDNA 18S (red signal) and 5S (green signal Results Baryancistrus xanthellus presents a diploid number of 52 chromosomes, (16m+28sm+8st), the fundamental number (FN) was equal 104 for males and females, and no differentiated sexual chromosomes were observed (Fig. 2). Active NOR sites were located at the interstitial portion of the short arm of the fourth metacentric pair of all the individuals analyzed. Size heteromorphism of the NOR was observed between the homologues in some specimens (Fig. 2). Constitutive heterochromatin was found in the centromeric region in the majority of the chromosomes, extending into the proximal region of both arms in some cases. Large blocks occupied the short arms completely on pair 1 and the long arms of pair 10. The NOR was C-band positive (Fig. 3a).i The mapping of 18S and 5S rDNA was obtained through fluorescence in situ hybridization (FISH), following Pinkel et al. (1986) with 77% strigency (2.5ng/ µl of probes, formamide 50%, dextran sulphate 10% and 2xSSC [saline sodium citrate solution] at 37°C for 18h). Chromosomes were contrasted with DAPI (2 mg/mL) in a Vectashield mounting medium (Vector). l Mapping of the 18S rDNA confirmed the results obtained by silver staining. As in the Ag-NOR, size heteromorphism was also observed. The 5S rDNA pattern was in the pericentromeric region of metacentric pair 7, which presented a conspicuous heterochromatic block on all analyzed specimens (Fig. 3b). The chromosomes were analyzed in an epifluorescence Olympus BX51 microscope and the images were captured Fig. 2. Karyotype of Baryancistrus xanthellus in conventional staining. The square indicates the pair that bears the nucleolus organizer region (NOR). Fig. 2. Karyotype of Baryancistrus xanthellus in conventional staining. The square indicates the pair that bears the nucleolus organizer region (NOR). Cytogenetic analysis of Baryancistrus xanthellus Neotropical Ichthyology, 14(2): e150108, 2016 4 Discussion Loricariidae, although with only 10% of species with plesiomorphic condition for this group of fish (Artoni & Bertollo, 2001; Alves et al., 2005; Kavalco et al., 2005). To the Ancistrini, the diploid number recorded Fig. 3. Karyotype of Baryancistrus xanthellus: a) C-banding; b) Mapping of rDNA 18S (red signal) and 5S (green signal) through double FISH. Cytogenetic analysis of Baryancistrus xanthellus Neotropical Ichthyology, 14(2): e150108, 2016 4 Neotropical Ichthyology, 14(2): e150108, 2016 y g y y Discussion plesiomorphic condition for this group of fish (Artoni Fig. 3. Karyotype of Baryancistrus xanthellus: a) C-banding; b) Mapping of rDNA 18S (red signal) and 5S (green signal) through double FISH. Fig. 3. Discussion plesiomorphic condition for this group of fish (Artoni & Bertollo, 2001; Alves et al., 2005; Kavalco et al., 2005). To the Ancistrini, the diploid number recorded so far is ≤ 54, indicating the presence of chromosomal rearrangements in the karyoevolution of this taxon (Oliveira et al., 2009; Mariotto et al., 2011). In this tribe, the most frequent diploid number is 52 chromosomes (Table 1). Loricariidae, although with only 10% of species with any cytogenetic published data (Kavalco et al., 2005), present a great karyotypic diversity in relation to the diploid number, ranging from 34 (Hypostominae) to 96 (Delturinae) (Kavalco et al., 2004; Oliveira et al., 2009). The diploid number of 54 chromosomes represents a Neotropical Ichthyology, 14(2): e150108, 2016 L. A. Medeiros, E. G. Ginani, L. M. Sousa, L. H. Rapp Py-Daniel & E. Feldberg p L. A. Medeiros, E. G. Ginani, L. M. Sousa, L. H. Rapp Py-Daniel & E. Feldberg p y gy, ( ) , 5 able 1. Survey of cytogenetic data of the species of Ancistrini. 2n (diploid number), FN (fundamental number), NOR (nucleolar organizer region), 18S (position that he rDNA 18S occupies in the karyotype) and 5S (pairs that have a rDNA 5S marking). pecies Locality 2n Karyotypic Formulae FN NOR 18S 5S References ncistrus ranunculus rio Xingu - PA 48 ♂ 20m + 8sm + 6st + 14a ♀ 19m + 9sm + 6st + 14a 82 (sm) 16q proximal (sm) 16q proximal 16 Oliveira et al. (2007); Favarato et al. (2016) ncistrus sp. Piagaçu lago Aiapuá - AM 52 ♂ 16m + 8sm + 2st + 26a ♀ 16m + 9sm + 2st + 25a ♂ 78 ♀ 79 (a) 26p terminal (a) 26p terminal 1, 5, 9, 14, 15, 20, 22, 24, 25, 26 Oliveira et al. (2007); Favarato et al. (2016) ncistrus sp. Purus rio Purus - AC 34 ♂21m + 11sm + 2st ♀ 20m + 12sm + 2st 68 (m) 4p distal (m) 4p distal 3, 5, 12, 13 Oliveira et al. (2009); Favarato et al. (2016) ncistrus sp. Macoari rio Branco - RR 46 ♂18m + 11sm + 6st + 11a ♀18m + 12sm + 6st + 10a ♂ 81 ♀ 82 (a) 19p distal - - Oliveira et al. (2009) ncistrus sp. Discussion Dimona fazenda Dimona - AM 52 16m + 8sm +2st + 26a 78 (st) 13q distal (st) 13q distal ♂1, 13 ; ♀ 13 Oliveira et al. (2009); Favarato et al. (2016) ncistrus sp. Vermelho rio Demeni - AM 42 26m + 6sm + 4st + 6a 78 (a) 20q terminal - - Oliveira et al. (2009) ncistrus sp. Trombetas rio Trombetas - PA 38 22m + 8sm + 5st + 3a 73 (m) 5 centromeric - - Oliveira et al. (2009) ncistrus sp. Balbina igarapé Barretinho - AM ♂ 39 ♀: 38 ♂ 27m + 10sm + 2st ♀ 26m + 10sm + 2st ♂ 78 ♀ 76 (m) 12q terminal (m) 12q terminal 4 Oliveira et al. (2008); Favarato et al. (2016) ncistrus sp. Barcelos rio Demeni - AM 52 ♂ 12m + 12sm + 4st + 24a ♀11m + 12sm + 4st + 25a ♂ 80 ♀ 79 (a) 23p terminal (a) 23p terminal 1, 2, 8, 9, 15, 16, 18, 19, 20, 22, 23, 24, 26 Oliveira et al. (2008); Favarato et al. (2016) ncistrus sp. Catalão lago Catalão - AM 34 22m + 8sm + 4st 68 (m) 4p distal (m) 4p distal 3, 6, 7, 12 Oliveira (2006); Favarato et al. (2016) ncistrus sp. Rio Branco igarapé Macoari- RR 46 ♂ 18m + 11sm + 6st + 11a ♀ 18m + 12sm + 6st + 10a ♂ 81 ♀ 82 (a) 19p distal (a) 19p distal 19 Oliveira (2006); Favarato et al. (2016) ncistrus sp. rio Iguaçu - PR 48 18m + 14sm + 12st + 4a 84 (st) p terminal - - Lara (1998) ncistrus sp. 1 igarapé São Francisco - AC 38 30m/sm + 8st 76 (m/sm) 5p interstitial - - Alves et al. (2003) ncistrus sp. 2 rio Betari - SP 52 32m/sm + 20st/a - (st/a) 24p terminal - - Alves et al. (2003) ncistrus multispinnis rio Itapocu - SC 52 28m/sm + 24st/a - (st/a) 17p terminal - - Alves et al. (2003) ncistrus sp. 1 rio Vermelho - GO ♂ 39 ♀ 40 ♂ 33m + 6sm ♀ 34m + 6sm ♂ 78 ♀ 80 (sm) 20q distal - - Alves et al. (2006) ncistrus sp. 2 rio Guaruva - SC 52 10m + 16sm + 12st + 14a 90 (st) 15p distal - - Alves et al. (2006) ncistrus sp. Discussion rio Alto Alegre - PR 50 12m + 14sm + 14st + 10a 90 - - - Tchaicka & Margarido (1999) ncistrus cf. dubius rio Coxipó; córrego Pari; córrego Flechas; Córrego Fundo - MT 42 24m + 10sm + 8st 84 (sm) 16p interstitial (sm) 16p interstitial 4, 14, 16 Mariotto & Miyazawa (2006); Mariotto et al. (2011) ncistrus claro rio Coxipó - MT 54 14m + 8sm + 8st + 24a 84 (a) 21q interstitial (a) 21q interstitial 4, 19, 21 Mariotto et al. (2011) ncistrus cuiabae baia Arrombado - MT 34 20m + 8sm + 6st 68 (m) 2p terminal (m) 2p terminal 3, 6, 9 Mariotto et al. (2009); (2011) ncistrus tombador rio Preto - MT 50 14m + 12sm + 8st + 16a 84 (a) 20p terminal - - Mariotto et al. (2013) ncistrus sp. 01 córrego Pipa - MT 54 14m + 8sm + 8st + 24a 84 (a) 21q interstitial - - Mariotto et al. (2013) ncistrus sp. 03 córrego Pari - MT 54 14m + 8sm + 8st + 24a 84 (a) 21q interstitial - - Mariotto et al. (2013) ncistrus sp. 04 rio Sepotuba - MT 52 16m + 8sm + 6st + 22a 82 (a) 22q proximal (a) 22q proximal 17, 25, 26 Mariotto et al. (2011) Neotropical Ichthyology, 14(2): e150108, 2016 6 Cytogenetic analysis of Baryancistrus xanthellus p y gy, ( ) , 6 Cytogenetic analysis of Baryancistrus xanthellus Table 1. Species Locality 2n Karyotypic Formulae FN NOR 18S 5S References Ancistrus sp. 06 rio Matrinxã - MT 50 18m + 10sm + 8st + 14a 86 (sm) 13p interstitial (sm) 13p interstitial 1, 13 Mariotto et al. (2011) Ancistrus sp. 08 rio Curupira - MT 44 18m + 10sm + 8st + 8a 80 (a) 21q proximal (a) 21q proximal 21 Mariotto et al. (2011) Ancistrus sp. 13 córrego Salgadinho - MT 40 26m + 10sm + 4st 80 (sm) 18q terminal (sm) 18q terminal 5, 15 Mariotto et al. (2011) Baryancistrus aff. Niveatus rio Xingu - PA 52 16m + 32sm + 4st 104 (m) 3p interstitial - - Souza et al. (2004) Baryancistrus sp. 1 rio Jarí - PA 52 8m + 34sm + 10st 104 (sm) 18p interstitial - - Souza (2003) Baryancistrus sp. 2 rio Xingu - PA 52 18m + 30sm + 4st 104 (m) 6p interstitial - - Souza (2003) Baryancistrus sp. Discussion 3 rio Xingu - PA 52 14m + 26sm + 12st 104 (m) 4p interstitial - - Souza (2003) Hemiancistrus sp. rio Araguaia - MT 52 20m + 20sm + 12st/a - (sm) q terminal - - Artoni & Bertollo (2001) Hemiancistrus spilomma rio Araguaia - MT 52 24m + 22sm + 6st 104 (m) 2q terminal 3p terminal/(sm 18q terminal - - Oliveira et al. (2006) Hemiancistrus spinosissimus rio Araguaia - MT 52 26m + 22sm + 4st 104 (sm) 17q terminal - - Oliveira et al. (2006) Hypancistrus cf. debilittera rio Uatumã - AM 52 34m/sm + 18st 104 1: (m) 2q terminal 1: (m) 2q terminal 1, 9 Silva et al. (2014) 2: (m) 2q terminal 2: (m) 2q terminal 1, 9 3: (m) 2q terminal; (st) 23q terminal 3: (m) 2q terminal (st) 23q terminal 1, 9 4: (m) 2q terminal/ interstitial (1 homologous) 4: (m) 2q terminal/ interstitial (1 homologous) 1, 9 5: (m) 2q terminal/ interstitia (2 homologous) 5: (m) 2q terminal/ interstitial (2 homologous) 1, 9 Hypancistrus zebra rio Xingu - PA 52 38m/sm + 14st 104 (m/sm) 13q terminal (m/sm) 13q terminal 1, 4 Silva et al. (2014) Lasiancistrus cf. schomburgkii rio Massangana - MT 54 26m + 16sm + 12st 108 (st) 25p terminal - - Mariotto (2009) Lasiancistrus sp. rio Cachoeira - MT 54 26m + 18sm + 10st 108 (st) 26p terminal - - Mariotto (2009) Megalancistrus aculeatus rio Paraná - PR 52 26m + 26sm 104 (sm) p interstitial - - Lara (1998) Panaque cf. nigrolineatus rio Araguaia - MT 52 26m + 20sm + 6st/a - (a) 26p terminal - - Artoni & Bertollo (2001) Parancistrus sp. 1 rio Xingu - PA 52 20m + 26sm + 6st 104 (m) 8p interstitial - - Souza (2003) Peckoltia sp. 1 rio Jarí - PA 52 18m + 26sm + 6st + 2a (+1B) 102 (m) 10q distal (st) 25q distal - - Souza et al. (2009) Peckoltia sp. 2 rio Jarí - PA 52 16m + 16sm + 8st + 2a 102 (st) 17q distal (st) 18q distal - - Souza et al. (2009) Peckoltia vittata rio Xingu - PA 52 16m + 20sm + 14st + 2a 102 (sm) 9q distal - - Souza et al. Discussion Large heterochromatic blocks are found in two or more chromosomal pairs in the genera Scobinancistrus, Hypancistrus and mainly in Peckoltia (Souza et al., 2009; Cardoso et al., 2013; Silva et al., 2014). This characteristic seems to be common to the Ancistrini and corroborates the suggestion proposed by Ziemniczak et al. (2012). In Siluriformes, the mapping of the ribosomal genes 18S and 5S is still very incipient (Kavalco et al., 2004; Centofante et al., 2006; Mendes-Neto et al., 2011). In Loricariidae, chromosomes with syntenic markings of the 18S and 5S rDNA were observed in species of Neoplecostominae, Hypoptopomatinae (Ziemniczak et al., 2012), Loricariinae (Kavalco et al., 2004), Hypostominae (Ancistrini and Hypostomini) (Mariotto et al., 2011; Traldi et al., 2013) and in the outgroup, Trichomycteridae family (Ziemniczak, 2011). Based on this data, Ziemniczak (2011) inferred that the synteny of these classes of rDNA is a plesiomorphic character in the family.i Baryancistrus xanthellus presented 2n=52, which was already verified in species of the genera Peckoltia, Hemiancistrus, Hypancistrus, Scobinancistrus and Panaque (Artoni & Bertollo, 2001; Oliveira et al., 2006; Souza et al., 2009; Cardoso et al., 2013; Silva et al., 2014). There was a single NOR located in the interstitial region of the short arm, in the third pair in B. aff. niveatus (Souza et al., 2004) and the fourth pair in B. xanthellus. This character cannot be considered specific for each genus, since species from the same genus may present sites in different locations. In Ancistrus and Hemiancistrus, for example, the single sites can be found in the short or long arms, and many times even multiple NORs were found (Oliveira et al., 2006, 2009). Like in B. aff. niveatus, the NOR was C-band positive differently from species of the genera Peckoltia and Scobinancistrus that have heterochromatic blocks adjacent to the NOR (Souza et al., 2004; Souza et al., 2009; Cardoso et al., 2013). For the Baryancistrus, this is the first record of the mapping of the rDNA 5S and 18S. The simultaneous hybridization of both probes (double-FISH) did not result in syntenic markings (Fig. 3b). This rDNA distribution in different chromosomal pairs constitutes an apomorphy in the Loricariidae (Ziemniczak, 2011). In the Ancistrini so far, only two genera have data on 5S rDNA: Ancistrus (Mariotto et al., 2011; Favarato et al., 2016) and Hypancistrus (Silva et al., 2014). Discussion (2009) Scobinancistrus aureatus rio Xingu - PA 52 22m + 20sm + 10st 104 (m)3q interstitial (m)3q interstitial - Cardoso et al. (2013) Scobinancistrus pariolispos rio Xingu - PA 52 24m + 18sm + 10st 104 (m) 3q distal (m) 3q distal - Cardoso et al. (2013) Table 1. L. A. Medeiros, E. G. Ginani, L. M. Sousa, L. H. Rapp Py-Daniel & E. Feldberg Neotropical Ichthyology, 14(2): e150108, 2016 7 L. A. Medeiros, E. G. Ginani, L. M. Sousa, L. H. Rapp Py-Daniel & E. Feldberg Neotropical Ichthyology, 14(2): e150108, 2016 7 L. A. Medeiros, E. G. Ginani, L. M. Sousa, L. H. Rapp Py-Daniel & E. Feldberg Neotropical Ichthyology, 14(2): e150108, 2016 7 With the exception of Ancistrus, for which the karyotypic differentiation is usually associated to a reduction in the diploid number (Oliveira et al., 2007, 2008, 2009; Mariotto et al., 2009), there is a tendency towards maintaining the diploid number in the tribe. However, a large amount of variation can be found in the karyotypic formulae promoted specially by pericentric inversions (Artoni & Bertollo, 2001; Alves et al., 2003, 2006; Bueno et al., 2012; Oliveira et al., 2007, 2008; Mariotto et al., 2009; Souza et al., 2004, 2009; Ziemniczak et al., 2012). Furthermore, chromosomal rearrangements like fusions, inversions, deletions, duplications and heterocromatinization may contribute in the differentiation of the group and may be associated to morphological speciation processes (Artoni & Bertollo, 2001; Milhomem et al., 2010; Mariotto et al., 2011; Ziemniczak, 2011; Bueno et al., 2012). According to Ziemniczak (2011), this great karyotypic diversity might have had an important role in the genetic and reproductive isolation of the Ancistrini species. (Fig. 3a). A similar pattern of C-banding was observed in B. aff. niveatus where pairs 1 and 10 also presented one of the arms almost completely heterochromatic (Souza et al., 2004), which could be a pattern for the genus. However, B. aff. niveatus also presented large blocks on pairs 11 and 22. Ziemniczak et al. (2012) suggested that the absence of large and numerous blocks of heterochromatin seem to be a plesiomorphic character in Loricariidae. This characteristic may be proven by comparing the basal genera with the derived ones (Artoni &Bertollo, 2001;Oliveira, 2006; Oliveira et al., 2008; Mariotto et al., 2009; Traldi et al., 2012). References pair is less frequent (Table 1), occurring in a few species of Ancistrus (Mariotto et al., 2011; Favarato et al., 2016) and in B. xanthellus. According to Martins & Galetti (1999), the localization of the ribosomal genes in different chromosomes may be advantageous if compared to the syntenic disposition because it might avoid unfavorable arrangements (Dover, 1986), since the occurrence of unequal crossing-over might be frequent in chromosomes with co-located ribosomal genes. Almeida-Toledo, L. F., F. Foresti & S. A. Toledo-Filho. 2000. Karyotipic evolution in Neotropical freshwater fish. Pp. 169-182. In: Olmo, E. & C. A. Redi (Eds.). Chromosomes today. Basel; Boston; Berlin; Birkhäuser Verlag/Switzerland. (chromosomes today series, v. 13). Alves, A. L., C. Oliveira & F. Foresti. 2003. Karyotype variability in eight species of the subfamilies Loricariinae and Ancistrinae (Teleostei, Siluriformes, Loricariidae). Caryologia, 56: 57-63. In B. xanthellus, it is possible to visualize the heterochromatin association with 18S and 5S rDNA. This is a recurrent characteristic in the evolutionary history of Neotropical fishes (Vicari et al., 2003). In some cases, the heterochromatic blocks might be adjacent to the nucleolar regions, while in other cases the markings may be overlapping or intercalated (Pendás et al., 1993a,b; Artoni & Bertollo, 2001). The presence of heterochromatin, which holds large quantities of satellite DNA and transposable elements (Dimitri et al., 2009), might facilitate transposition events, moving ribosomal genes to other regions of the genome (Moreira-Filho et al., 1984; Vicari et al., 2008; Gross et al., 2009, 2010), promoting genetic duplication and unequal crossing-over, and still lead to size variation of the heterochromatin segments as well as in the number of rDNA cistrons (Sola et al., 1988; Vicari et al., 2003, 2008). Alves, A. L., C. Oliveira & F. Foresti. 2005. Comparative cytogenetic analysis of eleven species of subfamilies Neoplecostominae and Hypostominae (Siluriformes: Loricariidae). Genetica, 124: 127-136. Alves, A. L., C. Oliveira, M. Nirchio, Á. Granado & F. Foresti. 2006. Karyotypic relationships among the tribes of Hypostominae (Siluriformes: Loricariidae) with description of X0 sex chromosome system in a Neotropical fish species. Genetica, 128: 1-9. Armbruster, J. W. 2004. Phylogenetic relationships of the suckermouth armoured catfishes (Loricariidae) with emphasis on the Hypostominae and the Ancistrinae. Zoological Journal of the Linnean Society, 141: 1-80. Artoni, R. F. & L. A. C. Bertollo. 2001. Trends in the karyotype evolution of Loricariidae fish (Siluriformes). Hereditas, 134: 201-210. Bertollo, L. A. C., C. S. Takahashi & O. Moreira Filho. References 1978. Citotaxonomic considerations on Hoplias lacerdae (Pisces: Erythrinidae). Revista Brasileira de Genética, 1: 103-120. In general, B. xanthellus conserves the karyotipic macrostructure of the Ancistrini. The maintenance of 2n=52 with a few heterochromatic blocks, a single NOR, and single rDNA sites are evidences that the genus occupies a basal position in the tribe. Our results can help to better understand the chromosomal evolution in this remarkable fish group, but the continuity of cytogenetic studies for the Baryancistrus is indispensable for a better comprehension of the evolutionary trends. Bueno, V., C. H. Zawadzki & V. P. Margarido. 2012. Trends in chromosome evolution in the genus Hypostomus Lacépède, 1803 (Osteichthyes, Loricariidae): a new perspective about the correlation between diploid number and chromosomes types. Reviews in Fish Biology and Fisheries, 22: 241-250. Cardoso, A. L., K. A. H. Sales, C. Y. Nagamachi, J. C. Pieczarka & R. C. R. Noronha. 2013. Comparative cytogenetics of two species of genus Scobinancistrus (Siluriformes, Loricariidae, Ancistrini) from the Xingu River, Brazil. Comparative Cytogenetics, 7: 43-51. A karyotypic diversity might result in great morphological diversity and color pattern in the species of Ancistrini endemic to the Xingu River. This species diversity represents an invaluable richness; therefore, it is important that there are efforts to understand the origin, evolution, behavior, ecology, and the subsequent preservation of such diversity, since many species are threatened by extinction due to the changes in their original habitats caused by the construction of hydropower dams. Centofante, L., L. A. C. Bertollo & O. Moreira-Filho. 2006. Cytogenetic characterization and description of an XX/ XY1Y2 sex chromosome system in catfish Harttia carvalhoi (Siluriformes, Loricariidae). Cytogenetic and Genome Research, 112: 320-324. Dimitri, P., R. Caizzi, E. Giordano, M. C. Accardo, G. Lattanzi & G. Biamonti. 2009. Constitutive heterochromatin: a surprising variety of expressed sequences. Chromosoma, 118: 419-435. Discussion As it has been observed for the 18S rDNA, the 5S rDNA also presented variable forms among the different species of Ancistrini. In a study by Mariotto et al. (2011), in which the 18S and 5S rDNA probes were hybridized in seven species of Ancistrus, all species presented 18S rDNA markings in a single pair. However, only one species, Ancistrus sp. 06, presented a single pair with 5S. This same species also presented synteny between the two ribosomal genes. The remaining species presented two or three pairs with the 5S rDNA. In regards to the position in the chromosome, 5S rDNA was found to be variable, occupying pericentromeric, interstitial, or terminal positions (Table 1). In some specimens of B. xanthellus, the NOR was heteromorphic in regards to size when compared the homologues, which was also observed by Souza et al. (2004) in some specimens of B. aff. niveatus. This heteromorphism is very frequent in Neotropical fish and has been explained as a duplication of ribosomal genes or by a process of accumulation of these genes in one of the homologues through unequal crossing-over (Foresti et al., 1981; Almeida-Toledo et al., 2000; Swarça et al., 2001). This might be due to the presence of constitutive heterochromatin between the ribosomal genes, which might have promoted unequal exchanges between the chromatids (Sola et al., 1988) or by accumulating constitutive heterochromatin in an adjacent position to the NOR (Vicari et al., 2008). The existence of multiple sites of the 5S rDNA in several species may be considered an important indication of the great karyotypic diversity present in Ancistrini and should correspond to an apomorphic condition in the group. Studies conducted so far in the group suggest that the localization of the 5S rDNA in a single chromosome In B. xanthellus, large blocks of heterochromatin were observed in the short arm of pair 1 and the long arm of pair 10, and conspicuous blocks were co-located with the 18S and 5S rDNA sites (pairs 4 and 7, respectively) of all specimens Neotropical Ichthyology, 14(2): e150108, 2016 8 Cytogenetic analysis of Baryancistrus xanthellus Cytogenetic analysis of Baryancistrus xanthellus Acknowledgements This study was supported by Instituto Nacional de Pesquisas da Amazônia/Genética, Conservação e Biologia Evolutiva (INPA/GCBEv), Fundação de Amparo a Pesquisas do Estado do Amazonas (PRONEX FAPEAM/ CNPq 003/2009), and Center for Studies of Adaptation to Environmental Changes in the Amazon (INCT ADAPTA, FAPEAM/CNPq 573976/2008-20). The authors are grateful to Laboratório de Genética e Morfofisiologia, Faculdade de Ciências Biológicas, UFPA, Altamira, PA, for support. Dover, G. A. 1986. 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Unpublished Ph.D. Submitted July 24, 2015 Accepted February 09, 2016 by Cláudio Oliveira Acknowledgements Bertollo & O. Moreira-Filho. 2005. Karyotypic diversity and evolution of Loricariidae (Pisces: Siluriformes). Heredity, 94: 180-186. Lara, M. C. S. 1998. Aspectos citogenéticos de quatro espécies de peixes da subfamília Ancistrinae (Siluriformes: Loricariidae) da bacia do rio Paraná. Unpublished Msc. Dissertation, Universidade Estadual de Maringá, Maringá, 46f. Oliveira, R. R. 2006. Diversidade cariotípica entre dez espécies do gênero Ancistrus (Siluriformes, Loricariidae) da Bacia Amazônica: estrutura e mecanismos de evolução cromossômica. Unpublished master’s thesis. Instituto Nacional de Pesquisas da Amazônia, Manaus, 96p. Levan, A., K. Fredga & A. A. Sandberg. 1964. Nomenclature for centromeric position on chromosomes. Hereditas, 52: 201- 220. Oliveira, R. R., E. Feldberg, M. B. Anjos & J. Zuanon. 2007. Karyotype characterization and ZZ/ZW sex chromosome heteromorphism in two species of the catfish genus Ancistrus Kner, 1854 (Siluriformes: Loricariidae) from the Amazon basin. Neotropical Ichthyology, 5: 301-306. Lujan, N. K., J. W. Armbruster, N. R. Lovejoy & H. López- Fernández. 2015. Multilocus molecular phylogeny of the suckermouth armored catfishes (Siluriformes: Loricariidae) with a focus on subfamily Hypostominae. Molecular Phylogenetics and Evolution, 82: 269-288. Mariotto, S. 2009. Estudo citogenético clássico e molecular em quinze espécies da tribo Ancistrini (Siluriformes, Loricariidae) de três bacias hidrográficas brasileiras. Unpublished Ph. D. Dissertation, Universidade Federal de São Carlos, São Carlos, SP, 101p. Oliveira, R. R., E. Feldberg, M. B. Anjos & J. Zuanon. 2008. Occurrence of multiple sexual chromosomes (XX/ XY1Y2 and Z1Z1Z2Z2/Z1Z2W1W2) in catfishes of the genus Ancistrus (Siluriformes: Loricariidae) from the Amazon Basin. Genetica, 134: 243-249. Neotropical Ichthyology, 14(2): e150108, 2016 10 Cytogenetic analysis of Baryancistrus xanthellus Cytogenetic analysis of Baryancistrus xanthellus Thesis, Universidade Estadual de Campinas, Campinas, 199f. Souza, A. C. P. 2003. Descrição cariotípica de peixes dos gêneros Baryancistrus, Parancistrus, Peckoltia e Ancistrus (Ancistrinae, Loricariidae) da Bacia Amazônica. Unpublished master’s thesis, Universidade Federal do Pará/Museu Paraense Emílio Goeldi, Belém, Pará, 130f., il. Submitted July 24, 2015 Accepted February 09, 2016 by Cláudio Oliveira Submitted July 24, 2015 Accepted February 09, 2016 by Cláudio Oliveira
https://openalex.org/W1829150188	https://www.repository.cam.ac.uk/bitstreams/14ff7485-f4b8-4b65-a1a2-2d5fe6e75894/download	English	null	Great earthquakes in low strain rate continental interiors: An example from SE Kazakhstan	Journal of geophysical research. Solid earth	2,015	cc-by	23,501	Citation: Received 2 FEB 2015 Accepted 9 JUN 2015 Accepted article online 16 JUN 2015 Published online 6 AUG 2015 ©2015. The Authors. 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. RESEARCH ARTICLE Great earthquakes in low strain rate continental interiors: An example from SE Kazakhstan Citation: Campbell, G. E., R. T. Walker, K. Abdrakhmatov, J. Jackson, J. R. Elliott, D. Mackenzie, T. Middleton, and J.-L. Schwenninger (2015), Great earthquakes in low strain rate continental interiors: An example from SE Kazakhstan, J. Geophys. Res. Solid Earth, 120, 5507–5534, doi:10.1002/2015JB011925. Key Points: Key Points: G. E. Campbell1, R. T. Walker2, K. Abdrakhmatov3, J. Jackson1, J. R. Elliott2, D. Mackenzie2, T. Middleton2, and J.-L. Schwenninger4 • The Lepsy fault is an E-W reverse right-lateral fault in the northern Tien Shan 1Bullard Laboratories, Department of Earth Sciences, University of Cambridge, Cambridge, UK, 2Department of Earth Sciences, University of Oxford, Oxford, UK, 3Kyrgyz Institute of Seismology, Bishkek, Kyrgyzstan, 4Research Laboratory for Archaeology and the History of Art, University of Oxford, Oxford, UK • It has generated two substantial earthquakes (up to Mw 8) in the Holocene • The fault connects a slowly deforming region to a faster deforming region Abstract The Lepsy fault of the northern Tien Shan, SE Kazakhstan, extends E-W 120 km from the high mountains of the Dzhungarian Ala-tau, a subrange of the northern Tien Shan, into the low-lying Kazakh platform. It is an example of an active structure that connects a more rapidly deforming mountain region with an apparently stable continental region and follows a known Palaeozoic structure. Field-based and satellite observations reveal an ∼10 m vertical oﬀset exceptionally preserved along the entire length of the fault. Geomorphic analysis and age control from radiocarbon and optically stimulated luminescence dating methods indicate that the scarp formed in the Holocene and was generated by at least two substantial earthquakes. The most recent event, dated to sometime after ∼400 years B.P., is likely to have ruptured the entire ∼120 km fault length in a Mw 7.5–8.2 earthquake. The Lepsy fault kinematics were characterized using digital elevation models and high-resolution satellite imagery, which indicate that the predominant sense of motion is reverse right lateral with a fault strike, dip, and slip vector azimuth of ∼110∘, 50∘S, and 317–343∘, respectively, which is consistent with predominant N-S shortening related to the India-Eurasia collision. In light of these observations, and because the activity of the Lepsy fault would have been hard to ascertain if it had not ruptured in the recent past, we note that the absence of known active faults within low-relief and low strain rate continental interiors does not always imply an absence of seismic hazard. Correspondence to: G. E. Campbell, gc416@cam.ac.uk Citation: Campbell, G. E., R. T. Walker, K. Abdrakhmatov, J. Jackson, J. R. Elliott, D. Mackenzie, T. Middleton, and J.-L. Schwenninger (2015), Great earthquakes in low strain rate continental interiors: An example from SE Kazakhstan, J. Geophys. Res. Solid Earth, 120, 5507–5534, doi:10.1002/2015JB011925. Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 Figure 1. Active faults, earthquake mechanisms and centroid depths, and GPS velocities of the Tien Shan region. The purple box shows the Lepsy fault (Figure 2). Major NW-SE oriented platform faults are dashed in black, and currently, their activity in the Quaternary is unknown. Named from SW to NE, these include the Karatau (Kt.), Dzhalair-Naiman (Dh.), Aktas (Ak.), and Chingiz (Ch.) faults [after Tapponnier and Molnar, 1979]. The major NW-SE range interior/range-bounding strike-slip faults are the Talas-Ferghana fault (TF) [Burtman et al., 1996] in the west and the Dzhungarian fault (Dz.) in the east [after Voitovich, 1969; Tapponnier and Molnar, 1979; Campbell et al., 2013]. All other active faults are mapped from this study (from remote observations of satellite imagery and ﬁeldwork) and existing studies [e.g., Suvorov, 1963; Tapponnier and Molnar, 1979; Avouac et al., 1993; Abdrakhmatov et al., 2001; Thompson et al., 2002; Arrowsmith et al., 2004]. The green dot marks part of the Chon-Kemin fault that ruptured in the 1911 Ms ∼8.2 earthquake, with the fault zone itself shown as a green approximately E-W line. GPS velocities relative to stable Eurasia are shown [after Zubovich et al., 2010]. Body waveform-modeled earthquakes are color coded by depth [after Sloan et al., 2011, and references therein], with centroid depths ≥30 km indicated. Rare earthquakes occur in the platform, e.g., depth 40 km (depth outlined in green). Earthquakes in the updated Engdahl et al. [1998] catalogue are shown as black dots. Lettered blue squares mark the locations of major cities: Bishkek and Almaty (B and A, respectively) and major towns Taldykorgan and Sarkand (T and S, respectively). Country boundaries are shown as solid black lines. Figure 1. Active faults, earthquake mechanisms and centroid depths, and GPS velocities of the Tien Shan region. The purple box shows the Lepsy fault (Figure 2). Major NW-SE oriented platform faults are dashed in black, and currently, their activity in the Quaternary is unknown. Named from SW to NE, these include the Karatau (Kt.), Dzhalair-Naiman (Dh.), Aktas (Ak.), and Chingiz (Ch.) faults [after Tapponnier and Molnar, 1979]. The major NW-SE range interior/range-bounding strike-slip faults are the Talas-Ferghana fault (TF) [Burtman et al., 1996] in the west and the Dzhungarian fault (Dz.) in the east [after Voitovich, 1969; Tapponnier and Molnar, 1979; Campbell et al., 2013]. Journal of Geophysical Research: Solid Earth All other active faults are mapped from this study (from remote observations of satellite imagery and ﬁeldwork) and existing studies [e.g., Suvorov, 1963; Tapponnier and Molnar, 1979; Avouac et al., 1993; Abdrakhmatov et al., 2001; Thompson et al., 2002; Arrowsmith et al., 2004]. The green dot marks part of the Chon-Kemin fault that ruptured in the 1911 Ms ∼8.2 earthquake, with the fault zone itself shown as a green approximately E-W line. GPS velocities relative to stable Eurasia are shown [after Zubovich et al., 2010]. Body waveform-modeled earthquakes are color coded by depth [after Sloan et al., 2011, and references therein], with centroid depths ≥30 km indicated. Rare earthquakes occur in the platform, e.g., depth 40 km (depth outlined in green). Earthquakes in the updated Engdahl et al. [1998] catalogue are shown as black dots. Lettered blue squares mark the locations of major cities: Bishkek and Almaty (B and A, respectively) and major towns Taldykorgan and Sarkand (T and S, respectively). Country boundaries are shown as solid black lines. recent decades (e.g., the 40 km depth event, outlined in green in Figure 1). GPS measurements indicate low though nonnegligible rates of shortening north of the Tien Shan (e.g.,≤2 mm/yr) [Zubovichetal., 2010]. Due to the sparse seismicity, and overall absence of obvious geomorphic evidence of late Quaternary active faulting, the foreland of the northern Tien Shan is usually considered as a SCR or low strain rate continent interior, and it is not clear whether these rare foreland earthquakes occur on faults that connect with the active structures of the mountains and indeed whether the Kazakh platform may be susceptible to larger, more damaging, earthquakes than those present in instrumental catalogues. The semiarid continental climate in the northern Tien Shan region has allowed exceptional preservation of historical earthquake ruptures, including the 1911 Ms ∼8.2 Chon-Kemin surface rupture [Bogdanovich et al., 1914; Delvaux et al., 2001; Abdrakhmatov et al., 2002] (with location marked by a green dot in Figure 1). We may therefore expect surface faulting associated with other, prehistoric, large earthquakes to be preserved in the landscape of this pristine environment. This paper examines the evidence for such an event on the Lepsy fault, in the Tien Shan forelands, and in an area that has no record of seismic activity. 1. Introduction This paper looks at seismic hazard in regions that may contain active faults, but which have no documented record of earthquakes. Large-magnitude earthquakes are rare though widespread in apparently stable continental regions (SCRs) [Sykes, 1978; Johnston and Kanter, 1990; Schulte and Mooney, 2005; Clark et al., 2012], and examples exist in parts of India, Australia, Africa, and North America [Fuller, 1912; Everingham and Gregson, 1969; McCaﬀrey, 1989; Johnston and Schweig, 1996; Seeber et al., 1996; Rajendran et al., 2001; Copley et al., 2012]. Often, but not always, such events cause widespread destruction because of their size, because thedamagingeﬀectsofshakingextendfurtherfromthesourcethaninmostcontinentalregions,andbecause the faults on which they occur are often unknown. In all of the examples of intraplate earthquakes listed above, long-term fault slip rates are relatively low (e.g., 1–2 mm/yr or less) compared with those in interplate regions or major collision boundaries within the conti- nents (such as the Himalayan frontal thrust), and the recurrence times of large earthquakes on the majority of active faults within low strain rate regions are likely to exceed the length of the documented historical record. With repeat times of many thousands of years, erosion may also remove any clear expression of surface fault- ing from the landscape, creating additional challenges in identifying and characterizing seismogenic faults in slowly deforming continent interiors. This paper is concerned with the foreland of the northern Tien Shan, central Asia (Figure 1). The Tien Shan itself is one of the worlds largest and most seismically active intracontinental orogens and accommodates up to 50% (15–20 mm/yr) of the total shortening related to the ongoing India-Eurasia collision [Abdrakhmatov etal., 1996]. GPS velocities relative to stable Eurasia indicatethat most of the shortening is concentrated within the mountain interior, which is where the majority of seismicity is conﬁned (Figure 1). However, in the ﬂat low-lying Kazakh platform, north of the Tien Shan, a few rare earthquakes of moderate size have occurred in ©2015. The Authors. 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. LOW STRAIN-RATE, CONTINENTAL FAULTING CAMPBELL ET AL. 5507 Journal of Geophysical Research: Solid Earth Journal of Geophysical Research: Solid Earth 2.1. Geological Setting Three types of active faulting dominate the mountains of the northern Tien Shan: NW-SE and NE-SW con- jugate strike-slip faults [e.g., Voitovich, 1969; Tapponnier and Molnar, 1979; Burtman et al., 1996; Selander et al., 2012; Campbell et al., 2013] and E-W oriented reverse faults [e.g., Avouac et al., 1993; Ghose et al., 1997; Burbank et al., 1999; Thompson et al., 2002]. The Lepsy fault scarp (Figure 1), which is the subject of this study, is oriented approximately E-W and extends for ∼120 km from the high Chirindy Mountains, a subrange of the Dzhungarian Ala-tau, into the ﬂat, low-lying Kazakh platform, where it terminates abruptly in a N-S oriented fault (Figure 2). An ∼10 m high vertical scarp is observed almost continuously in late Quaternary sediments along 120 km of the fault trace in the ﬁeld. Despite having being originally recognized as potentially active in the Quaternary [Kurdiukov, 1956], it is unreported on active fault maps [e.g., Trifonov, 1978; Tapponnier and Molnar, 1979; Thomas et al., 1996]. The Lepsy fault scarp appears to be on a structure that may continue even further into the platform (see Figure 2 and section 2.5) and into a region where erosion and depo- sition have removed most of its topographic expression. This westernmost section does not display clear evidence of late Quaternary activity. At its eastern end, the fault is ∼10 km SSW from the central section of the Dzhungarian strike-slip fault, a major active NW-SE trending right-lateral fault, over ∼300 km long, which bounds the eastern extent of the Dzhungarian Ala-tau [Voitovich, 1969; Tapponnier and Molnar, 1979; Campbell et al., 2013], and it is likely that the Lepsy fault is a splay from the active Dzhungarian fault. At its westernmost end the Lepsy fault is ∼80 km from the NW-SE Aktas fault, whose potential Quaternary activity is unknown (Figure 1). GPS velocities relative to stable Eurasia indicate that 1–3 mm/yr of N-S shortening must be accommodated north of the Tien Shan (Figure 1) [Zubovich et al., 2010]. Despite low strain rates (10−16 s−1) within the plat- form where the Lepsy fault is sited, the instrumental earthquake record reveals that occasional earthquakes have occurred (Figure 1), but these are all relatively small, with magnitudes ranging from Mw 5.3–6.4 [Sloan et al., 2011]. Journal of Geophysical Research: Solid Earth Identiﬁcation and characterization of structures like the Lepsy fault are increasingly signiﬁcant for hazard evaluation, especially given that it is only ∼250 km NE from the highly populated former capital city of Kazakhstan, Almaty (Figure 1). The Lepsy fault is also only ∼100 km from two signiﬁcant administrative capitals, Sarkand and Taldykorgan (Figure 1), with a combined population of over 114,000 people. We describe remote sensing and ﬁeld observations, and Quaternary dating results, which show that the Lepsy fault has generated at least two substantial earthquakes in the Holocene, the latter of which may have been Mw 8 and is dated to within the last ∼400 years B.P. We then characterize the geometry and kinematics of the Lepsy fault, as well as its late Holocene seismic activity, and discuss the role of the fault in accommodating regional N-S shortening related to the ongoing India-Eurasia collision. LOW STRAIN-RATE, CONTINENTAL FAULTING CAMPBELL ET AL. CAMPBELL ET AL. 5508 Journal of Geophysical Research: Solid Earth Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 Figure 2. Shaded relief map of the structural and geomorphological features observed along the Lepsy fault from ﬁeldwork and analysis of satellite imagery. The scarp shows both right-lateral and vertical (uplift to the south) components of slip along its entire length. Sites discussed in detail in the text are marked by numbered black dashed boxes. Sample locations are shown by yellow circles. The N-S, dashed white lines delimit the east, central, and west Lepsy fault sections. Figure 2. Shaded relief map of the structural and geomorphological features observed along the Lepsy fault from ﬁeldwork and analysis of satellite imagery. The scarp shows both right-lateral and vertical (uplift to the south) components of slip along its entire length. Sites discussed in detail in the text are marked by numbered black dashed boxes. Sample locations are shown by yellow circles. The N-S, dashed white lines delimit the east, central, and west Lepsy fault sections. 2.1. Geological Setting Well-determined earthquake centroid depths in the Tien Shan forelands and Kazakh platform (Figure 1) indicate that the seismogenic layer is relatively thick (≥35 km) [Chen and Molnar, 1977; Nelson et al., 1987; Sloan et al., 2011]. If any of the Palaeozoic structures within the foreland are reactivated in future earth- quakes, then the large downdip width of the faults within the platform increases their potential maximum earthquake magnitude. Figure 2 shows an annotated shaded relief map of the geomorphological features we interpret as fault scarps alongtheLepsyfaultandwhichwehavemappedfromsubmeterresolutionWorldviewsatelliteimagery,addi- tional imagery accessed on Google Earth and Bing Maps, and ﬁeldwork. Figures 3–19 show ﬁeld observations, which are described in the following sections, beginning in the east and ending in the west. We assess the amount and direction of slip, which are essential in order to establish the fault kinematics and are useful in the LOW STRAIN-RATE, CONTINENTAL FAULTING CAMPBELL ET AL. CAMPBELL ET AL. 5509 Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 Figure 3. East Lepsy fault map and the Jaxa-Kol ﬁeld site. (a) Google Earth (source for all Google Earth images: earth.google.co.uk/) image showing the main E-W scarp (red triangles) and a secondary scarp (yellow triangles) that splays to the NE crossing N-S oriented ridges in the high Chirindy mountains of the Dzhungarian Ala-tau, SE Kazakhstan. The scarp uplifts the south side relative to the north side and right laterally oﬀsets a series of channels (solid and dashed blue arrows) and ridge crests (solid black-white lines). Wind gaps are preserved on the south side, and a series of (mostly dried) ponds have formed against the scarp. The location of the ﬁeld photo in Figure 3b is marked (black arrow), and the sampling site is shown (yellow circle); (b) View south of ridges C and D showing ∼10 m vertical oﬀset and signiﬁcant right-lateral oﬀset. Figure 3. East Lepsy fault map and the Jaxa-Kol ﬁeld site. (a) Google Earth (source for all Google Earth images: earth.google.co.uk/) image showing the main E-W scarp (red triangles) and a secondary scarp (yellow triangles) that splays to the NE crossing N-S oriented ridges in the high Chirindy mountains of the Dzhungarian Ala-tau, SE Kazakhstan. The scarp uplifts the south side relative to the north side and right laterally oﬀsets a series of channels (solid and dashed blue arrows) and ridge crests (solid black-white lines). 2.1. Geological Setting Wind gaps are preserved on the south side, and a series of (mostly dried) ponds have formed against the scarp. The location of the ﬁeld photo in Figure 3b is marked (black arrow), and the sampling site is shown (yellow circle); (b) View south of ridges C and D showing ∼10 m vertical oﬀset and signiﬁcant right-lateral oﬀset. Figure 3. East Lepsy fault map and the Jaxa-Kol ﬁeld site. (a) Google Earth (source for all Google Earth images: earth.google.co.uk/) image showing the main E-W scarp (red triangles) and a secondary scarp (yellow triangles) that splays to the NE crossing N-S oriented ridges in the high Chirindy mountains of the Dzhungarian Ala-tau, SE Kazakhstan. The scarp uplifts the south side relative to the north side and right laterally oﬀsets a series of channels (solid and dashed blue arrows) and ridge crests (solid black-white lines). Wind gaps are preserved on the south side, and a series of (mostly dried) ponds have formed against the scarp. The location of the ﬁeld photo in Figure 3b is marked (black arrow), and the sampling site is shown (yellow circle); (b) View south of ridges C and D showing ∼10 m vertical oﬀset and signiﬁcant right-lateral oﬀset. assessment of earthquake hazard. The following sections present our observations. We discuss the fault kine- matics in section 3.1. The details of our methods for estimating vertical and horizontal displacements, and all Quaternary dating procedures, are described in appendices. 2.2. East: Jaxa-Kol Section 2.2.1. Overview People for scale (white circles) stand in the channel thalweg on the north side where there is a dried pond and on the (upthrown) south side where there is now a wind gap. (b) Digital elevation model (DEM) of ridges A and B generated from gridded Diﬀerential Global Positioning System (DGPS) points from a ﬁeld survey (individual survey points have horizontal and vertical uncertainties of ∼10 cm, and the DEM is gridded at a 50 cm pixel spacing). The DEM shows the traces of fault-parallel elevation proﬁles a-a′ (light blue) and b-b′ (dark blue) on the hanging wall and footwall, respectively. (c) Fault-parallel elevation proﬁles generated from the DEM shown in Figure 4b, also shown in Figure 5. Restoration of ∼6 m right-lateral slip realigns the well-deﬁned channel thalweg (the center point of which is deﬁned by the vertical red line between ridges A and B) on either side of the fault. Around 5 m of vertical oﬀset then totally realigns the topography on either side of the fault to form a single continuous “prefaulted” surface. Note that the vertical oﬀset required to align the topography underestimates the vertical displacement across the scarp, as it does not account for the southward topographic slope. Figure 4. (a) View south of ridges A and B (see Figure 3 for regional location). The south side is uplifted relative to the north side. Right-lateral displacement is revealed by the asymmetry of the oﬀset ridges across the fault. People for scale (white circles) stand in the channel thalweg on the north side where there is a dried pond and on the (upthrown) south side where there is now a wind gap. (b) Digital elevation model (DEM) of ridges A and B generated from gridded Diﬀerential Global Positioning System (DGPS) points from a ﬁeld survey (individual survey points have horizontal and vertical uncertainties of ∼10 cm, and the DEM is gridded at a 50 cm pixel spacing). The DEM shows the traces of fault-parallel elevation proﬁles a-a′ (light blue) and b-b′ (dark blue) on the hanging wall and footwall, respectively. (c) Fault-parallel elevation proﬁles generated from the DEM shown in Figure 4b, also shown in Figure 5. Restoration of ∼6 m right-lateral slip realigns the well-deﬁned channel thalweg (the center point of which is deﬁned by the vertical red line between ridges A and B) on either side of the fault. 2.2. East: Jaxa-Kol Section 2.2.1. Overview Around 5 m of vertical oﬀset then totally realigns the topography on either side of the fault to form a single continuous “prefaulted” surface. Note that the vertical oﬀset required to align the topography underestimates the vertical displacement across the scarp, as it does not account for the southward topographic slope. LOW STRAIN-RATE, CONTINENTAL FAULTING 2.2. East: Jaxa-Kol Section 2.2.1. Overview The DEM shows the traces of -parallel elevation proﬁles a-a′ (light blue) and b-b′ (dark blue) on the hanging wall and footwall, respectivel t-parallel elevation proﬁles generated from the DEM shown in Figure 4b, also shown in Figure 5. Restoration o t-lateral slip realigns the well-deﬁned channel thalweg (the center point of which is deﬁned by the vertical re ween ridges A and B) on either side of the fault. Around 5 m of vertical oﬀset then totally realigns the topogra er side of the fault to form a single continuous “prefaulted” surface. Note that the vertical oﬀset required to a graphy underestimates the vertical displacement across the scarp as it does not account for the southward 10.1002/2015JB011925 re 4. (a) View south of ridges A and B (see Figure 3 for regional location). The south side is uplifted relative t h side. Right-lateral displacement is revealed by the asymmetry of the oﬀset ridges across the fault. People fo te circles) stand in the channel thalweg on the north side where there is a dried pond and on the (upthrown where there is now a wind gap. (b) Digital elevation model (DEM) of ridges A and B generated from gridded rential Global Positioning System (DGPS) points from a ﬁeld survey (individual survey points have horizonta cal uncertainties of ∼10 cm, and the DEM is gridded at a 50 cm pixel spacing). The DEM shows the traces of -parallel elevation proﬁles a-a′ (light blue) and b-b′ (dark blue) on the hanging wall and footwall, respective -parallel elevation proﬁles generated from the DEM shown in Figure 4b, also shown in Figure 5. Restoration -lateral slip realigns the well-deﬁned channel thalweg (the center point of which is deﬁned by the vertical re ween ridges A and B) on either side of the fault. Around 5 m of vertical oﬀset then totally realigns the topogr er side of the fault to form a single continuous “prefaulted” surface. Note that the vertical oﬀset required to a h d ti t th ti l di l t th it d t t f th th d Figure 4. (a) View south of ridges A and B (see Figure 3 for regional location). The south side is uplifted relative to the north side. Right-lateral displacement is revealed by the asymmetry of the oﬀset ridges across the fault. 2.2. East: Jaxa-Kol Section 2.2.1. Overview In the east, the fault scarp crosses N-S trending ridges in high (∼1450 m elevation) mountain topography of the Dzhungarian Ala-tau (Figures 2 and 3). A regionally extensive south tilted peneplain surface separating Paleozoic and overlying Cenozoic rocks [Chediya, 1986; Mikolaichuk et al., 2003; Cunningham et al., 2003] is preserved throughout the Dzhungarian Ala-tau. Abandoned drainage channels within the peneplain surface south of the Lepsy fault, which are preserved as what we interpret to be wind gaps, attest to cumulative fault movement of ≥100 m (Figure 3). The main scarp strikes E-W and follows a bedding plane in resistant Paleozoic bedrock, which dips steeply north (80∘N) and is covered by a thin veneer of soil stabilized with thick vegetation (Figure 3b). Vertical and right-lateral displacement of a series of ridge crests and adjacent channels has resulted in ponding of drainage against the upthrown southern side (Figures 3–5) and smaller-scale ponding of channel thalwegs against reactivated distributed bedding planes with oﬀsets of typically tens of centimeters. Although the main scarp is concentrated on a single bedding plane, a secondary scarp extends northeast of the main scarp trace for 1.5 km (Figure 3). Analysis of satellite imagery indicates that the main fault scarp continues eastward for a further 4 km from the view shown in Figure 3 to ∼81∘05′E with a more southeasterly strike of ∼110∘ LOW STRAIN-RATE, CONTINENTAL FAULTING CAMPBELL ET AL. 5510 CAMPBELL ET AL. Journal of Geophysical Research: Solid Earth Journal of Geophysical Research: Solid Earth of Geophysical Research: Solid Earth 10.1002/2015JB011 re 4. (a) View south of ridges A and B (see Figure 3 for regional location). The south side is uplifted relative to h side. Right-lateral displacement is revealed by the asymmetry of the oﬀset ridges across the fault. People fo te circles) stand in the channel thalweg on the north side where there is a dried pond and on the (upthrown) where there is now a wind gap. (b) Digital elevation model (DEM) of ridges A and B generated from gridded rential Global Positioning System (DGPS) points from a ﬁeld survey (individual survey points have horizontal cal uncertainties of ∼10 cm, and the DEM is gridded at a 50 cm pixel spacing). LOW STRAIN-RATE, CONTINENTAL FAULTING The lateral proﬁles are shown in Figure 4c. (bottom) Proﬁles along the crests of ridges A and B show similar gradients on both sides of the fault and yield estimates of ∼13 ± 2 m and ∼11 ± 2 m vertical oﬀset, respectively. Figure 5. Oﬀset ridges, east Lepsy fault. (top) Digital elevation model (DEM) of ridges A and B generated from gridded Diﬀerential Global Positioning System (DGPS) points. The x, y, z points from which the DEM was constructed are shown as brown dots. Each survey point has uncertainties in horizontal and vertical locations of ∼10 cm. The black arrow shows the direction of streamﬂow to the south, which is ponded against the fault scarp. A wind gap is preserved on the south side of the fault. To estimate the vertical displacement across the scarp, we are restricted to making measurements across the oﬀset ridge crests, as the gradient of channel bottoms has been altered by erosion and deposition, and the large lateral component makes matching the sloping hillsides diﬃcult. The tracks of the vertical (orange and yellow lines) and lateral (light and dark blue lines) fault elevation proﬁles are shown on either side of the fault. The vertical proﬁles are shown in Figure 5 (bottom). The lateral proﬁles are shown in Figure 4c. (bottom) Proﬁles along the crests of ridges A and B show similar gradients on both sides of the fault and yield estimates of ∼13 ± 2 m and ∼11 ± 2 m vertical oﬀset, respectively. (see Figure 2). At its easternmost extent, the scarp bends southeastward and terminates in a series of smaller secondary splays. LOW STRAIN-RATE, CONTINENTAL FAULTING 5511 CAMPBELL ET AL. Journal of Geophysical Research: Solid Earth Journal of Geophysical Research: Solid Earth Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 10.1002/2015JB011925 Figure 5. Oﬀset ridges, east Lepsy fault. (top) Digital elevation model (DEM) of ridges A and B generated from gridded Diﬀerential Global Positioning System (DGPS) points. The x, y, z points from which the DEM was constructed are shown as brown dots. Each survey point has uncertainties in horizontal and vertical locations of ∼10 cm. The black arrow show the direction of streamﬂow to the south, which is ponded against the fault scarp. A wind gap is preserved on the south side of the fault. To estimate the vertical displacement across the scarp, we are restricted to making measurements across the oﬀset ridge crests, as the gradient of channel bottoms has been altered by erosion and deposition, and the large lateral component makes matching the sloping hillsides diﬃcult. The tracks of the vertical (orange and yellow lines) and lateral (light and dark blue lines) fault elevation proﬁles are shown on either side of the fault. The vertical proﬁles are shown in Figure 5 (bottom). The lateral proﬁles are shown in Figure 4c. (bottom) Proﬁles along the crests of ridges A and B show similar gradients on both sides of the fault and yield estimates of ∼13 ± 2 m and ∼11 ± 2 m vertic oﬀset, respectively. Figure 5. Oﬀset ridges, east Lepsy fault. (top) Digital elevation model (DEM) of ridges A and B generated from gridded Diﬀerential Global Positioning System (DGPS) points. The x, y, z points from which the DEM was constructed are shown as brown dots. Each survey point has uncertainties in horizontal and vertical locations of ∼10 cm. The black arrow shows the direction of streamﬂow to the south, which is ponded against the fault scarp. A wind gap is preserved on the south side of the fault. To estimate the vertical displacement across the scarp, we are restricted to making measurements across the oﬀset ridge crests, as the gradient of channel bottoms has been altered by erosion and deposition, and the large lateral component makes matching the sloping hillsides diﬃcult. The tracks of the vertical (orange and yellow lines) and lateral (light and dark blue lines) fault elevation proﬁles are shown on either side of the fault. The vertical proﬁles are shown in Figure 5 (bottom). 2.2.2. Geomorphology We generated digital elevation models (DEMs) of three adjacent shutter ridges (labeled A, B, and C, from east to west along strike, shown in Figure 3) using gridded Diﬀerential Global Positioning System (DGPS) measure- ments (Figures 4, 5, and 8). Uncertainties in horizontal positions and heights of the individual survey points are less than 10–20 cm and are negligible compared with small-scale relief on the ridge surfaces surveyed at this site and alluvial surfaces surveyed at later sites. To generate a DEM, a regular grid was constructed from the irregularly spaced x, y, z points. The pixel spacing in the DEM is 0.5 m. Between ridges A and B (Figures 4 and 5), a well-deﬁned channel thalweg indicates ∼6 ± 2 m of right-lateral slip. Topographic proﬁles along ridge crests A and B show a vertical oﬀset of 13 ± 2 m and 11 ± 2 m, respec- tively (Figure 5). There is some ambiguity in trying to determine the fault dip direction and hence the nature of fault motion at this site, because the fault follows bedding that dips very steeply, 80∘N, and is unlikely to represent the fault dip at depth. We also observe distributed bedding plane slip manifest as decimeter verti- cal oﬀsets (south side down), on a series of almost vertical beds, over a distance of ∼100 m to the south of the main scarp (e.g., Figure 6). Ridge C (Figures 3, 7, and 8) is 160 m west of ridges A and B and records 4 ± 2 m of right-lateral slip and ∼9 m of vertical slip (Figure 8), considerably less than the measurements from A and B, but with a similar ratio of vertical and horizontal displacements. 2.2.3. Scarp Age At this eastern site, three samples (OSL1, OSL2, and RC1; Tables 1 and 2) were collected from an ∼1.20 m deep pit in dried pond sediments, which have accumulated between ridges A and B after scarp formation, and LOW STRAIN-RATE, CONTINENTAL FAULTING 5512 CAMPBELL ET AL. CAMPBELL ET AL. Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 Figure 6. View SW from ridge B of steeply north dipping Paleozoic bedding planes (white arrows). The Lepsy fault trace is marked with red triangles with people for scale (white circles) standing on ridge A. 2.2.2. Geomorphology Black dashed lines show potential vertical oﬀsets on consecutive bedding planes over a distance of ∼100 m to the south of the ∼10 m scarp. Figure 6. View SW from ridge B of steeply north dipping Paleozoic bedding planes (white arrows). The Lepsy fault trace is marked with red triangles with people for scale (white circles) standing on ridge A. Black dashed lines show potential vertical oﬀsets on consecutive bedding planes over a distance of ∼100 m to the south of the ∼10 m scarp. blocking of a small south ﬂowing channel (Figure 5). The pit stratigraphy revealed ∼30 cm of stony dark top soil, which graded into a homogeneous silt unit. This silt was underlain by interbedded medium sand-gravel and homogeneous silt layers and a dark organic-rich soil with angular ∼1 cm bedrock fragments. We infer that the sand and silt units represent ponding against the scarp and were therefore deposited after some amount of oﬀset on the fault. The dark soil beneath these former units might represent the original prefaulted surface or alternatively a dry period during which a soil layer formed on top of older pond units formed against a preexisting scarp. OSL1 was collected from silt unit at a depth of 0.85 m and yielded an age of 1770 ± 510 years B.P. provid- ing a minimum age estimate for scarp formation. OSL2 was taken from silt unit B at a depth of 0.65 m and yielded an age of 910±520 years B.P. and also provides a minimum age estimate for scarp formation. A single Figure 7. View south of oﬀset ridge C with people standing on the ridge crest either side of the fault (white circles) for scale. A DEM generated from ﬁeld DGPS survey of the same area. See Figure 8 for the elevation proﬁles measuring fault vertical and lateral oﬀsets. Figure 7. View south of oﬀset ridge C with people standing on the ridge crest either side of the fault (white circles) for scale. A DEM generated from ﬁeld DGPS survey of the same area. See Figure 8 for the elevation proﬁles measuring fault vertical and lateral oﬀsets. LOW STRAIN-RATE, CONTINENTAL FAULTING CAMPBELL ET AL. 5513 Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 (a) Field DGPS-generated DEM of ridge C. The x, y, z points from which the DEM was constructed are shown as brown dots. Each survey point ha ties in horizontal and vertical locations of ∼10 cm. (b) Fault-parallel elevation proﬁles on either side of the fault record 4 ± 2 m of right-lateral oﬀ ately 7 m vertical slip restores the total amount of oﬀset and restores the topography back to an original prefaulted surface. Note that the vertic uired to align the topography underestimates the vertical displacement across the scarp, as it does not account for the southward topographic s -perpendicular elevation proﬁle along the ridge crest shows diﬀerent slopes on either side of the fault, introducing additional uncertainty in esti al displacement. We assign an uncertainty of ±2 m to the 8.8 m displacement measured across the center point of the scarp, which reﬂects varia ment depending on whether it is measured at the base or at the top of the scarp; see Appendix A for more details. Figure 8. (a) Field DGPS-generated DEM of ridge C. The x, y, z points from which the DEM was constructed are shown as brown dots. Each survey point has uncertainties in horizontal and vertical locations of ∼10 cm. (b) Fault-parallel elevation proﬁles on either side of the fault record 4 ± 2 m of right-lateral oﬀset. Approximately 7 m vertical slip restores the total amount of oﬀset and restores the topography back to an original prefaulted surface. Note that the vertical oﬀset required to align the topography underestimates the vertical displacement across the scarp, as it does not account for the southward topographic slope. (c) A fault-perpendicular elevation proﬁle along the ridge crest shows diﬀerent slopes on either side of the fault, introducing additional uncertainty in estimating the vertical displacement. We assign an uncertainty of ±2 m to the 8.8 m displacement measured across the center point of the scarp, which reﬂects variability in displacement depending on whether it is measured at the base or at the top of the scarp; see Appendix A for more details. bulk radiocarbon sample (RC1) collected from the dark, organic-rich soil at a depth of 1.05 m gave an age of between 1988 and 1806 calibrated radiocarbon years B.P. The increase in age with depth is consistent with stratigraphic position. CAMPBELL ET AL. We interpret the basal dark soil beneath the interbedded gravel-sand-silt units as rep- resenting periods of downslope transport of gravel followed by settling of ﬁnes in a pond. Because we cannot be sure that the dark soil is the true base of the pond, these dates allow us to infer that the scarp has been present in this location, at least in some form, since at least ∼2000 years ago. 2.3. Central Section: West of the Tentek River 2.3.1. Overview Between the north ﬂowing Tentek and Shingildy Rivers, (80∘54′E and 80∘34′E, respectively) the Lepsy scarp continues for 25 km along the Chirindy range front, with peak heights ∼800–900 m above the range front (Figure 2). Several parallel lines of surface rupturing are observed, deﬁning a fault zone up to 1 km wide, and LOW STRAIN-RATE, CONTINENTAL FAULTING 2.3. Central Section: West of the Tentek River 2.3.1. Overview Between the north ﬂowing Tentek and Shingildy Rivers, (80∘54′E and 80∘34′E, respectively) the Lepsy scarp continues for 25 km along the Chirindy range front, with peak heights ∼800–900 m above the range front (Figure 2). Several parallel lines of surface rupturing are observed, deﬁning a fault zone up to 1 km wide, and LOW STRAIN-RATE, CONTINENTAL FAULTING 5514 CAMPBELL ET AL. Journal of Geophysical Research: Solid Earth Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 Figure 9. The central Lepsy fault. (a) Google Earth satellite image centered at 45∘55′N/80∘41′E shows a characteristic section of the central Lepsy fault where it bounds the lower relief of the Chirindy range front (see Figure 2 for the regional ﬁgure location). A 1 km wide zone of distributed deformation is marked out by several subparallel lines of surface rupturing (red triangles). The black dashed box shows the lobate morphology and wrinkle ridge surface texture, which is characteristic of landslide deposits (in alluvium and loess). (b) View south from 45∘56′N/80∘51′E, showing continuous ∼10 m vertical oﬀset preserved in Quaternary alluvium (red triangles) also characteristic of the central Lepsy fault. A small scallop landslide in the fault scarp is shown (white arrow shows the direction of motion in landsliding). (c) View west of landsliding in loess (the white solid arrow shows the direction of sliding, and the white dashed line marks the landslide toe), also characteristic of the central fault section. Vehicle circled for scale. Figure 9. The central Lepsy fault. (a) Google Earth satellite image centered at 45∘55′N/80∘41′E shows a characteristic section of the central Lepsy fault where it bounds the lower relief of the Chirindy range front (see Figure 2 for the regional ﬁgure location). A 1 km wide zone of distributed deformation is marked out by several subparallel lines of surface rupturing (red triangles). The black dashed box shows the lobate morphology and wrinkle ridge surface texture, which is characteristic of landslide deposits (in alluvium and loess). (b) View south from 45∘56′N/80∘51′E, showing continuous ∼10 m vertical oﬀset preserved in Quaternary alluvium (red triangles) also characteristic of the central Lepsy fault. A small scallop landslide in the fault scarp is shown (white arrow shows the direction of motion in landsliding). (c) View west of landsliding in loess (the white solid arrow shows the direction of sliding, and the white dashed line marks the landslide toe), also characteristic of the central fault section. Vehicle circled for scale. slumping in thick loess cover is pervasive (Figure 9). In places, two parallel scarps are visible in satellite imagery (Figure 10a). The Chirindy range ends east of the Shingildy River, but the late Quaternary fault scarp continues westward through the steppe (Figure 12). LOW STRAIN-RATE, CONTINENTAL FAULTING The white box shows the area of the DEM in Figure 11b. The location of ﬁeld photos are shown: (b) view SSW of the main Lepsy scar ed in late Quaternary fan material with people circled for scale and (c) view SW from 45∘55.706′N/80∘53.847′E, showing the fault dip of ∼50∘S prese ock in a gorge west of the Tentek River gorge. The slopes are vegetated, obscuring the view of the fault in the photograph. However, the fault can b from a subtle color change (arising from a juxtaposition of two bedrock types) between the north side and south side of the fault (marked by the r ine) and a height change at the ridge crest. (d) Scarp elevation proﬁles i–iv, derived from a series of NNE-SSW proﬁle lines from the DGPS DEM show 1b and numbered in order from east to west. Proﬁles show variable vertical oﬀset that we interpret to arise from the presence of steeper colluvial s on the upthrown side. We estimate that the actual scarp height is within the range ∼5–8 m. 0. (a) Google Earth satellite image showing an overview of the Tentek River ﬁeld site. The Lepsy fault is observed as two north and south subpara red triangles). The white box shows the area of the DEM in Figure 11b. The location of ﬁeld photos are shown: (b) view SSW of the main Lepsy sca d in late Quaternary fan material with people circled for scale and (c) view SW from 45∘55.706′N/80∘53.847′E, showing the fault dip of ∼50∘S pres ck in a gorge west of the Tentek River gorge. The slopes are vegetated, obscuring the view of the fault in the photograph. However, the fault can b from a subtle color change (arising from a juxtaposition of two bedrock types) between the north side and south side of the fault (marked by the ne) and a height change at the ridge crest. (d) Scarp elevation proﬁles i–iv, derived from a series of NNE-SSW proﬁle lines from the DGPS DEM sho 1b and numbered in order from east to west. Proﬁles show variable vertical oﬀset that we interpret to arise from the presence of steeper colluvial on the upthrown side. We estimate that the actual scarp height is within the range ∼5–8 m. Figure 10. (a) Google Earth satellite image showing an overview of the Tentek River ﬁeld site. LOW STRAIN-RATE, CONTINENTAL FAULTING Cenozoic red beds exposed south of the fault scarp, in cuttings of the Shingildy River, are folded into a gentle anticline over a wavelength of ∼2 km and contain minor south dipping reverse faults (Figure 13). We observe a fault dip of ∼50∘S preserved in poorly exposed bedrock in a smaller river gorge west of the Tentek River at 45∘55.706′N/80∘53.847′E (Figure 10c). 2.3.2. Geomorphology Although more discontinuous in this central section relative to the eastern part, the main scarp trace still uplifts the south side (range front) relative to the north side (foreland) as a 5–10 m vertical oﬀset in Quaternary material (Figure 10). At 45∘55′N/80∘52′E, where there is no evidence of slumping in the loess cover, the main scarp is preserved as a vertical oﬀset in an alluvial fan surface (Figure 10). The local strike at this site is ∼110∘. The estimated vertical displacement varies between ∼6 m and ∼9 m within the four individual topographic proﬁles shown in Figure 10d. The variation may arise from the locally steeper slopes found on the southern, upthrown, side of the scarp, where the alluvium grades into steeper hillslope colluvial deposits. Given that the proﬁle showing the largest scarp height of ∼9 m also shows steep slopes on its southern side, we exclude it and estimate the likely full range of vertical displacement at this site to be 5–8 m. A well-preserved N-S oriented ﬂuvial riser also records 7 ± 2 m of right-lateral oﬀset at this same location (Figure 11). We did not collect samples for dating from this site, but the scarp here is preserved in alluvium, as opposed to the resistant bedrock in which it is preserved in the east at Jaxa-Kol; therefore, we suggest that the scarp here is at least comparable in age to the scarp at Jaxa-Kol, if not younger. We also note that postuplift stream incision has only migrated upstream by ∼50 m, where a prominent knickpoint is visible (Figures 11a and 11b). There is no evidence of older oﬀsets, in the form of higher river terraces, recorded in the geomorphology south of LOW STRAIN-RATE, CONTINENTAL FAULTING CAMPBELL ET AL. 5515 CAMPBELL ET AL. Journal of Geophysical Research: Solid Earth Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 10. (a) Google Earth satellite image showing an overview of the Tentek River ﬁeld site. The Lepsy fault is observed as two north and south subparal (red triangles). LOW STRAIN-RATE, CONTINENTAL FAULTING OSL3 consisted mainly of ﬁne silt, with some evidence of ﬂuvial reworking in the form of ﬁne- to medium-grained sand interbedded on a 5 cm scale and yielded an age of 2190 ± 470 years B.P. OSL3 provides a postdate on the major period of ﬂuvial gravel deposition that formed the terrace. OSL4 was collected from a medium-grain sand unit within coarser river CAMPBELL ET AL. LOW STRAIN-RATE, CONTINENTAL FAULTING 5516 At the Shingildy River, we collected samples OSL3, OSL4, and RC2 (Tables 1 and 2, respectively) from the 7.4 ± 1 m high scarp where it is preserved in a terrace on the west side of the Shingildy River (Figure 12). OSL3 was collected from the base of the Loess cap, 5 cm above the contact with the river gravels (Figure 12b), at a depth of 1.05 m below the surface, or ∼6.5 m above the river bed. OSL3 consisted mainly of ﬁne silt, with some evidence of ﬂuvial reworking in the form of ﬁne- to medium-grained sand interbedded on a 5 cm scale and yielded an age of 2190 ± 470 years B.P. OSL3 provides a postdate on the major period of ﬂuvial gravel deposition that formed the terrace. OSL4 was collected from a medium-grain sand unit within coarser river LOW STRAIN-RATE, CONTINENTAL FAULTING The Lepsy fault is observed as two north and south subparallel strands (red triangles). The white box shows the area of the DEM in Figure 11b. The location of ﬁeld photos are shown: (b) view SSW of the main Lepsy scarp preserved in late Quaternary fan material with people circled for scale and (c) view SW from 45∘55.706′N/80∘53.847′E, showing the fault dip of ∼50∘S preserved in bedrock in a gorge west of the Tentek River gorge. The slopes are vegetated, obscuring the view of the fault in the photograph. However, the fault can be inferred from a subtle color change (arising from a juxtaposition of two bedrock types) between the north side and south side of the fault (marked by the red dotted line) and a height change at the ridge crest. (d) Scarp elevation proﬁles i–iv, derived from a series of NNE-SSW proﬁle lines from the DGPS DEM shown in Figure 11b and numbered in order from east to west. Proﬁles show variable vertical oﬀset that we interpret to arise from the presence of steeper colluvial deposits on the upthrown side. We estimate that the actual scarp height is within the range ∼5–8 m. the fault. At the western end of the central section, at the Shingildy River (Figure 12), we measured a scarp height of 7.4 ± 1 m using DGPS. 2 3 3 Scarp Age the fault. At the western end of the central section, at the Shingildy River (Figure 12), we measured a scarp height of 7.4 ± 1 m using DGPS. 2.3.3. Scarp Age At the Shingildy River we collected samples OSL3 OSL4 and RC2 (Tables 1 and 2 respectively) from the the fault. At the western end of the central section, at the Shingildy River (Figure 12), we measured a scarp height of 7.4 ± 1 m using DGPS. 2.3.3. Scarp Age At the Shingildy River, we collected samples OSL3, OSL4, and RC2 (Tables 1 and 2, respectively) from the 7.4 ± 1 m high scarp where it is preserved in a terrace on the west side of the Shingildy River (Figure 12). OSL3 was collected from the base of the Loess cap, 5 cm above the contact with the river gravels (Figure 12b), at a depth of 1.05 m below the surface, or ∼6.5 m above the river bed. LOW STRAIN-RATE, CONTINENTAL FAULTING CAMPBELL ET AL. 5516 Journal of Geophysical Research: Solid Earth Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 Figure 11. (a) View south of a right laterally displaced ﬂuvial riser (people for scale) from the location shown in Figure 11b. (b) DGPS-derived DEM of the Lepsy fault scarp at the site area marked in Figure 10a. See the main text for DEM resolution and details of oﬀset and error measurements. The DGPS survey track is shown in brown dots, scarp-perpendicular elevation proﬁles i–iv are marked by labeled black triangles and are shown in Figure 10c. The fault strike at this site is ∼110∘. Note that a knickpoint has only migrated ∼50 m into the uplifted fault hanging wall. (c) Scarp-parallel elevation proﬁles showing the original (top), lateral (middle), and total (bottom) oﬀsets across the Lepsy fault. The thick black line (Figure 11c, bottom) represents the original, prefaulted surface after the total slip is restored. Figure 11. (a) View south of a right laterally displaced ﬂuvial riser (people for scale) from the location shown in Figure 11b. (b) DGPS-derived DEM of the Lepsy fault scarp at the site area marked in Figure 10a. See the main text for DEM resolution and details of oﬀset and error measurements. The DGPS survey track is shown in brown dots, scarp-perpendicular elevation proﬁles i–iv are marked by labeled black triangles and are shown in Figure 10c. The fault strike at this site is ∼110∘. Note that a knickpoint has only migrated ∼50 m into the uplifted fault hanging wall. (c) Scarp-parallel elevation proﬁles showing the original (top), lateral (middle), and total (bottom) oﬀsets across the Lepsy fault. The thick black line (Figure 11c, bottom) represents the original, prefaulted surface after the total slip is restored. gravel at ∼0.50 m below the base of the loess cap and yielded an age of 5010 ± 700 years B.P., which is consis- tent with its lower stratigraphic position. A radiocarbon sample of charred gravel (RC2) was collected ∼0.40 m lower in the stratigraphy than OSL3, but at the same sampling site, from a burned layer preserved in the river gravels (Figures 12b and 12d). However, the 14C concentration in this sample was not high enough to yield a radiocarbon age. 2.4. West: Ayak-Kol 2.4.1. Overview The Chirindy Mountains end at the Shingildy River (Figure 2). West of this point there is no sharp relief other than the vertical oﬀset preserved along the Lepsy fault in sand dunes and loess cover. Between the Shingildy River and ∼80∘13′E the fault strikes E-W for ∼25 km, where the fault changes strike to ∼110∘(black cross in Figure 2) and continues for a further 30 km. This ∼55 km long western fault section is characterized by a single 9–10 m vertical step, with the south side uplifted relative to the north. Ayak-Kol, near the western end of the fault scarp at (45∘59.931′N/79∘54.901′E, Figures 2 and 14a), is a small (∼120 m2) lake formed by a spring on the fault. At Ayak-Kol the 9–10 m high scarp splits into two parallel scarps with diﬀerent amounts of vertical oﬀset, a northern one with height 5.6 ± 1.5 m and a southern one ∼2 m high (Figure 14a). Several north ﬂowing river channels are abandoned (Figure 2 dashed black arrows) and/or diverted westward, including the Lepsy River itself. Although these river diversions may simply be due to channel switching, rather than being caused by an earthquake, the age of the ﬂuvial sediments places a valuable constraint on the maximum age of the most recent event, as we discuss below. At ∼79∘50′E the LOW STRAIN-RATE, CONTINENTAL FAULTING LOW STRAIN-RATE, CONTINENTAL FAULTING The available age constraint suggests that this terrace was uplifted at least ∼5000 years B.P., which is consistent with the minimum age estimates of scarp formation dated by both radiocarbon and OSL dating methods in the east at Jaxa-Kol (∼2000 years B.P.; section 2.2). We have no direct age constraints on the scarp at the Tentek River site (Figures 10 and 11), though there are suggestions that it is relatively young and also that it is potentially formed in a single event. The scarp crosses several stream beds, one of which is shown in Figure 11. Although the scarp has uplifted the upstream parts of the channels, their beds are relatively intact, with narrow incisions that have migrated only ∼50 m upstream. LOW STRAIN-RATE, CONTINENTAL FAULTING CAMPBELL ET AL. CAMPBELL ET AL. 5517 Journal of Geophysical Research: Solid Earth Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 12. The Shingildy River site (a) Google Earth satellite image, centered at 45∘54′/80∘34′, showing an overview of the Shingildy River site. The Lepsy fa marked by red triangles, and the trace of the DGPS west river terrace elevation proﬁle track a-a′ is shown; see Figure 12e for scarp oﬀset measureme west of the sample OSL3, taken from the base of the loess cap terrace river gravel contact. (c) View NW showing the north ﬂowing Shingildy River a race sampling site (dashed white box). The south tilted peneplain is also shown in the far distance. (d) View west of a burned layer containing charre RC2, Table 2) taken at ∼0.50 m depth within the river terrace gravels. (e) N-S scarp proﬁle a-a′ showing 7.4 ± 1 m (total measurement error; see ix A) oﬀset preserved in the late Quaternary river terrace. The left-hand panel shows the full topographic proﬁle, whereas the right-hand panel show ew of the terrace adjacent to the fault scarp. e 12. The Shingildy River site (a) Google Earth satellite image, centered at 45∘54′/80∘34′, showing an overview of the Shingildy River site. The Lepsy is marked by red triangles, and the trace of the DGPS west river terrace elevation proﬁle track a-a′ is shown; see Figure 12e for scarp oﬀset measure ew west of the sample OSL3, taken from the base of the loess cap terrace river gravel contact. (c) View NW showing the north ﬂowing Shingildy Rive terrace sampling site (dashed white box). The south tilted peneplain is also shown in the far distance. (d) View west of a burned layer containing ch l (RC2, Table 2) taken at ∼0.50 m depth within the river terrace gravels. (e) N-S scarp proﬁle a-a′ showing 7.4 ± 1 m (total measurement error; see ndix A) oﬀset preserved in the late Quaternary river terrace. The left-hand panel shows the full topographic proﬁle, whereas the right-hand panel sh Figure 12. The Shingildy River site (a) Google Earth satellite image, centered at 45∘54′/80∘34′, showing an overview of the Shingildy River site. The Lepsy fault scarp is marked by red triangles, and the trace of the DGPS west river terrace elevation proﬁle track a-a′ is shown; see Figure 12e for scarp oﬀset measurements. LOW STRAIN-RATE, CONTINENTAL FAULTING (b) View west of the sample OSL3, taken from the base of the loess cap terrace river gravel contact. (c) View NW showing the north ﬂowing Shingildy River and west terrace sampling site (dashed white box). The south tilted peneplain is also shown in the far distance. (d) View west of a burned layer containing charred gravel (RC2, Table 2) taken at ∼0.50 m depth within the river terrace gravels. (e) N-S scarp proﬁle a-a′ showing 7.4 ± 1 m (total measurement error; see Appendix A) oﬀset preserved in the late Quaternary river terrace. The left-hand panel shows the full topographic proﬁle, whereas the right-hand panel shows a closer view of the terrace adjacent to the fault scarp. 5.6 ± 1.5 m high E-W scarp changes strike by 90∘to trend N-S. The scarp height decays southward over a distance of ∼22 km (Figures 2 and 14b). 2.4.2. Geomorphology Along the 55 km length of the Lepsy fault in the platform, a 9–10 m of vertical oﬀset (e.g., Figure 15) is preserved in the sand dunes and loess cover. Left-stepping, en echelon pull-apart basins are also preserved along strike of the main scarp, with basin dimensions ranging between 0.3 to 3 km length (Figure 15). The en echelon arrangement of surface faulting has generated these basins and is consistent with right- lateral faulting. At Ayak-Kol 45∘59′N/79∘46′E, the single 9–10 m scarp splits into two parallel segments, which have heights of 5.6 ± 1.5 m (northern branch) and 2 ± 1 m (southern branch). Both scarps have a south side up sense of vertical motion. The southern branch of the scarp is left stepping and segmented and can be traced westward for 3.6 km to 45∘59′N/79∘52′E where it ends (Figure 14), whereas the 5.6±1.5 m high scarp continues a further 4 km and terminates along a N-S fault, which we discuss in section 3.2. The southern, ∼2 m high, scarp crosses 2.4.3. Main Scarp Age We collected an OSL sample and two radiocarbon samples (OSL5, RC3, and RC4; Tables 1 and 2) from an ∼1.40 m deep soil pit excavated into a small pull-apart basin at (45∘59′N/79∘57′E, Figures 2 and 15) along the main 9–10 m scarp. The pit stratigraphy revealed 10 cm of black top soil, underlain by ﬁne beige sands and clay until ∼0.60 m depth, where there was an ∼5 cm thick dark soil. Beneath this soil unit, dark silt-clay contin- ued to the base of the pit, which was waterlogged. RC3 was a bulk radiocarbon sample taken from the 5 cm thick soil unit at a depth of 0.55 m from which charcoal of a gymnosperm small branch yielded a radiocarbon age of 558 to 380 calibrated radiocarbon years B.P. OSL5 was taken from a pale beige ﬁne sand unit directly below the dark soil at 0.60 m and yielded an age 300 ± 150 years B.P. An additional OSL sample was collected from the dark silt-clay material at the base of the pit at a depth of 1.30 m. However, this sample did not yield an OSL signal and is not reported in Table 1. Bulk sample RC4 was taken from 1.30 m depth from dark grey clay, but this sample yielded a date indistinguishable from modern. Although we have no constraint on the depth of the base of the pond, samples RC3 and OSL5 indicate that the Lepsy scarp was present in some form here since at least ∼300 to 500 years B.P. Further west at 45∘59′N/79∘54′E (Figures 14 and 16), where the fault consists of two parallel strands, a quarry exposure excavated through the northern, 5.6 ± 1.5 m high, scarp revealed a ﬂuvial sequence of well-sorted cross-bedded sands, capped with 40 cm of wind-blown loess. Two samples from a sedge bed (Figures 17b and 17c) preserved within this ﬂuvial sequence, which include Cyperaceae leaves (sedges) covered with a mineral coating, and a fragment of pine bark, yielded radiocarbon ages of 375 to modern and 348–66 calibrated radiocarbon years B.P., respectively (samples RC5 and RC7, Table 2). A small fragment of plant resin (RC6) from ∼5 cm below the sedge bed yielded an age >57,800 years B.P. The hard nature of the resin material and its much greater age indicates that this sample has residual age and we do not consider it further. LOW STRAIN-RATE, CONTINENTAL FAULTING LOW STRAIN-RATE, CONTINENTAL FAULTING CAMPBELL ET AL. 5518 Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 Figure 13. South dipping red beds at the Shingildy River site. (a) View south of south dipping Cenozoic red beds and the well-preserved terrace surface. (b) View east of minor reverse faulting in Cenozoic red beds. (c) View east to south tilted Cenozoic red beds. All photos taken from a 2 km walk south along the Shingildy River. Figure 13. South dipping red beds at the Shingildy River site. (a) View south of south dipping Cenozoic red beds and the well-preserved terrace surface. (b) View east of minor reverse faulting in Cenozoic red beds. (c) View east to south tilted Cenozoic red beds. All photos taken from a 2 km walk south along the Shingildy River. Figure 13. South dipping red beds at the Shingildy River site. (a) View south of south dipping Cenozoic red beds and the well-preserved terrace surface. (b) View east of minor reverse faulting in Cenozoic red beds. (c) View east to south tilted Cenozoic red beds. All photos taken from a 2 km walk south along the Shingildy River. the abandoned channel of the Lepsy River (Figures 14 and 19) but is not preserved in the bed of the channel itself. Fault-parallel elevation proﬁles across the abandoned channel indicate that it is more deeply incised on the south side of the ∼2 m scarp than on the north side (Figure 19c), indicating that the river was able to continue ﬂowing after this 2 m step-forming event. This southern scarp, which the river has been able to erode into and retain its course, hence formed in an earlier event (or events) than the main 5.6 ± 1.5 m high scarp, which is not eroded even within the old river bed. 2.4.3. Main Scarp Age A single OSL sample of the ﬁne quartz sand from 5 cm below the sedge deposit gave an age of 430 ± 120 years B.P. (OSL6, Table1).Theseagesandtheircontextareverysigniﬁcantasthesedgesareoverlainbyﬂuvialchanneldeposits, indicating that the river was still ﬂowing northward <400 years ago (Figure 18). If the main scarp formed at any measurable time prior to abandonment of the stream, it would have been incised into by the river, but this is not the case. The scarp must, therefore, have formed during or after stream LOW STRAIN-RATE, CONTINENTAL FAULTING LOW STRAIN-RATE, CONTINENTAL FAULTING CAMPBELL ET AL. CAMPBELL ET AL. 5519 Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 Figure 14. West Lepsy fault termination. (a) Worldview satellite image of the main Lepsy fault trace (5.6 ± 1.5 m vertical oﬀset) at Ayak-Kol. The yellow dashed boxes show the locations of Figures 15, 16, and 19. The main scarp crosses the abandoned N-S oriented Lepsy River ﬂood plain (bright white pixels and black dashed boundary). The most recent active channel within the ﬂood plain is also shown (dotted in black). We infer that the N-S river course was abandoned after the 5.6 ± 1.5 m oﬀset event (see section 2.2). Ayak-Kol has formed against the uplifted south side of the Lepsy fault. A secondary subparallel segmented, left-stepping scarp is also preserved to the south of the 5.6 ± 1.5 m fault scarp. (b) The 90 m resolution Shuttle Radar Topography Mission (SRTM) DEM of the western fault termination. The yellow dashed box denotes the area shown in Figure 14a. The Lepsy River now ﬂows westward (solid white arrow); the abandoned northward river course is also seen clearly (black dashed arrow). Possible older drainage deﬂections are also noted (white dashed arrows), which may relate to older events. It is possible that such older events only generated h i l ﬀ i ll di h ﬂ f h L Ri Figure 14. West Lepsy fault termination. (a) Worldview satellite image of the main Lepsy fault trace (5.6 ± 1.5 m vertical oﬀset) at Ayak-Kol. The yellow dashed boxes show the locations of Figures 15, 16, and 19. The main scarp crosses the abandoned N-S oriented Lepsy River ﬂood plain (bright white pixels and black dashed boundary). The most recent active channel within the ﬂood plain is also shown (dotted in black). We infer that the N-S river course was abandoned after the 5.6 ± 1.5 m oﬀset event (see section 2.2). Ayak-Kol has formed against the uplifted south side of the Lepsy fault. A secondary subparallel segmented, left-stepping scarp is also preserved to the south of the 5.6 ± 1.5 m fault scarp. (b) The 90 m resolution Shuttle Radar Topography Mission (SRTM) DEM of the western fault termination. The yellow dashed box denotes the area shown in Figure 14a. The Lepsy River now ﬂows westward (solid white arrow); the abandoned northward river course is also seen clearly (black dashed arrow). LOW STRAIN-RATE, CONTINENTAL FAULTING Possible older drainage deﬂections are also noted (white dashed arrows), which may relate to older events. It is possible that such older events only generated enough vertical oﬀset to partially divert the ﬂow of the Lepsy River. LOW STRAIN-RATE, CONTINENTAL FAULTING LOW STRAIN-RATE, CONTINENTAL FAULTING 5520 CAMPBELL ET AL. CAMPBELL ET AL. Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 Figure 15. West Lepsy fault, main scarp; no other relief is observed in this part of the Kazakh platform except the ∼10 m vertical fault oﬀset. (a) Worldview satellite image showing the main Lepsy scarp (red triangles) and location of a small pull-apart basin, which is also the location of samples OSL5 and OSL6 (yellow circle and Table 1) and RC3 and RC4 (Table 2). The small white arrows point to grave circles that appear to be sited along the east bank of the abandoned Lepsy River channel. The inset shows two scarp-perpendicular DGPS proﬁles, oﬀsets shown in Figure 15c. (b) The pull-apart basin sample pit stratigraphy and the location of sample OSL5 (see section 2.4 for details). (c) DGPS scarp elevation proﬁles, see Figure 15a for proﬁle locations, record 9–10 m of vertical oﬀset of a very gently dipping surface and with uplift of the south side relative to the north side. (d) Google Earth satellite image centered at 45∘57′N/80∘04′E, showing left-stepping, en echelon pull-apart basins, which are consistent with right-lateral slip. Figure 15. West Lepsy fault, main scarp; no other relief is observed in this part of the Kazakh platform except the ∼10 m vertical fault oﬀset. (a) Worldview satellite image showing the main Lepsy scarp (red triangles) and location of a small pull-apart basin, which is also the location of samples OSL5 and OSL6 (yellow circle and Table 1) and RC3 and RC4 (Table 2). The small white arrows point to grave circles that appear to be sited along the east bank of the abandoned Lepsy River channel. The inset shows two scarp-perpendicular DGPS proﬁles, oﬀsets shown in Figure 15c. (b) The pull-apart basin sample pit stratigraphy and the location of sample OSL5 (see section 2.4 for details). (c) DGPS scarp elevation proﬁles, see Figure 15a for proﬁle locations, record 9–10 m of vertical oﬀset of a very gently dipping surface and with uplift of the south side relative to the north side. (d) Google Earth satellite image centered at 45∘57′N/80∘04′E, showing left-stepping, en echelon pull-apart basins, which are consistent with right-lateral slip. abandonment—hence in the last 400 years. LOW STRAIN-RATE, CONTINENTAL FAULTING Given the young age of the uplifted ﬂuvial sands, we ﬁnd it most plausible that the 5.6 ± 1.5 m scarp preserved in the river bed formed in a single large-magnitude event, rather than forming in multiple earthquakes, for which there is no evidence in the documented historical record [Mushketov, 1785]. Additionally, given the very low relief of the Kazakh platform, it is possible that this amount of oﬀset prompted the switching of the Lepsy River. We discuss this possibility more in section 3.2. A row of grave mounds (Kurgans; typical structures of the Bronze Age in central Asia) is also aligned along the abandoned channel (Figure 15a; white arrows), suggesting that water was still ﬂowing northward until at least ∼2–3 ka [Sala and Deom, 2005]. Two further radiocarbon samples (RC8 and RC9, Table 2) were collected from deeper in the ﬂuvial stratigra- phy at the same quarry site, where a sand boil has deformed the overlying channel deposits (Figure 17d), LOW STRAIN-RATE, CONTINENTAL FAULTING LOW STRAIN-RATE, CONTINENTAL FAULTING CAMPBELL ET AL. CAMPBELL ET AL. 5521 Journal of Geophysical Research: Solid Earth Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 Figure 16. West Lepsy fault quarry site and Ayak-Kol. (a) Worldview satellite image centered on 45∘59.972′N/79∘54.437′E of a quarry excavation through the main Lepsy scarp (red triangles), showing sample locations (yellow circle), DGPS scarp proﬁles (black-white lines), and the location of ﬁeld photo in Figure 16b. (b) View south of the main scarp (people for scale), which has a south side up sense of motion. (c) Two DGPS scarp elevation proﬁles show variable scarp heights of ∼4.7 m and ∼6.5 m. We therefore estimate a range of uncertainty on the scarp height at this location of 5.6 ± 1.5 m. Figure 16. West Lepsy fault quarry site and Ayak-Kol. (a) Worldview satellite image centered on 45∘59.972′N/79∘54.437′E of a quarry excavation through the main Lepsy scarp (red triangles), showing sample locations (yellow circle), DGPS scarp proﬁles (black-white lines), and the location of ﬁeld photo in Figure 16b. (b) View south of the main scarp (people for scale), which has a south side up sense of motion. (c) Two DGPS scarp elevation proﬁles show variable scarp heights of ∼4.7 m and ∼6.5 m. We therefore estimate a range of uncertainty on the scarp height at this location of 5.6 ± 1.5 m. but not aﬀected the uppermost units above that are dated to ∼400 years B.P. by the preserved sedge bed. Wood and charcoal samples collected from two deformed sediment layers contained fern, gymnosperm, bark fragments, charcoal fragments, and horsetail (Equisetum). RC8 (gymnosperm) yielded an age of 5328–4931 calibrated radiocarbon years B.P. RC9 (horsetail) failed to yield a radiocarbon age. The soft-sediment deformation hence predates deposition of the uplifted sedge bed, and its formation is not related to the scarp-forming earthquake. 2.4.4. Southern Scarp Age The southern scarp is preserved as an ∼2 m step in Quaternary alluvium and can be traced westward for 3.6 km to 45∘59′N/79∘52′E where it ends (Figures 14 and 19). The scarp crossed the abandoned ﬂood plain of the Lepsy River (Figures 14 and 19) but is not preserved in the bed of the most recent abandoned channel itself. Fault-parallel elevation proﬁles across this abandoned channel indicate that it is more deeply incised on the south side of the 2 m scarp than on the north side (Figure 19c) indicating that the river was able to continue ﬂowing after this 2 m step-forming event and subsequently incised through the uplifted southern block. The 2 m high southern scarp, through which the river has been able to erode into and retain its course, hence formed in an earlier event (or events) than the main 5.6 ± 1.5 m high scarp, which postdated or was synchronous with deﬂection of the river, and abandonment of the channel. Two luminescence samples, OSL7 and OSL8 (Table 1), were collected at depths of 0.30 m and 0.65 m respec- tively from a pit excavated into the abandoned channel on the downthrown north side of the southern scarp (Figure 19a). The stratigraphy revealed rounded, well-sorted, medium-grained quartz-rich river sands. OSL7 and OSL8 gave ages of 2245 ± 480 years and 1810 ± 400 years B.P., respectively. However, they are from the upper 1 m of ﬂuvial sand and hence should be at a similar stratigraphic level to the sedge bed exposed at the quarry, within the same ﬂuvial sequence, and which we dated using both radiocarbon and OSL to within the last∼400years(RC5,RC7,andOSL6).Wesuggestthatﬂuvialreworkingofsedimentfromtheincisedupthrown side of the southern scarp has led to anomalously old ages for OSL7 and OSL8. 2.5. Possible Western Continuation of the Lepsy Fault In addition to the 120 km of Holocene fault scarp, we suggest a possible geological continuation of the Lepsy fault for a further ∼25 km west of the 5.6 ± 1.5 m scarp termination (shown as a purple line in Figure 2). However, we did not investigate this inferred continuation in the ﬁeld, and we do not know if it shows evidence for Quaternary reactivation. The projected trace of the fault follows a linear color change in Qua- ternary sediment cover that may result from weathering of a subtle step in the landscape that is too small to resolve in 90 m resolution Shuttle Radar Topography Mission (SRTM) data (Figure 14b). In the far west at Kokterek town (∼79∘36′E, Figure 2), the westward ﬂowing Lepsy River passes an apparent abandoned east ﬂowing river channel preserved in the Goldybek Valley (Figure 2). At this abandoned conﬂuence, outcrops of approximately E-W striking Paleozoic bedrock are observed, with apparent uplift to the north. This sense LOW STRAIN-RATE, CONTINENTAL FAULTING CAMPBELL ET AL. CAMPBELL ET AL. 5522 Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 Figure 17. Quarry sampling site, west Lepsy. (a) View west of the quarry excavation, through the main scarp, which exposes a sequence of ﬂuvial sands. The dashed white box shows the area of photo in Figure 17d. (b) Zoom of south facing exposure wall through the main scarp, showing cross-bedded ﬂuvial sands, the location of the sedge bed preserved within the ﬂuvial sequence (sample OSL7 and Table 1and samples RC5–RC7, Table 2). Dating materials from the sedge bed include Cyperaceae leaves (sedge plant family), with a mineral coating (likely to be related to the spring at this site along the fault, which has formed Ayak-Kol), wood, Pinus (conifer), and plant resin. (c) Close-up photo of the sedge bed, interpreted as a low-energy channel deposit. (d) Close-up photo of the sand boil soft-sediment deformation structure and location of radiocarbon samples RC8 (fern) and RC9 (gymnosperm charcoal) (Table 2). These radiocarbon samples include also unspeciﬁed bark fragments and equisetum (horsetail). The sand boil does not deform the overlying i h d d b l RC5 d OSL6 Figure 17. Quarry sampling site, west Lepsy. (a) View west of the quarry excavation, through the main scarp, which exposes a sequence of ﬂuvial sands. The dashed white box shows the area of photo in Figure 17d. 2.5. Possible Western Continuation of the Lepsy Fault (b) Zoom of south facing exposure wall through the main scarp, showing cross-bedded ﬂuvial sands, the location of the sedge bed preserved within the ﬂuvial sequence (sample OSL7 and Table 1and samples RC5–RC7, Table 2). Dating materials from the sedge bed include Cyperaceae leaves (sedge plant family), with a mineral coating (likely to be related to the spring at this site along the fault, which has formed Ayak-Kol), wood, Pinus (conifer), and plant resin. (c) Close-up photo of the sedge bed, interpreted as a low-energy channel deposit. (d) Close-up photo of the sand boil soft-sediment deformation structure and location of radiocarbon samples RC8 (fern) and RC9 (gymnosperm charcoal) (Table 2). These radiocarbon samples include also unspeciﬁed bark fragments and equisetum (horsetail). The sand boil does not deform the overlying stratigraphy dated by samples RC5 and OSL6. Figure 17. Quarry sampling site, west Lepsy. (a) View west of the quarry excavation, through the main scarp, which exposes a sequence of ﬂuvial sands. The dashed white box shows the area of photo in Figure 17d. (b) Zoom of south facing exposure wall through the main scarp, showing cross-bedded ﬂuvial sands, the location of the sedge bed preserved within the ﬂuvial sequence (sample OSL7 and Table 1and samples RC5–RC7, Table 2). Dating materials from the sedge bed include Cyperaceae leaves (sedge plant family), with a mineral coating (likely to be related to the spring at this site along the fault, which has formed Ayak-Kol), wood, Pinus (conifer), and plant resin. (c) Close-up photo of the sedge bed, interpreted as a low-energy channel deposit. (d) Close-up photo of the sand boil soft-sediment deformation structure and location of radiocarbon samples RC8 (fern) and RC9 (gymnosperm charcoal) (Table 2). These radiocarbon samples include also unspeciﬁed bark fragments and equisetum (horsetail). The sand boil does not deform the overlying stratigraphy dated by samples RC5 and OSL6. LOW STRAIN-RATE, CONTINENTAL FAULTING 5523 3.1. Kinematics 3.1.1. Fault Geometry and the Dominant Type of Faulting 1640 and A.D. 1682. Sample OSL6, which was taken from ﬁne quartz sands adjacent to the sedge bed, yielded an age of 430 ± 120. This independent age constraint provides conﬁdence that the radiocarbon sample is in the older A.D. 1640 to A.D. 1682 age range (333–375 years B.P.). There is some ambiguity in trying to determine the fault dip direction, and hence the nature of fault motion, mainly because in the east the reactivated bedding dips very steeply, 80∘N, and hence suggests a normal component. However, elsewhere several further observations, both at a regional scale and at a local scale, suggest that the fault is south dipping with a dominant reverse component. In particular, (1) earthquake mechanisms on E-W faults throughout the Tien Shan indicate dominantly reverse faulting consistent with N-S shortening (Figure 1); (2) these earthquake mechanisms are also consistent with a regional N-S short- ening revealed by GPS measurements (Figure 1); (3) a fault dip of 50∘S was observed close to the Tentek River site (Figure 10c); (4) the scarp is not likely to be a sackung [e.g., Zischinsky et al., 1966; Varnes et al., 1989; Gutiérrez-Santolalla et al., 2005] as for most of its length it runs through the low-relief Kazakh platform (Figure 2); (5) reverse-faulted and folded Cenozoic red beds exposed at the Shingildy River immediately to the south of the scarp (Figure 13) suggest that the most recent period of activity in this region is compres- sional; and (6) in the east we infer that the fault has steepened at shallow depth to follow bedding, yielding an apparent north dip. Such depth-dependent variation in the type of fault is often observed in reverse-faulting earthquakes [e.g., Yielding et al., 1981; Liu-Zeng et al., 2010]. From the horizontal and vertical oﬀset values (7 ± 2 m and 5–9 m, respectively) measured from our DEM at the Tentek River ﬁeld site in Figures 10 and 11, the local fault strike of ∼110∘and using the ∼50∘S fault dip measured nearby (Figure 10c), we calculate a slip vector azimuth of 317∘to 343∘, which is similar to the azimuth of the GPS velocities relative to Eurasia that show NNW-SSE shortening at this latitude (∼330∘) (Figure 1). The overall fault motion is hence oblique reverse right lateral. LOW STRAIN-RATE, CONTINENTAL FAULTING CAMPBELL ET AL. CAMPBELL ET AL. Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 Figure 18. Radiocarbon sample RC5 (Cyperaceae leaves) from the sedge bed, west Lepsy quarry exposure. The left-hand axis shows radiocarbon concentration expressed in years “before present,” and the bottom axis shows calendar years (derived from tree ring data). The pair of blue curves shows the radiocarbon measurements on the tree rings (plus and minus one standard deviation), and the red curve on the left indicates the radiocarbon concentration in the sample. The grey histogram shows possible ages for the sample (the higher the histogram, the more likely that age is). There is 95% conﬁdence that the sample has an age range between AD 1640 and modern, with a 49% probability that its age is between A.D. 1640 and A.D. 1682. Sample OSL6, which was taken from ﬁne quartz sands adjacent to the sedge bed, yielded an age of 430 ± 120. This independent age constraint provides conﬁdence that the radiocarbon sample is in the older A.D. 1640 to A.D. 1682 age range (333–375 years B.P.). l h d i P l i l d d i i lik l h i of uplift is contrary to that mapped along the main Lepsy scarp but indica- tive of the predominant orientation of preexisting structure throughout this platform region. 3.1. Kinematics 3.1.1. Fault Geometry and the Dominant Type of Faulting 3.1. Kinematics 3.1.1. Fault Geometry and the Dominant Type of Faulting adiocarbon sample RC5 (Cyperaceae leaves) from the sedge psy quarry exposure. The left-hand axis shows radiocarbon n expressed in years “before present,” and the bottom axis dar years (derived from tree ring data). The pair of blue curves diocarbon measurements on the tree rings (plus and minus d deviation), and the red curve on the left indicates the concentration in the sample. The grey histogram shows s for the sample (the higher the histogram, the more likely There is 95% conﬁdence that the sample has an age range 1640 and modern, with a 49% probability that its age is . 1640 and A.D. 1682. Sample OSL6, which was taken from ﬁne adjacent to the sedge bed, yielded an age of 430 ± 120. This t age constraint provides conﬁdence that the radiocarbon the older A.D. 1640 to A.D. 1682 age range (333–375 Dominant Type of Faulting Our observations and analysis of satellite imagery and ﬁeldwork indi- cate that the overall sense of motion along the Lepsy fault is dip slip right lateral, with vertical motion as the dominant component in the east and with no direct constraint on hor- izontal motion in the west. In the Kazakh platform, the fault strikes WNW-ESE (∼110–290∘) and is hence likely to have a greater component of right-lateral strike slip than in its eastern section. This lateral motion is manifested in the left-stepping eche- lon pull-apart basins observed in the west (Figure 15). The Lepsy fault fol- ominant Palaeozoic structural trend, and it is likely that it represents a reactivated basement fault kh platform [e.g., Suvorov, 1963, 1973; Tapponnier and Molnar, 1979; Allen and Vincent, 1997]. Figure 18. Radiocarbon sample RC5 (Cyperaceae leaves) from the sedge bed, west Lepsy quarry exposure. The left-hand axis shows radiocarbon concentration expressed in years “before present,” and the bottom axis shows calendar years (derived from tree ring data). The pair of blue curves shows the radiocarbon measurements on the tree rings (plus and minus one standard deviation), and the red curve on the left indicates the radiocarbon concentration in the sample. The grey histogram shows possible ages for the sample (the higher the histogram, the more likely that age is). There is 95% conﬁdence that the sample has an age range between AD 1640 and modern, with a 49% probability that its age is between A.D. 3.1. Kinematics 3.1.1. Fault Geometry and the Dominant Type of Faulting From the measurements of fault dip, strike slip, and vertical slip we also calculate that 8.2–13.8 m of slip along the fault plane would be required to produce the observed vertical and lateral displacements across the scarp at the Tentek River site. At its western end, the Lepsy scarp changes strike by 90∘to trend N-S, and its height decays southward over a distance of ∼22 km (Figures 2 and 14b). We interpret this terminal N-S fault to represent a tear, accommodat- ing the uplift of the Lepsy fault hanging wall relative to the unfaulted plain to the west. Such orthogonal tear LOW STRAIN-RATE, CONTINENTAL FAULTING CAMPBELL ET AL. CAMPBELL ET AL. 5524 Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 ure 19. (a) Worldview satellite image of the subparallel southern fault scarp (see Figure 14 for image area location). Left-stepping segments of up to several dred meters long are clear. The locations of samples OSL7 and OSL8 are shown (yellow circle). (b) DGPS scarp-perpendicular elevation proﬁles show 2 m of ical oﬀset (to which we add an uncertainty of ±1 m) preserved in late Quaternary ﬂuvial deposits (proﬁle line location shown in Figure 19a). (c) DGPS t-parallel elevation proﬁles on either side of the fault show that the river continued ﬂowing northward after this 2 m oﬀset event. The hanging wall (south ) of the fault is more deeply incised (proﬁle b-b′) than the north side (proﬁle c-c′), which is consistent with uplift of the south side and continued northward r incision to reach an equilibrium base level. Figure 19. (a) Worldview satellite image of the subparallel southern fault scarp (see Figure 14 for image area location). Left-stepping segments of up to several hundred meters long are clear. The locations of samples OSL7 and OSL8 are shown (yellow circle). (b) DGPS scarp-perpendicular elevation proﬁles show 2 m of vertical oﬀset (to which we add an uncertainty of ±1 m) preserved in late Quaternary ﬂuvial deposits (proﬁle line location shown in Figure 19a). (c) DGPS fault-parallel elevation proﬁles on either side of the fault show that the river continued ﬂowing northward after this 2 m oﬀset event. The hanging wall (south side) of the fault is more deeply incised (proﬁle b-b′) than the north side (proﬁle c-c′), which is consistent with uplift of the south side and continued northward river incision to reach an equilibrium base level. 3.1. Kinematics 3.1.1. Fault Geometry and the Dominant Type of Faulting fault terminations have been observed along other large intraplate reverse faults (e.g., the 1897 Mw 8.1 Assam earthquake, India [BilhamandEngland, 2001], the 1999 Mw 7.3 Chi Chi earthquake, Taiwan [LeeandShih, 2011], and the 2008 Mw 7.9 Wenquan earthquake [Xu et al., 2009]). The distance over which the vertical oﬀset in the hanging wall of a fault decays is related to the depth extent of faulting [SavageandHastie, 1966; Wallace, 1989; Contreras et al., 2000]. In the case of the Lepsy fault, a depth extent of faulting of >20 km is consistent with well-constrained earthquake centroid depths, which indicate a seismogenic layer thickness of up to 40 km in the Kazakh platform (e.g., Figure 1). Alternatively, the geometry of the western Lepsy fault termination may be related to its strike-slip component. Similar fault terminations have been observed along other long intraplate faults with a strike-slip component elsewhere, for example, in Mongolia [Bayasgalan et al., 1999] and Iran [Berberian et al., 2000; Walker et al., 2004; Wesnousky, 2005; Hollingsworth et al., 2006]. The right-lateral com- ponent of motion and the fault conﬁguration at the termination of the main Lepsy scarp are compatible with either interpretation. Considering that the Lepsy fault seems to be a splay from the Dzhungarian fault, which extends into a low-relief part of the platform at its northern end, it is possible that the Lepsy fault has a similar role to that of the Dzhungarian fault. Campbell et al. [2013] determined a late Quaternary slip rate of 2.2 ± 0.8 mm/yr for the Dzhungarian fault and, using a simple kinematic model, determined that its slip rate is consistent with the accommodation of N-S shortening by counterclockwise rotation. The geometry of the Dzhungarian fault may, therefore, indicate that both right-lateral strike-slip and counterclockwise rotations about a vertical axis play a role in accommodating N-S shortening in the northern Tien Shan. Vertical axis block rotations have been documented in many places around the world and have been shown to be an eﬃcient mechanism to LOW STRAIN-RATE, CONTINENTAL FAULTING LOW STRAIN-RATE, CONTINENTAL FAULTING CAMPBELL ET AL. CAMPBELL ET AL. 5525 Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 Table 1. Luminescence Ages for the Lepsy Fault Radioisotopesb Labcode Site Latitude Longitude Depth (m) Watera(%) K (%) Th (ppm) U (ppm) Cosmic (Gy/ka) Total Dose Rate (Gy/ka) Dec(Gy) OSL Aged (ka) X6030e (OSL1) Jaxa-Kol 45∘55′07.80 81∘25′51.56 0.85 7.6 2.88 9.0 3.1 0.24 ± 0.03 3.98 ± 0.24 7.04 ± 2.00 1.77 ± 0.51 X6031e (OSL2) Jaxa-Kol 45∘55′18.96 80∘34′20.76 0.65 10.6 2.84 10.1 3.0 0.25 ± 0.04 3.86 ± 0.23 3.50 ± 2.01 0.91 ± 0.52 X6019 (OSL3) Shingildy 45∘55′18.96 80∘34′20.76 1.05 1.8 2.56 6.0 1.4 0.20 ± 0.02 3.30 ± 0.21 7.23 ± 1.48 2.19 ± 0.47 X6018 (OSL4) Shingildy 45∘55′18.96 80∘34′20.76 1.55 0.9 2.17 5.8 1.6 0.20 ± 0.02 2.97 ± 0.18 14.88 ± 1.84 5.01 ± 0.70 X6020 (OSL5) Pull-apart basin 45∘59′08.63 79∘58′00.17 0.60 5.7 2.24 7.1 1.5 0.21 ± 0.04 2.93 ± 0.18 0.87 ± 0.43 0.30 ± 0.15 X6023 (OSL6) Quarry, sedge bed 45∘59′08.63 79∘58′00.17 0.80 7.2 2.40 6.4 1.6 0.20 ± 0.03 2.97 ± 0.19 1.27 ± 0.36 0.43 ± 0.12 X6021 (OSL7) Channel 45∘59′08.63 79∘58′00.17 0.30 0.3 2.12 6.8 1.8 0.22 ± 0.05 3.05 ± 0.20 6.85 ± 1.38 2.24 ± 0.48 X6022 (OSL8) Channel 45∘59′08.63 79∘58′00.17 0.65 8.9 2.65 5.3 1.2 0.21 ± 0.03 3.03 ± 0.20 5.49 ± 1.15 1.81 ± 0.40 aMeasured water contents are expressed as a percentage of the dry mass of the sample. A long-term mean water content of 3.0 ± 3.0% was assumed for the dose rate determinations of samples X6018, X6019, and X6021. bMeasurements were made on dried, homogenized, and powdered material by ICP-MS/AES with an assigned systematic uncertainty of ±5%. Dry beta dose rates calculated from these activities were adjusted for the ﬁeld water content. cOSL measurements were made with an automated TL/DA-15 Risø luminescence reader [Bøtter-Jensen, 1997; Bøtter-Jensen et al., 2000] and conducted on 180–250 μm diameter quartz grains mounted as multigrain aliquots. The equivalent dose (De) was obtained using a single-aliquot regeneration measurement protocol [Murray and Wintle, 2000; Wintle and Murray, 2006]. dThe age datum refers to A.D. 2013 when the samples were measured and the luminescence dates are based on central age model estimates of the weighted mean De values. LOW STRAIN-RATE, CONTINENTAL FAULTING The total uncertainty (1𝜎), calculated as the quadratic sum of the random and systematic errors, includes all measurement uncertainties as well as a relative error of 2% to account for possible bias in the calibration of the laboratory beta source. eOSL samples X6030 and X6031 had low quartz sensitivity. The dose rates of these samples rely on a small number of measurements, hence the relatively larger error estimates for these samples. 3.2. Earthquake Chronology, Scaling, and Magnitudes In this section we use our observations of the geomorphology in combination with OSL and radiocarbon ages to discuss the possible seismic history of the Lepsy fault. We then use the mapped sense of oﬀset, the lateral extent of surface faulting, and the measurements of slip to speculate on potential earthquake magnitudes. 3.2.1. Seismic History y Our dating indicates evidence for at least two substantial earthquakes along the Lepsy fault during the Holocene. In the west, a 5.6±1.5 m vertical scarp at the quarry site (Figures 14 and 16) appears to have formed since ∼400 years B.P. We suggest that this 4–7 m scarp was generated by a single large earthquake in the early part of this age range, rather than by multiple earthquakes within the last 400 years, as it is more consistent with the lack of documented historical earthquakes in this region [Mushketov, 1785]. If the 5.6 ± 1.5 m scarp at Ayak-Kol formed in a single earthquake, then the Lepsy River may well have been diverted from its course by this event. The hanging wall is on the southern, upstream, side of the fault, and considering that hanging wall uplift is typically 10 times footwall subsidence in large reverse-faulting earthquakes [Oglesby et al., 1998], the river would have experienced a signiﬁcant decrease in the gradient of the river. Given that it is sited in the low-relief Kazakh platform, if any slip failed to reach the surface, additional folding and warping of the hang- ing wall (e.g., as observed in the Coalinga, Northridge, and Tabas earthquakes) [Berberian, 1979; SteinandKing, 1984; Hauksson et al., 1995] could also cause the same signiﬁcant decrease in the gradient of the river. In the very low relief of the Kazakh platform this decrease in gradient would have aﬀected the ﬂow of the river and may have prompted the river to change its course. South of the quarry site, a second parallel scarp ∼2 m high must date from an earlier event, as the Lepsy River eroded through this oﬀset and was not diverted from its N-S course until later, but prior to the last earthquake. Journal of Geophysical Research: Solid Earth Journal of Geophysical Research: Solid Earth Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 Table 2. Radiocarbon Material, Ages, and Calibration for Samples Collected Along the Lepsy Faulta Lab Codea Site Latitude Longitude Material 14C Age (B.P.)b,c Calibrated Range (95%)d Age (Cal Years B.P.) OxA-27911 (RC1) Jaxa-Kol 45∘55′07.80 81∘02′51.56 Organic-rich soil 1, 904 ± 26 A.D. 75 to A.D. 209 1,988–1,806 OxA-27910 (RC2) Shingildy 45∘55′18.96 80∘34′20.76 Charred gravel – – – OxA-27700 (RC3) Pull-apart basin 45∘59′08.63 79∘58′00.17 Charcoal (gymnosperm, 351 ± 24 A.D. 1482 to A.D. 1626 558–380 small branch) OxA-27909 (RC4) Pull-apart basin 45∘59′08.63 79∘58′00.17 Grass stems and leaves – – – OxA-27701 (RC5) Quarry A 45∘59′57.60 79∘54′25.16 Cyperaceae leaves (sedges), 231 ± 26 A.D. 1640 to modern 375 to modern with mineral coating OxA-29866 (RC6) Quarry B 45∘59′57.60 79∘54′25.16 Plant resin >57,800 OxA-29383 (RC7) Quarry Aa 45∘59′57.60 79∘54′25.16 Wood: Pinus (conifer) 149 ± 24 A.D. 1667 to A.D. 1949 348–66 OxA-27702 (RC8) Quarry C 45∘59′57.60 79∘54′25.16 Plant remains (fern) 4, 409 ± 33 B.C. 3,313 to B.C. 2,916 5,328–4,931 P-33197 (RC9) Quarry D 45∘59′57.60 79∘54′25.16 Gymnosperm charcoal, – – – Equisetum (horsetail) aSamples prepared and run at the accelerator mass spectroscopy, Research Laboratory for Archaeology and the History of Art, University of Oxford. For details of the chemical pretreatment, target preparation and AMS measurement, see Radiocarbon 46 , 17–24 and 155–63, and Archaeometry44 (3 Supplement 1) and 1–149. bDelta 13C values are measured independently on a stable isotope mass spectrometer (to ± 0.3‰ relative to VPDB). cThe quoted ages are in radiocarbon years using the Libby half-life of 5568 years. dCalibration with IntCal13: Northern Hemisphere [after Reimer et al., 2013]. aSamples prepared and run at the accelerator mass spectroscopy, Research Laboratory for Archaeology and the History of Art, University of Oxford. For details of the chemical pretreatment, target preparation and AMS measurement, see Radiocarbon 46 , 17–24 and 155–63, and Archaeometry44 (3 Supplement 1) and 1–149. bDelta 13C values are measured independently on a stable isotope mass spectrometer (to ± 0.3‰ relative to VPDB). cThe quoted ages are in radiocarbon years using the Libby half-life of 5568 years. dCalibration with IntCal13: Northern Hemisphere [after Reimer et al., 2013]. accommodate and transfer strain on the continents [e.g., Freund, 1970; Ron et al., 1984; McKenzie and Jackson, 1986; Nur et al., 1986; Jackson and Molnar, 1990; Goldsworthy et al., 2002; Walker and Jackson, 2004]. Journal of Geophysical Research: Solid Earth In the northern Tien Shan, without better GPS coverage and paleomagnetic data, it is diﬃcult to further quantify the role of the Lepsy fault in accommodating N-S shortening by vertical axis rotation. LOW STRAIN-RATE, CONTINENTAL FAULTING CAMPBELL ET AL. 5526 Journal of Geophysical Research: Solid Earth We have no direct measurement of fault dip in this western area, and so to estimate the slip along the fault plane at Ayak-Kol, we assume that it is unchanged from the 50∘S measured in the Tentek River gorge in the east. Taking that fault dip, the slip vector azimuth of 317–343∘, and the local fault trend of 110∘at Ayak-Kol, we estimate between 2.6 m and 11.6 m of right-lateral strike slip at Ayak-Kol and a total slip along the fault plane of 6 m to 14.7 m. We suspect that the fault dip is not particularly gentle due to the large component of strike slip. If the local dip is steeper, the total slip will be smaller. If our interpretations are correct, the ∼120 km length of the Lepsy fault ruptured in the last 400 years in a single eventwithslipof8.2–13.8m.Giventhe120kmlength,ourestimatesyieldasliptolengthratioof∼10−4,which is larger than the majority of global earthquakes [Wells and Coppersmith, 1994], but examples with similar ratios are known. Most recently, the Mw 7.6 2001 Bhuj earthquake in India involved slip of ∼10 m on a rupture with a length of only 20–30 km by 20 km [Schmidt and Bürgmann, 2006; Copley et al., 2011]. The 1897 Mw 8.1 Assam earthquake, India, is also thought to have involved slip of 11–25 m of slip on a fault 110 km in length [Bilham and England, 2001]. Well-constrained prehistoric examples also exist, for example, the ∼4 ka Egiin Davaa paleo-earthquake rupture in Mongolia, which involved almost uniform slip of ∼8 m along an 80 km length [Walker et al., 2015]. These examples are all from intraplate regions, where the seismogenic thickness may be larger than typical, and the large slip at Lepsy may be related to the structural immaturity of the fault, which appears to be a major factor in controlling the magnitude of slip in an earthquake, with “immature” faults tending to fail in shorter though more energetic ruptures [Manighetti et al., 2007]. To estimate the magnitude of this event, we use the empirical relationships of Wells and Coppersmith [1994]. Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 formed in a single event. We note that there are two parallel scarps visible in the geomorphology adjacent to the western bank of the Tentek River (Figure 10), and we speculate that these two scarps represent ruptures from two separate earthquakes. We have no direct constraint on the age of the∼2 m high scarp at Ayak-Kol, other than it is older than 400 years (section 2.4), though it may be related to soft-sediment deformation we found in ﬂuvial sediments exposed in the Ayak-Kol quarry, and dated to less than ∼5000 years. It is unclear whether the penultimate event at the western quarry site ruptured most of the length of the Lepsy fault or whether the fault ruptured in a number of smaller events along its length. In the easternmost section of the fault, near Jaxa-Kol, an earthquake must have occurred before ∼2000 years B.P. in order for sediments to be ponded at the scarp (section 2.2). Also, at the Shingildy River initial uplift of the lowest observed terrace occurred between ∼5 ± 0.7 and 2.2 ± 0.5 ka. Scarp formation along these eastern and central fault sections may be related to the same older event scarp observed in the west or may relate to other older events. 3.2.2. Fault Scaling and Potential Earthquake Magnitudes Our observations of the geomorphology and age control indicate that the Lepsy fault has generated at least two large earthquakes in the Holocene. The fault scarp appears very fresh along its entire length, from which we infer that it has been reactivated along its entire length in the recent past, but for most of its length the scarp is likely to represent cumulative displacement in at least two earthquakes. However, we have identiﬁed two sites where the geomorphology suggests displacement arising solely from a single earthquake. The ﬁrst of these sites is at the Tentek River, where we were able to estimate 8.2–13.8 m of slip with an azimuth of 317–343∘. The second location is at Ayak-Kol, near the western end of the fault, where we interpret a 5.6 ± 1.5 m vertical scarp to have formed in a single earthquake within the last ∼400 years. 3.2. Earthquake Chronology, Scaling, and Magnitudes Therefore, where the northern (5.6 ± 1.5 m high) and southern (2 ± 1 m high) fault scarps merge to form a single 9–10 m high scarp, we know that this maximum oﬀset did not occur in a single earthquake, at least in the west, despite forming a single steep and continuous scarp. Following this reasoning, it is not clear whether the scarps we observed farther east were generated by a single event or whether they are composite scarps formed in multiple events. The older 2 m step-forming event, or other events, may contribute to the observed scarp heights in these more easterly sections. The Tentek River site (section 2.3.3) is the only location other than Ayak-Kol where we are conﬁdent from our interpretation of the geomorphology that the scarp was LOW STRAIN-RATE, CONTINENTAL FAULTING CAMPBELL ET AL. CAMPBELL ET AL. 5527 3.3. Implications for Faulting in Intraplate Regions 3.3. Implications for Faulting in Intraplate Regions The Tien Shan is one of the most seismically active intracontinental mountain belts in the world, with many earthquakes occurring on reactivated faults within the high mountains, where rates of shortening are fastest (e.g., ∼15–20 mm/yr; Figure 1). Less well known are the rare earthquakes that occur in the Tien Shan foreland, where rates of shortening are much less (≤2 mm/yr). The Lepsy fault provides a clear example, through its ∼10 m high Holocene escarpment, of a fault that extends from the more rapidly deforming mountainous region to the slowly deforming foreland. The fault appears to follow a Palaeozoic structure, and given that along the western half of the scarp there is relatively little evidence for Cenozoic reactivation other than the ∼10 m scarp, we suggest that it has been reactivated relatively recently. There is evidence that the geological Lepsy fault continues even farther west into the low strain rate platform, though we do not know whether these sections have been active in the Quaternary. It is possible that other structures within the mountain ranges of central Asia may actually extend into regions that are thought of as “stable” and that these structures may also remain active in the Quaternary, but we are unable to observe them because the long recurrence times allow processes of erosion and/or deposition that remove or bury the expression of surface faulting in the landscape [e.g., Walker et al., 2008, 2013]. In summary, there are two key issues. First is whether low strain rate continent interiors should always be thought of as stable, considering their potential for large unexpected earthquakes, as evidenced by this study and a growing number of studies from other apparently aseismic continent interiors worldwide [e.g., Crone andLuza, 1990; Walkeretal., 2008; Clarketal., 2012, 2013; Copleyetal., 2014]. Second is whether our framework for viewing such earthquakes is one of isolated seismogenic faults within absolutely stable and undeforming continental interiors or whether these faults are on a network of interconnected faults, perhaps linked to a distant, more rapidly deforming region, but whose connectivity is obscured by erosional and depositional processes operating rapidly compared to the long earthquake recurrence times. Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 same calculations yield seismic moments between 1.31 × 1021 Nm and 2.27 × 1021 and moment magnitudes of Mw 8.1–8.2. The oldest event described in the documented historical record for the region [Mushketov and Orlov, 1893] indicates that a large earthquake, located in the Dzhungaria region of SE Kazakhstan, in the vicinity of Lake Balkhash, occurred in 1715. Assuming that our magnitude estimate for the most recent earthquake on the Lepsy fault is correct (Mw 7.5–8.2) and that it occurred sometime in the last 400 years, this documented historical earthquake of 1715 may be a candidate for the last earthquake on the Lepsy fault. 4. Conclusions Our observations and analysis of satellite imagery and ﬁeldwork indicate that the Lepsy fault is a reverse right-lateral fault that extends approximately E-W for ∼120 km from the mountains of the Dzhungarian Ala-tau, in the northern Tien Shan, into the low-lying Kazakh platform. Dating of the most recent surface faulting events on the Lepsy fault suggests that two large earthquakes have occurred: the ﬁrst, since at least 5000 years B.P. in the west, and the second, after ∼400 years B.P., in an event which likely ruptured the entire ∼120 km fault length. Earthquake scaling relationships suggest that the last event would have generated a maximum magnitude Mw 7.5–8.2 earthquake. Although the documented historical earthquake record for this region is relatively short (several hundred years), this last earthquake may correspond to a large destructive earthquake that occurred in 1715 in the region of Lake Balkhash, Dzhungaria. The Holocene activity on the Lepsy fault illustrates two important points: (1) the fact that large-magnitude earthquakes can and do occur in regions that have been free of large earthquakes in the instrumental period, such as the Kazakh platform, and provide a reminder that seismic behavior should be assessed over much longer periods of time when considering long-term deformation and associated seismic hazard, and (2) the example of the Lepsy fault reveals that structures in regions that are considered stable may be connected directly with distant, more rapidly deforming regions and may follow networks of interconnected faults inherited from earlier tectonic periods. Journal of Geophysical Research: Solid Earth We have only two estimates of the slip in the most recent earthquake and so perform a range of calculations making either the assumption that the slip of ∼8–14 m at the Tentek River site represents the maximum sur- face slip or considering that the scarp height is similar at sites in both the east and west, and to acknowledge that not all slip may have reached the surface, we also calculate the magnitude using 8–14 m as an aver- age slip. Applying the scaling relationship between Mw (moment magnitude) and fault displacement yields an Mw of 7.5–7.7 using the mean coeﬃcients for strike-slip faulting. Treating the 8–14 m as a maximum slip yields a magnitude of 7.8–8.1. Applying the scaling relationship between Mw (moment magnitude) and rup- ture length (120 km) yields an Mw of 7.5 using the mean coeﬃcients for strike-slip faulting. We also calculate seismic moment (Mo) using the relationship Mo = 𝜇Āu [Scholz, 1982], assuming a value of 3 × 1010 Nm−2 for the shear modulus (𝜇), slip (̄u) of either 8 or 14 as above, and a rupture area (A) computed from a scarp length of 120 km, a fault dip of 50∘, and a range of seismogenic depths. With a mean slip of 8 m, we estimate seismic moment between 7.5 × 1020 Nm and 1.3 × 1021, depending on whether the seismogenic depth is taken to be 20 km or 35 km, which in turn yield moment magnitudes of Mw 7.8–8.0. Using slip of 14 m, the LOW STRAIN-RATE, CONTINENTAL FAULTING CAMPBELL ET AL. CAMPBELL ET AL. CAMPBELL ET AL. 5528 Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 observed scarp. The uncertainty in displacement can be estimated by taking multiple topographic proﬁles at many sites. However, the ﬁeld surveys that we present in this paper form a reconnaissance that was performed in a relatively short time and with restricted access along much of the fault length. Here we brieﬂy describe the approaches we took in measuring the vertical and strike-slip components of slip at those sites where we were able to doso. observed scarp. The uncertainty in displacement can be estimated by taking multiple topographic proﬁles at many sites. However, the ﬁeld surveys that we present in this paper form a reconnaissance that was performed in a relatively short time and with restricted access along much of the fault length. Here we brieﬂy describe the approaches we took in measuring the vertical and strike-slip components of slip at those sites where we were able to doso. Vertical fault oﬀsets at each DGPS-surveyed site (with the exception of Figure 5, which was interpolated from the gridded DEM) were calculated from a linear inversion of the proﬁle points sited at a distance from the degraded scarp [e.g., Walker et al., 2015]. Typically, only points measured beyond 15–30 m on either side of the fault scarp were used in the oﬀset/slope calculations, with the choice of oﬀset distance chosen on a case-by-case basis. For instance, in Figure 19, a wide shallow depression is present at the base of the scarp, and survey points within this depression were not used in the calculation. On longer proﬁles, heights beyond where the fan slope ﬂattens downslope or steepens upslope are discounted. A value of vertical displacement is then calculated at the midpoint of the scarp, where the scarp gradient is greatest. The error on the vertical displacement is estimated from the ﬁt of the modeled upper and lower surfaces and includes a weighting of the GPS points assuming a constant error of 6 cm for each. (The reported error in the heights of the individual points on the DGPS proﬁles is variable but typically within ∼5–6 cm.) The errors reported from this approach, though relatively small, do not account for uncertainties arising from changes in gradient between the upper and lower surfaces nor do they account for variability in scarp height along strike. Appendix B: Quaternary Dating Methods In total, 17 samples were dated. Nine of these samples were collected for radiocarbon dating, which provides an age on organic material incorporated within the sediment layers. Eight samples were collected for opti- cally stimulated luminescence (OSL), which determines the time at which quartz-bearing sediment was last exposed to sunlight. Where possible, both radiocarbon and OSL samples were collected at the same site for age comparison, and we found good agreement in the three sites where we used both dating methods. OSL and radiocarbon ages and sample details are reported in Tables 1 and 2, respectively. Detailed descriptions of the dating sites, the sample context, and sample materials are provided on a site-by-site basis. Journal of Geophysical Research: Solid Earth In the text we hence apply a uniform uncertainty of ± ∼1 m to all measurements of vertical displacement, as this range encompasses the range of heights. Exceptions are the scarps shown in Figures 5, 10, and 15, where variability between displacement measurements on duplicate closely spaced proﬁles exceeds 2 m and may reﬂect a real variation of vertical displacement along strike. In these cases we assign an uncertainty that is suﬃcient to cover the range of heights measured from each set of closely spaced proﬁles. Lateral oﬀsets were estimated at the few sites where we were able to observe well-deﬁned ridge crests or ﬂu- vialrisersthatareorthogonaltothefaulttrace.Itisnecessarytoonlyattemptlateralreconstructionswherethe oﬀset landform is orthogonal to the fault scarp, as the large shortening component would introduce apparent lateral displacements of any nonorthogonal feature. To estimate lateral displacement, we ﬁrst gridded indi- vidual DGPS survey points to produce digital elevation models (with a pixel spacing of ∼50 cm). Topographic proﬁles parallel to the fault, and on either side of it, were then extracted from the DEM. A piercing point for the channel thalweg or ridge crest was determined on either side of the fault by projecting the channel slopes to a crossover point. Lateral oﬀsets were then estimated visually by realigning the piercing points. For each measurement of lateral displacement we show the original topographic proﬁles, the proﬁles with lateral dis- placement restored, and also a panel with the vertical displacement removed to provide a visual measure of the degree of ﬁt. For each measurement of lateral displacement we state a conservative uncertainty of ±2 m, as outside this range there is a visible degradation in the restoration. Appendix A: Estimation of Vertical and Lateral Displacements The measurements of vertical and horizontal displacements across the Lepsy scarps are important both for estimating the direction of slip and also for estimating the amount of slip required to produce the LOW STRAIN-RATE, CONTINENTAL FAULTING CAMPBELL ET AL. 5529 Journal of Geophysical Research: Solid Earth B1. Radiocarbon All radiocarbon samples were collected either from newly dug excavations or from refreshed cuttings. Samples were extracted from the sediment and collected either in glass vials or in metal foil, which were then placed in sealed plastic sample bags. Prior to analysis, all plant material was identiﬁed, any root mate- rial or other contaminants removed, and if possible the most rapid growing species selected from each site to reduce the potential for inheritance (S. Harris, Oxford University, personal communication, 2013). Radio- carbon dating was performed at the University of Oxford, following the standard procedures for chemical pretreatment, target preparation, and AMS measurement given in Radiocarbon46 (1), 17–24 and 155–63, and Archaeometry44 (3 Supplement 1), 1–149. Isotopic fractionation has been corrected for using the mea- sured 𝛿13C values measured on the accelerator mass spectrometer. The quoted 𝛿13C values are measured LOW STRAIN-RATE, CONTINENTAL FAULTING B2. OSL We thank Barry Parsons, Oxford University, for providing Worldview satellite imagery, through COMET+. Great thanks to Ivan, Atyr, and Ainagul for their support in the ﬁeld and helping with the logistics of the ﬁeldwork and to Natalya Mikhailova at the Kazakhstan National Data Centre (KNDC). This work beneﬁted from discussions with Magali Rizza, JeﬀRitz, and Tom Rockwell. We also thank T. Higham and R. Staﬀ at the Research Laboratory for Archaeology and the History of Art, Oxford, for their help with radiocarbon sample preparation and analysis and S. Harris at the Department of Plant Sciences, University of Oxford, for his expertise in identifying our organic sample material. We thank Olaf Zielke, an anonymous reviewer, and the Associate Editor for their detailed comments that have led to a much improved paper. The natural and regenerative doses were preheated at 26∘C for 10 s, and the ﬁxed test doses (which are used to correct for any sensitivity changes) were preheated at a reduced temperature of 240∘C for 10 s, before opti- cal stimulation. The absence of infrared-sensitive minerals (e.g., feldspars) was checked and conﬁrmed using an infrared bleach (50 s) provided by clusters of 870 nm IR light-emitting diodes providing >135 m W/cm2 a solid-state laser diode (83,010 nm; 1 W cm2) at 50∘C for 100 s before blue light stimulation [Banerjee et al., 2001]. The ultraviolet OSL emission at 370 nm was detected using an Electron Tubes Ltd. 9235QB15 photo- multiplier tube ﬁtted with a blue-green sensitive bialkali photocathode and 7.5 mm of Hoya U-340 glass ﬁlter. Laboratory doses used for constructing dose response curves were given using a calibrated 90Sr/90Y beta source housed within the reader. At least 12 disks were prepared from each sample, except for samples OSL1 and OSL2, as marked in Table 1. Following measurement of the natural signal, a dose response curve was con- structed from six to eight dose points including a zero dose point and a replicate measurement of the lowest regenerative dose. The environmental gamma and beta components of the dose rate result from the radioactive decay series of 238U, 232Th, and 40K within the sediment. The concentrations of these parent isotopes were obtained by inductively coupled plasma mass spectroscopy (ICP-MS/AES) using a lithium metaborate/tetraborate fusion. Abdrakhmatov, K., et al. (1996), Relatively recent construction of the Tien Shan inferred from GPS measurements of present-day crustal deformation rates, Nature, 384, 450–453. Abdrakhmatov, K., K. D. Djanuzakov, and D. Delvaux (2002), Active tectonics and seismic hazard of the Issyk-Kul basin in the Kyrgyz Tian-Shan, in Lake Issyk-Kul: Its Natural Environment, vol. 13, edited by J. Klerkx and B. Imanackunov, pp. 147–160, NATO Science Series IV, Earth and Environmental Sciences, Kluwer Acad., Dordrecht, Netherlands. Abdrakhmatov, K. Y., R. J. Weldon, S. C. Thompson, D. W. Burbank, C. Rubin, M. M. Miller, and P. Molnar (2001), Onset, style and current rate of shortening in the central Tien Shan, Kyrgyz Republic, Geol. Geoﬁz., 42, 1585–1609. Adamiec, G., and M. Aitken (1998), Dose-rate conversion factors: New data, Ancient Therm. Lum., 16(2), 37–50. Allen, M. B., and S. J. Vincent (1997), Fault reactivation in the Junggar region, northwest China: The role of basement structures during Mesozoic-Cenozoic compression, J. Geol. Soc. London, 154(1), 151–155. B2. OSL The OSL samples were collected in steel tubes, hammered into the sediment, and sealed at the ends with layers of metal foil and opaque tape. The samples were opened and prepared under low-intensity light-emitting diode lighting (emitting at ∼588 nm). Sediment in the outer thirds of each tube was removed and used for dose rate measurements. The inner third of the sediment was prepared for dating. The OSL sample preparation and analysis procedures are as reported in Campbell et al. [2013]. The dating was per- formed at the University of Oxford and was based on quartz grains extracted from the sediment samples. Laboratory procedures were designed to yield clean, sand-sized (180–250 μm) grains of quartz for opti- cal dating according to standard preparation methods, including wet sieving, HCl acid digestion, heavy liquid ﬂotation using sodium polytungstate, and etching in concentrated hydroﬂuoric acid (40%) to dis- solve potassium feldspar minerals and to clean and remove the outer alpha-dosed layer of quartz grains. The latter residual grains were resieved to the original grain size range and mounted as multigrain mono- layers of approximately 4 mm diameter onto aluminium disks using a silicone oil adhesive. To determine the equivalent dose (De), a single-aliquot regeneration measurement protocol was used [Murray and Wintle, 2000; WintleandMurray, 2006] and OSL measurements were conducted using an automated TL/DA-15 Risøluminescence reader [Bøtter-Jensen, 1997; Bøtter-Jensenetal., 2000]. Optical stimulation for single aliquots was provided by clusters of blue light-emitting diodes (42 Nichia 470 nm) providing a sample stimulation power of >80 m W cm2. Acknowledgments We thank NERC for a small grant (NE/G010978/1) awarded to R.T.W. and the Royal Society International Travel Grant, Mike Coward Fund of the Geological Society of London, Percy Sladen Fund of the Linnean Society, the Gilchrist Educational Trust, and the Earth and Space Foundation for their support in funding the ﬁeld component of this project. G.E.C.’s doctoral studentship is funded by the National Environmental Research Council (NERC). R.T.W. is supported by a University Research Fellowship awarded by the Royal Society. We also thank the National Centre for Earth Observation (NCEO), the Centre for the Observation and Modelling of Earthquakes, Volcanoes and Tectonics (COMET+), and the NERC-ESRC funded project, Earthquakes without Frontiers (EwF), all of which have contributed to the funding of this project. DigitalGlobe high-resolution satellite imagery was obtained from Google Earth (www.earth.google.co.uk/). Shuttle Radar Topography Mission (SRTM) was obtained from CGIAR-CSI. B2. OSL These concentrations were converted to dose rates according to attenuation factors proposed by Adamiec and Aitken [1998], using corrections for grain size [Mejdahl, 1979] and water content [Zimmerman, 1971]. The cosmic ray dose was calculated according to standard data reported by Prescott and Hutton [1994], taking into account the height and density of the overburden, as well as the geomagnetic latitude and elevation of the site. Quartz yields and sensitivities were low for two samples (OSL1 and OSL2), which would decrease conﬁdence in the ages obtained for these two, but radiocarbon samples taken from the same sample pit give concordant ages. LOW STRAIN-RATE, CONTINENTAL FAULTING CAMPBELL ET AL. CAMPBELL ET AL. 5530 Journal of Geophysical Research: Solid Earth 10.1002/2015JB011925 independently on a stable isotope mass spectrometer (to ±0.3‰ relative to Vienna Peedee belemnite (VPDB)).UncalibratedagesaregiveninradiocarbonyearsB.P.(B.P.toA.D.1950)usingthehalf-lifeof5568years. Calibrations were performed using Oxcal (v4.2) of Bronk [1994], using the “INTCAL13” data set [Reimer et al., 2013]. independently on a stable isotope mass spectrometer (to ±0.3‰ relative to Vienna Peedee belemnite (VPDB)).UncalibratedagesaregiveninradiocarbonyearsB.P.(B.P.toA.D.1950)usingthehalf-lifeof5568years. Calibrations were performed using Oxcal (v4.2) of Bronk [1994], using the “INTCAL13” data set [Reimer et al., 2013]. 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Wood (1981), Relations between surface deformation, fault geometry, seismicity, and rupture characteristics during the El Asnam (Algeria) earthquake of 10 October 1980, Earth Planet. Sci. Lett., 56, 287–304. Zimmerman, D. W. (1971), Thermoluminescent dating using ﬁne grains from pottery, Archaeometry, 13(1), 29–52. Zi hi k U t l (1966) O th d f ti f hi h l i P di Fi t C f th I t ti l S i t f R k M h i LOW STRAIN-RATE, CONTINENTAL FAULTING 5534 CAMPBELL ET AL. CAMPBELL ET AL.
https://openalex.org/W2760950957	https://epigeneticsandchromatin.biomedcentral.com/track/pdf/10.1186/1756-8935-6-S1-P126	English	null	Spatial compartmentalization at the nuclear periphery characterized by genome-wide mapping	Epigenetics & chromatin	2,013	cc-by	1,100	Results We have identified ~15, 000 sequencing-based Lamina- Associated Domains (sLADs) in both mouse 3T3 fibro- blasts and C2C12 myoblasts. These genomic regions range from a few kb to over 1 Mb and cover ~30% of the gen- ome. We have used immunofluorescence staining and DNA-FISH to verify that all of the tested regions are spa- tially proximal to the NL. We have analyzed the sLAD regions relative to genome-wide distributions of histone modifications in C2C12 myoblasts [3-5]. We found that genomic regions covered by sLADs are characterized by extremely low levels of active histone modifications such as H3K4me2/3, H3K9Ac, H3K36me3 and H3K79me2, but Background How gene positioning to the nuclear periphery regulates transcription remains largely unclear. By high-resolution cell imaging, we have previously observed the differential compartmentalization of transcription factors and histone modifications at the nuclear periphery in mouse C2C12 myoblasts [1]. Here, we aim to identify DNA sequences associated with the nuclear lamina (NL) and examine this compartmentalization at the genome-wide level. We have also cross-analyzed the available gene expres- sion data in C2C12 myoblasts [6] with the NL-association map generated in this study, and identified 272 expressed genes in significant association with the NL that we named as active sLAD genes. Genomic regions around transcription start sites of active sLAD genes display reduced associations with the NL and possess active his- tone modifications. Surprisingly, we noticed that gene bodies of active sLAD genes possess very low levels of active histone modifications such as H3K36me3. © 2013 Wu and Yao; 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. Wu and Yao Epigentics & Chromatin 2013, 6(Suppl 1):P126 http://www.epigeneticsandchromatin.com/content/6/S1/P126 Materials and methods We cultured NIH-3T3 mouse fibroblasts and C2C12 mouse myoblasts, performed DNA adenine methyltrans- ferase identification (DamID) assay on mouse Lamin B1 as previously described [2], and sequenced the methylated DNA fragments using high-throughput DNA sequencing at Yale Center for Genome Analysis. We have developed a set of bioinformatic analyses to identify and compare genomic regions and/or genes with enriched adenine methylation. Spatial compartmentalization at the nuclear periphery characterized by genome-wide mapping Feinan Wu, Jie Yao* From Epigenetics & Chromatin: Interactions and processes Boston, MA, USA. 11-13 March 2013 sLAD and non-sLAD regions contain similar densities of repressed histone modifications such as H3K27me3. Conclusions Our experimental results and bioinformatic analyses in mouse C2C12 myoblasts suggest that chromatin at the nuclear periphery is characterized by the paucity of active histone modifications rather than the enrichment of repressive histone modifications. The distinct histone mod- ification profiles among active sLAD genes may give clues on how gene positioning to the nuclear periphery (via NL association) affects transcription regulation in mammalian cells. Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA 1. Yao J, Fetter RD, Hu P, Betzig E, Tjian R: Subnuclear segregation of genes and core promoter factors in myogenesis. Genes Dev 2011, 25(6):569-580. 2. Vogel MJ, Peric-Hupkes D, van Steensel B: Detection of in vivo protein- DNA interactions using DamID in mammalian cells. Nat Protoc 2007, 2(6):1467-1478. 3. Asp P, Blum R, Vethantham V, Parisi F, Micsinai M, Cheng J, Bowman C, Kluger Y, Dynlacht BD: Genome-wide remodeling of the epigenetic landscape during myogenic differentiation. Proc Natl Acad Sci USA 2011, 108(22):E149-E158. References 1. Yao J, Fetter RD, Hu P, Betzig E, Tjian R: Subnuclear segregation of genes and core promoter factors in myogenesis. Genes Dev 2011, 25(6):569-580. 2. Vogel MJ, Peric-Hupkes D, van Steensel B: Detection of in vivo protein- DNA interactions using DamID in mammalian cells. Nat Protoc 2007, 2(6):1467-1478. 3. Asp P, Blum R, Vethantham V, Parisi F, Micsinai M, Cheng J, Bowman C, Kluger Y, Dynlacht BD: Genome-wide remodeling of the epigenetic landscape during myogenic differentiation. Proc Natl Acad Sci USA 2011, 108(22):E149-E158. 4. ENCODE Project Consortium: A user’s guide to the encyclopedia of DNA elements (ENCODE). PLoS Biol 2011, 9(4):e1001046. 5. ENCODE/Caltech. [http://genome.ucsc.edu/cgi-bin/ igTables? db=mm9&hgta_group=regulation&hgta_ References References 1. Yao J, Fetter RD, Hu P, Betzig E, Tjian R: Subnuclear segregation of genes and core promoter factors in myogenesis. Genes Dev 2011, 25(6):569-580. 2. Vogel MJ, Peric-Hupkes D, van Steensel B: Detection of in vivo protein- DNA interactions using DamID in mammalian cells. Nat Protoc 2007, 2(6):1467-1478. 3. Asp P, Blum R, Vethantham V, Parisi F, Micsinai M, Cheng J, Bowman C, Kluger Y, Dynlacht BD: Genome-wide remodeling of the epigenetic landscape during myogenic differentiation. Proc Natl Acad Sci USA 2011, 108(22):E149-E158. 4. ENCODE Project Consortium: A user’s guide to the encyclopedia of DNA elements (ENCODE). PLoS Biol 2011, 9(4):e1001046. 5. ENCODE/Caltech. [http://genome.ucsc.edu/cgi-bin/ igTables? db=mm9&hgta_group=regulation&hgta_ p y g , ( ) 2. Vogel MJ, Peric-Hupkes D, van Steensel B: Detection of in vivo protein- DNA interactions using DamID in mammalian cells. Nat Protoc 2007, 2(6):1467-1478. 3. Asp P, Blum R, Vethantham V, Parisi F, Micsinai M, Cheng J, Bowman C, Kluger Y, Dynlacht BD: Genome-wide remodeling of the epigenetic landscape during myogenic differentiation. Proc Natl Acad Sci USA 2011, 108(22):E149-E158. 4. ENCODE Project Consortium: A user’s guide to the encyclopedia of DNA elements (ENCODE). PLoS Biol 2011, 9(4):e1001046. 5. ENCODE/Caltech. [http://genome.ucsc.edu/cgi-bin/ igTables? db=mm9&hgta group=regulation&hgta db=mm9&hgta_group=regulation&hgta_ © 2013 Wu and Yao; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Page 2 of 2 Page 2 of 2 Wu and Yao Epigentics & Chromatin 2013, 6(Suppl 1):P126 http://www.epigeneticsandchromatin.com/content/6/S1/P126 track=wgEncodeCaltechHist&hgta_table=wgEncodeCaltechHistC2c12Ab35 94FCntrl50bPcr1xPkRep1&hgta_doSchema=describe+table+schema]. 6. Liu Y, Chu A, Chakroun I, Islam U, Blais A: Cooperation between myogenic regulatory factors and SIX family transcription factors is important for myoblast differentiation. Nucleic Acids Res 2010, 38(20):6857-6871. doi:10.1186/1756-8935-6-S1-P126 Cite this article as: Wu and Yao: Spatial compartmentalization at the nuclear periphery characterized by genome-wide mapping. Epigentics & Chromatin 2013 6(Suppl 1):P126. doi:10.1186/1756-8935-6-S1-P126 Cite this article as: Wu and Yao: Spatial compartmentalization at the nuclear periphery characterized by genome-wide mapping. Epigentics & Chromatin 2013 6(Suppl 1):P126. doi:10.1186/1756-8935-6-S1-P126 Cite this article as: Wu and Yao: Spatial compartmentalization at the nuclear periphery characterized by genome-wide mapping. Epigentics & Chromatin 2013 6(Suppl 1):P126. References Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
W2062583931.txt	https://dugi-doc.udg.edu/bitstream/10256/8463/1/RetrievingCloud.pdf	en		Retrieving Cloud Characteristics from Ground-Based Daytime Color All-Sky Images	Journal of atmospheric and oceanic technology	2,006	public-domain	12,822	VOLUME 23 JOURNAL OF ATMOSPHERIC AND OCEANIC TECHNOLOGY MAY 2006 Retrieving Cloud Characteristics from Ground-Based Daytime Color All-Sky Images C. N. LONG Pacific Northwest National Laboratory, Richland, Washington J. M. SABBURG Department of Biological and Physical Sciences, Faculty of Sciences, University of Southern Queensland, Toowoomba, Australia J. CALBÓ Department of Physics, and Institute of the Environment, University of Girona, Girona, Spain D. PAGÈS Institute of the Environment, University of Girona, Girona, Spain (Manuscript received 27 May 2005, in final form 17 October 2005) ABSTRACT A discussion is presented of daytime sky imaging and techniques that may be applied to the analysis of full-color sky images to infer cloud macrophysical properties. Descriptions of two different types of skyimaging systems developed by the authors are presented, one of which has been developed into a commercially available instrument. Retrievals of fractional sky cover from automated processing methods are compared to human retrievals, both from direct observations and visual analyses of sky images. Although some uncertainty exists in fractional sky cover retrievals from sky images, this uncertainty is no greater than that attached to human observations for the commercially available sky-imager retrievals. Thus, the application of automatic digital image processing techniques on sky images is a useful method to complement, or even replace, traditional human observations of sky cover and, potentially, cloud type. Additionally, the possibilities for inferring other cloud parameters such as cloud brokenness and solar obstruction further enhance the usefulness of sky imagers. 1. Introduction In recent years, atmospheric researchers have become increasingly interested in quantifying clouds. Clouds are a major meteorological phenomena related to the hydrological cycle and affect the energy balance on both local and global scales through interaction with solar and terrestrial radiation. It is broadly recognized that clouds (and cloud–aerosol interaction) are responsible for the largest uncertainties in climate models and climate predictions (Houghton et al. 2001). In addition, clouds affect our everyday lives, for example, by modi- Corresponding author address: Dr. Charles N. Long, Atmospheric Radiation Measurement Program, Pacific Northwest National Laboratory, P.O. Box 999, MSIN: K9-38, Richland, WA 99352. E-mail: Chuck.Long@pnl.gov © 2006 American Meteorological Society JTECH1875 fying the amount of ultraviolet (UV) radiation that reaches the earth’s surface (Calbó et al. 2005). Most cloud-related studies require some sort of cloud observations, such as the amount and type of clouds that are present. These macrophysical observations have been performed historically by human observers who recorded cloud cover and cloud type at several meteorological stations and at given time intervals (typically hourly at many U.S. sites, 3 hourly at many other sites worldwide). However, high costs associated with human observers have led observations toward automatic devices to detect and quantify cloud amount and type. There is, of course, satellite information, but satellite retrievals have known weaknesses in quantifying small and/or low cloud features due to their limited spatial resolution and unknown surface influences on the measured radiances. One option for obtaining continuous information on 633 634 JOURNAL OF ATMOSPHERIC AND OCEANIC TECHNOLOGY sky conditions is the use of sky-imaging devices. In two recent publications (Calbó et al. 2005; Parisi et al. 2004) there are overviews on atmospheric cloud detection, the importance of clouds with respect to the earth’s climate and human health, and how ground-based sky imagers can complement the coverage of equivalent satellite instruments. These papers highlight the increased number of ground-based sky imagers being developed in several countries. This development is partly due to the dramatic improvements in technology in recent years, both with respect to the hardware, for example, charge-coupled devices (CCDs) and digital image processing techniques. Most of these imagers have been purposely constructed with a specific application in mind, for example, the first integrated sun-centered sky camera (SCSC) for solar UV research (Sabburg and Wong 1999), while other imagers have the general purpose of measuring cloud macroscopic characteristics (Pagès et al. 2002; Lu et al. 2004; Long and DeLuisi 1998). A family of Whole Sky Imagers (WSIs), developed by the Scripps Institution of Oceanography at the University of California, San Diego, are designed to measure radiances at distinct wavelength bands across the hemisphere (Johnson et al. 1989; Shields et al. 2003). The various models of the WSI include a highquality temperature-controlled CDD, high-quality optics including spectral filtering, detailed mapping of the sky dome to CCD element, and careful calibration of dark current and stray light influences needed to make the scientific-quality spectral radiance measurements. The WSI data, besides having many other interesting scientific capabilities, can be used to estimate fractional sky cover (Tooman 2003; Johnson et al. 1989). Unfortunately, because of the high-quality components and sophisticated engineering involved, the significantly higher cost puts the WSI beyond the means of many individual researchers and research groups whose only interest lies in inferring daytime fractional sky cover. Commercially, there are very few nonradiance sky camera systems available. One of the better-known sky imagers to the atmospheric science community is the total sky imager (TSI) manufactured by Yankee Environmental Systems, Inc. (YES), Massachusetts. There are advantages to developing specific-purpose imagers; for example, the user has a detailed knowledge of the prototype system and can choose components that best suit a specific task. However, this development process requires extensive knowledge of detector and optical systems, as well as development of the interface algorithms needed to construct actual images in a format using the readings of the detector array. In addition, given the implications inherent in the word “prototype,” the reliability of a commercial instrument VOLUME 23 may likely be an advantage if the instrument operates for extended periods (years) with minimal operational problems. With this in mind, Sabburg and Long (2004) developed three new image-processing algorithms for the TSI, similar functionally to the algorithms used with the SCSC mentioned previously. The SCSC had few operational problems during the first year of operation, but a more robust design was needed to investigate the effects of clouds on UV enhancement over a time period greater than 2 yr. One solution was the purchase of a commercially available sky imager. Our paper discusses image-processing techniques that can be used to obtain standard (i.e., fractional sky cover) and other sky characteristics from daytime ground-based, all-sky images. This paper provides information for researchers to use with images obtained from similar all-sky camera systems. Our examples use two sky imagers: the TSI and the whole sky camera (WSC) developed by Spain’s University of Girona (Pagès et al. 2002). Technical information for both imagers are presented including elaboration on the TSItype methodology previously reported by Long et al. (2001) and Pfister et al. (2003). Topics and concepts include shadowband and horizon masking, pixel classification (e.g., thresholding), geometric corrections, and other sky characteristics such as solar obstruction, cloud brokenness (CB), and cloud patterns. Results are presented to show how some of these concepts are applied to sample images from the TSI and WSC compared to direct sky observations as well as visual inspections of the images. 2. Sky imagers used a. Hemispheric Sky Imager/total sky imager The prototype Hemispheric Sky Imager (HSI), which was the precursor to the commercial TSI, was developed at the National Oceanic and Atmospheric Administration Surface Radiation Research Branch (SRRB) located in Boulder, Colorado (Long and DeLuisi 1998). The development was a joint effort between SRRB and the U.S. Department of Energy’s Atmospheric Radiation Measurement (ARM) Program. This instrument was developed into a commercial product named the TSI in a cooperative effort with YES under a Small Business Innovative Research grant from the U.S. Department of Agriculture. The basic design of both the HSI and TSI includes a digital camera mounted to look down on a curved mirror (Fig. 1) to provide a horizon-to-horizon view of the sky. The mirror rotates to keep a dull black strip on the mirror aligned with the solar azimuth angle to block the direct sun from the camera. The mirror rotation is ac- MAY 2006 LONG ET AL. 635 greater than 10° (zenith angles less than 80°), and images are processed for a maximum 160° field of view (FOV), ignoring the 10° of sky near the horizon. The user can configure the time between image captures, overall processing FOV, and adjustments to the basic image-processing limits to differentiate between cloudless, thin, and opaque cloud retrievals. The TSI software also allows setting a separate FOV for processing, centered at zenith and of lesser angular width than the overall setting. In addition, the software processes the relative brightness along the sun-blocking strip to act as a “sunshine meter,” thus suggesting whether or not the sun is blocked by a cloud. An example of a TSI sky image and the corresponding cloud decision image is shown in Fig. 2 (top). The cloud decision image displays the 160° FOV of the retrieval, and a second retrieval circle centered on zenith covering a 100° FOV is outlined in green. (Other areas outlined in green are discussed in the next section.) The yellow dot on the sunblocking strip mask represents the location of the sun in the image, with the color yellow indicating that the sun is not completely obscured by a cloud [similar to the World Meteorological Organization (WMO) specifications of sun obscuration (Pfister et al. 2003)]. If the sun is blocked by a cloud, the dot is colored white. FIG. 1. YES TSI deployed at the ARM Climate Research Facility Southern Great Plains site in Oklahoma. tively controlled by a small onboard computer. The TSI captures images of the sky during daylight hours and can be set to do so as often as every 10 s (depending on ancillary image-processing computer processor speed). The sky images are 24-bit color JPEG format at 352 ⫻ 288 pixel resolution captured from a camera based on a commercially available digital camera. More information on TSI specifications is given in Table 1. The raw sky images are processed to suggest fractional sky cover. Note that the TSI sky cover retrievals are generally valid only for solar elevation angles TABLE 1. Technical specifications of the TSI. Characteristic Imager resolution Sampling rate Operating temperature Weight Power requirements Data storage Specification 352 ⫻ 288, 24-bit color Variable, with a maximum of one image every 10 s ⫺30° to 34°C Approximately 50 lbs (23 kg) 110/220 VAC Disk on local computer or remote computer over a full-time TCP/IP connection b. Whole sky camera The WSC was developed at University of Girona (UdG) to collect a continuous record of the sky conditions at low cost and was used for research associated with radiative transfer phenomena in the atmosphere. The WSC consists of three different components: the CCD camera and optics, the graphics card and corresponding software, and the enclosure and various protections. A picture of the device and a schematic drawing showing its parts are presented in Fig. 3. The main component is a commercial CCD color video camera equipped with a “fish eye” zoom lens. The focal length is fixed to 1.6 mm to project a 180° FOV onto the 1/3-in. (8.3 mm) CCD matrix. Both the focus and the iris are manually operated and were fixed at the optimum values for sky viewing after initial tests. Various technical specifications of the system are listed in Table 2. The camera sends an analog signal to the graphics card, which captures and stores the image in digital format. Based upon the software package that comes with the graphics card, we developed software to control the capture and recording of the images, including switching on and off the capture function in relation to the time of sunrise and sunset. In addition, the same code controls the time interval between image capture and the storage format of the captured images. Currently, 636 JOURNAL OF ATMOSPHERIC AND OCEANIC TECHNOLOGY VOLUME 23 FIG. 2. (top left) TSI sky image and (top right) corresponding 160° FOV cloud decision image, (bottom left) WSC sky image, and (bottom right) corresponding 160° FOV cloud decision image. For the TSI cloud decision image, blue represents retrieved cloud-free pixels, gray represents thin cloud, white represents opaque cloud, and black represents masked pixels that are not counted in determining fractional sky cover. The green outlines denote special retrieval areas discussed in the text. The yellow dot on the sun-blocking strip mask denotes the sun location in the image. For the WSC processed image, retrieved cloud-free pixels are shown in gray, while cloudy pixels are in white. Black represents masked pixels that are not counted in determining fractional sky cover. The red line represents the path of the sun through the image FOV, and the yellow dot denotes the sun location in this image. one bitmap (BMP) image is recorded every 15 min and one JPEG (JPG) image every minute. The CCD camera is placed inside an enclosure, which is built in two layers. The first one covers the camera itself and contains two thermostats and connections for the power cable and signal cables. The second one protects the whole device from rain and other environmental factors. One thermostat controls a Peltier cell to refrigerate the air around the camera if needed. The Peltier cell has a maximum working temperature of 50°C. This cooling is needed when the camera is exposed to high summer temperatures (air temperature around 35°C) with accompanying direct sunlight, and the camera also generating some heat of its own. This Peltier system is turned on when the first thermostat records temperatures higher than 35°C. The second thermostat is for backup, that is, to protect the camera if temperatures reach 40°C. In this case, the camera is also switched off. Low temperatures are not a problem in the climate of Girona, where temperatures below 0°C are rare, and the heat produced by the camera is enough to avoid freezing temperatures. A hemispherical glass dome is used to protect the lens from the environment, while allowing a view of the sky. A shadowband, similar to those used for diffuse radiation measurements, is used to protect the CCD sensor from the direct sun. This sun-blocking arrangement makes it difficult for the detection of some sky characteristics, such as sun obstruction, because in this design the sun is always occulted behind the shadowband. However, the shadowband is required, because the CCD could be damaged by a continuous exposure to high radiation intensities. Due to the seasonal change of sun height, the position of the shadowband is manually adjusted periodically (approximately once a week). Since the summer of 2001, the WSC has been operated on the roof of a building of the University of Girona (41.97°N, 2.82°E; 100-m altitude), along with other meteorological and radiometric instrumentation. This site enjoys an open horizon, meaning that no obstacle in the horizon MAY 2006 LONG ET AL. 637 FIG. 3. (left) Picture of the WSC with shadowband, and (right) a schematic drawing of the main parts. subtends an angle greater than 10°, so there are no obstructions over the 160° FOV currently analyzed. An example WSC image and the corresponding processed image are shown in Fig. 2 (bottom). 3. Image processing a. Previous treatments In processing raw sky images, several areas need to be identified before the actual cloud/clear pixel accounting is performed. The overall images themselves are generally rectangular in shape, whereas the mapping of the sky dome onto the image is circular. In addition, due to the natural radiative scattering characteristics of the atmosphere and the long pathlengths, unambiguous determination between clear sky and cloudy sky is difficult near the horizon. For this reason, both the WSC and TSI process only a 160° FOV of the sky image centered on zenith, resulting in a loss of about 17% of the hemispherical solid angle of the sky dome. The rest of the pixels in the image area outside TABLE 2. Technical characteristics of the WSC. Camera and optics Color camera CCD: 1/3⬙ Minimum illumination: 0.5 lx at f 1.2 Image resolution: 752(H) ⫻ 582(V) Working temperature: ⫺10° to ⫹50°C Lens: fish-eye zoom, 1.6–3.4 mm at f 1.4 FOV: up to 180° Enclosure and other Protecting container with glass dome Shadowband (radius, 635 mm; width, 73 mm) Temperature controlled by two thermostats and a Peltier cell this 160° FOV circle is set to a “mask” value corresponding to “black” and are ignored in the clear/cloudy pixel accounting for all TSI/HSI/WSC processing presented here. Of the remaining 160° FOV, part of the area is covered by the shadowband (WSC) or the black sunblocking strip (TSI) that is used to protect the camera from the direct sun. The TSI additionally must ignore the camera arm and the camera housing directly overhead. The pixels corresponding to these objects are also masked and not included in the clear/cloudy pixel accounting. For the TSI, because the black strip moves during the day with the azimuthal position of the sun, a different mask must be applied to each image depending on the time of day. However, the same masks are valid for all days in the year. The fraction of the 160° FOV image that is lost when masking the black strip, and camera arm and housing, is about 8%. For the WSC, the shadowband is manually adjusted every 2 to 5 days. We have defined a different mask to hide the shadowband for each day in the year, allowing the same mask to be applied to all images for a given day. The masks were obtained by drawing a strip of sufficient width (around 60 pixels in our case) that follows the daily trajectory of the solar disk derived from astronomical formulas. The fraction of area of the image that is hidden for these masks is between approximately 10% in winter and 15% in summer. See Fig. 2 for examples of how this masking affects the image processing. In the cloudless sky, the area near the sun is most often whiter and brighter than the rest of the hemisphere due to the forward scattering by aerosols and haze. Even a slight haze or moderate aerosol loading 638 JOURNAL OF ATMOSPHERIC AND OCEANIC TECHNOLOGY VOLUME 23 FIG. 4. (top left) Clear-sky image taken by the TSI, (top second from left) corresponding relative red/blue ratio “image,” (lower left) separated blue, and (lower second from left) red pixel value amount images. (top third from left) Cloudy-sky image, (top right) corresponding relative red/blue ratio “image,” (lower third from left) separated blue, and (lower right) red pixel value amount images. will make a large angular area of the horizon whiter and brighter when the sun is low on the horizon. The human eye has an amazing ability to handle a range of light intensity spanning orders of magnitude. One of the problems in using commercial digital cameras such as those used in the TSI and WSC is the intensity range limitations of the camera detector. It is desirable to have images bright enough to detect thin clouds, yet this might lead to the part of the image near the sun and near the horizon for low sun appearing whiter in the images than they actually are, not because that is the color perceived by the human eye, but because the commercial CCD elements could produce an exaggerated relative signal. But even for high-quality detectors such as those used in the WSI, these areas of the image are naturally whiter than other parts of the cloudless sky in the image due to the forward scattering. With no a priori knowledge of the aerosol or haze loading that can be used in some way to predict an increased brightness, these pixels are often interpreted as “cloudy” in the sky-imager retrievals when a human observer would label them as “cloudless.” The TSI software allows user-configurable settings to keep separate additional accounting of clear/thin/opaque determinations for these two “problem areas.” The “sun circle” setting is in terms of an angular FOV centered on the sun position in the sky image. For the “horizon area” there are two settings, one in terms of angular height above the horizon, and the other in angular width centered on the solar azimuth. These two special areas, along with a sample zenith circle retrieval discussed in section 2, are shown in the cloud decision image in Fig. 2 outlined in green. For this retrieval, the zenith circle is set to an FOV of 100°, the sun circle radius is set to 25°, and the horizon area is set to an elevation of 40° with a total angular width of 100°. While the TSI software itself only produces separate total, thin, and opaque pixel counts for these areas, an additional analysis, such as that described in Pfister et al. (2003), can be performed to help determine if these areas should be counted as cloudy or not [see section 4a(1)]. b. Pixel classification For molecular scattering (clear skies, no aerosols), more blue light is scattered than red, which is why the clear sky appears blue to our eyes. A sample TSI image of clear sky is shown in Fig. 4. Below this sample sky image are two images that show the corresponding extracted red–green–blue (RGB) color channel blue and red pixel values that make up the sample image. The red pixel values are relatively small (dark) in the sky portion of the image because little red light is scattered by this clear atmosphere compared to the correspondingly greater blue scattering and greater blue pixel val- MAY 2006 639 LONG ET AL. ues, except near the horizon where the increased atmospheric pathlength makes the original sky image appear white to our eyes, and somewhat near the sun in the image. The corresponding relative red/blue ratio values are shown in the upper second from left image in Fig. 4. For clear sky the red/blue ratio is small, that is, dark in the image, but increasing near the sun and near the horizon. Additionally, although not shown here, the clear-sky relative red/blue ratio value for any given pixel changes with solar elevation. Thus a clear-sky limit for a given pixel should, for better results, be based as a function of solar elevation, the pixels’ distance from zenith, and the pixels’ distance from the sun location in the image. Clouds, unlike the clear sky, generally scatter both the blue and red visible light more equally. A sample TSI image of a partly cloudy sky is also shown in Fig. 4. As in the Fig. 4 clear-sky case, below this sample cloudy-sky image are two images that show the corresponding extracted blue and red pixel values that make up the sample image. In this case, where there are clouds present, the red pixel values are much greater than where there are not clouds. The blue pixel image shows far less contrast in pixel values. The relative ratio of red/blue pixel values (Fig. 4, upper right) clearly shows that the ratio is greater for clouds than for clear sky. The concept of the red/blue ratio for cloud algorithms was first developed at Scripps Institution of Oceanography with the WSI. A variable threshold algorithm similar to that used with the TSI is described in Koehler et al. (1991). A lower limit is set for a clear-sky ratio value for each pixel in the image, and the pixels for which the red/blue ratio exceeds the clear limit are counted as “cloudy.” It must be noted that the particular limit function is climate and camera dependent. For example, if one takes three digital cameras (even of the same make and model), takes an image of the sky with the three cameras simultaneously, and then simultaneously displays all three images on the same screen, one will note that each image displays a slightly different color rendering. In addition, how white tinted the “blue” of the sky appears, which is considered to be “cloud free,” relates to such factors as the typical aerosol loading and pressure depth of the atmosphere at a given location. One way to account for these effects is to tailor the clear/cloud limit specifically for a given camera and location, as is the case for WSC at the Girona site. Alternatively, as was the case with the SCSC, a solar sensor might be used to adjust the threshold limit based on the brightness of the sky. But because the TSI is a commercial instrument intended to be deployed at many locations, a generic baseline clear- sky function has been established using the pixel distance from center, distance from the sun, and solar zenith angle (SZA) as independent variables. The user then uses configurable settings to set the clear/thin and thin/opaque limits as desired, to a first approximation as a percentage offset from the baseline value for that pixel. The results of the above TSI type of processing are depicted in the cloud decision image shown in Fig. 2. For the case of the WSC, pixel classification is based on the same grounds, but with a simpler approach. A single threshold is used in the whole nonmasked area of the images. A fixed threshold algorithm like that used with the WSC is presented in Johnson et al. (1988). Specifically, pixels with a red-to-blue signal ratio (R/B) greater that 0.6 are classified as cloudy, while lower values of R/B are labeled as cloud-free. The value of the threshold was set based on several tests performed on training images. The set of training images contained some 100 images and is a subset of the images used to assess the performance of the WSC automatic retrieval (see section 4b). Initially, some images were analyzed by using different thresholds and subsequently visually compared with the raw images. Second, a specific software package for image processing was used. In these latter tests, different areas of some images were classified subjectively as cloudy or cloudless. Then, R/B was computed for all pixels in these areas. We found that the value that distinguished the best between cloudy and cloudless areas was R/B ⫽ 0.6. With the use of a unique threshold, as expected, some problems occur with circumsolar pixels (especially in high aerosol conditions) and misdetection of thin clouds. c. Fractional sky cover Once the pixel-by-pixel determination of clear/cloud is made, the estimated fractional sky cover is then calculated, typically as the number of cloudy pixels divided by the total number of pixels in the 160° FOV (ignoring any masked pixels) for both the TSI and WSC. As a first approximation, fractional sky cover f is f⫽ Ncloudy Ncloudy ⫽ , Ntotal Ncloudy ⫹ Nclear 共1兲 where N denotes the number of pixels of each type, according with the subindices. This is the approximation used in the commercial TSI retrievals. Note that this fractional sky cover is the fractional sky cover in the area of sky that has not been masked in the image. However, this should be a good approximation to the actual fractional sky cover over time, provided that (a) the masking process does not hide a large fraction of 640 JOURNAL OF ATMOSPHERIC AND OCEANIC TECHNOLOGY VOLUME 23 FIG. 5. Fractional radial distance vs SZA for HSI/TSI images. Black line is the HSI/TSI image radial distortion fitted function by SZA, and light gray line is the difference from the 1:1 line (dark gray). the sky, and (b) the probability that clouds are systematically behind the masked sections is low. Condition (a) is usually met by typical all-sky imagers such as the TSI or the WSC. Condition (b) is difficult to demonstrate, but seems generally plausible given the typical behavior of clouds. Fractional sky cover, which is typically defined from the point of view of a ground-based observer, is an angular measurement. That is, fractional sky cover is the ratio between the solid angle occulted by clouds and the total solid angle of the visible sky hemisphere (i.e., 2␲ sr if the horizon is absolutely free of obstacles). Therefore, Eq. (1) is correct only if all pixels correspond to the same solid angle in the sky. This condition is not usually met by typical all-sky images. Note that the TSI retrieval algorithm does not attempt to correct pixels for their view angle bias, that is, where each pixel in the imager has a slightly different solid angle view of the sky compared to adjacent pixels. The TSI retrieval does not attempt to bias or weight any pixels during processing for sky cover. One relevant issue for image processing is geometrical distortion. Distortion is in general a complex issue, but for our purposes the primary issues have two forms. First, are equal angles (in the zenithal coordinate) in the real world represented as equal distances in the circularly mapped image? Second, is any object represented equally in the image, independently of its azimuthal position? If, from this definition, there is no significant distortion (as has been determined for the WSC), no correction is needed. For the HSI/TSI, with the camera looking down from a finite distance onto a convex mirror, there is some small amount of radial distortion due to geometry. If there is significant distortion, images could be corrected by a transformation that adjusts the position of the pixels. The distortion and the suitable transformation depend on the exact optics and geometry of the specific device. Either a theoretical analysis of the optics or an empirical study of the device must be performed to estimate the distortion. An empirical analysis of the HSI/TSI radial distortion (and mapping as discussed below) is shown in Fig. 5. Here, the position of the sun in the image is compared to the calculated actual position of the sun in the sky, and the relative difference is plotted (along with the “true” line), as well as the relative difference from “true.” In general, distortion only in the radial (zenithal) direction (from the image center) is to be expected, making it simple to perform the corresponding transformation: all pixels must be mapped into a new image slightly modifying their distance to the center. Even when the image is not distorted or when it has already been corrected, the same areas close to the zenith, or close to the horizon, correspond to different solid angles. In other words, equal areas (or equal number of pixels) in the image correspond to different solid angles in reality. The correction to be applied for a single pixel covering an infinitesimal area, dA, corresponding to an infinitesimal solid angle, d⍀, is [sin(␪)]/ ␪, because the solid angle is proportional to the sine of the zenith angle ␪, while the area is proportional to the MAY 2006 641 LONG ET AL. where the subindex i refers to each of the portions, and the superindex means that Eq. (4) has been applied by counting only the cloudy pixels or all pixels in the portion, respectively. Note that the denominator should be equal to 2␲ sr if the image would cover the whole sky (180° FOV). Because we only use a 160° FOV and part of the sky image is hidden by the shadowband or the shadow strip, the effective total solid angle is always less than 2␲ sr. Combining Eqs. (4) and (5), we have f⫽ FIG. 6. Diagram showing the relationship between angular coordinates in the sky and polar coordinates in the image, used to obtain the relationship between solid angle and area of a portion of the sky in the sky image. angle itself. The maximum difference between area and solid angle associated with this effect is about 35% for ␪ ⫽ 90°. For the more usual maximum zenith angle analyzed in our images (␪ ⫽ 80°), the difference is about 30%. The corresponding differences for smaller zenith angles are 10% at 45°, 1% at 10°, and 0% at 0°. To calculate the fractional sky cover, it is more convenient to apply similar corrections to portions or sectors of the sky rather than to single pixels. From Fig. 6 and from the definition of solid angle, it is easy to demonstrate that the solid angle of the portion of the sky that is highlighted is ⍀ ⫽ 共␭2 ⫺ ␭1兲共cos␪1 ⫺ cos␪2兲, 共2兲 while the area of the corresponding projection in the image is (note angles in radians) 1 A ⫽ 共␭2 ⫺ ␭1兲q2共␪22 ⫺ ␪21兲 2 共3兲 for a distortion-free image, that is, r1 ⫽ q␪1 and r2 ⫽ q␪2, with q being the proportionality constant between actual zenith angle and projected distances. Obviously, area and solid angle are not proportional to each other. Combining Eqs. (2) and (3), and considering that A ⫽ mN, where m is the proportionality constant between the number of pixels and corresponding area, we can write ⍀⫽ 2共cos␪1 ⫺ cos␪2兲mN q2共␪22 ⫺ ␪21兲 . 共4兲 If, in order to calculate the fractional sky cover, we divide the image (or the real sky) in a finite number of portions, we have f⫽ 兺⍀icloudy 兺⍀itotal , 共5兲兺⍀icloudy 兺⍀itotal 兺li m Nicloudy q2 兺liNicloudy ⫽ ⫽ , m total 兺liNitotal 兺li 2 Ni q 共6兲 where li is li ⫽ 2共cos␪i1 ⫺ cos␪i2兲 . 2 2 ⫺ ␪i1 兲共␪i2 共7兲 Equation (6) makes the difference between a correct estimation of fractional sky cover and the first approximation given by Eq. (1) apparent. Note that in Eq. (6), and everywhere in this mathematical development, both the number of pixels N or solid angles refer to the visible part of the image (i.e., masked pixels are not considered). For the case of the WSC, we have divided the image of the sky into portions that cover 10° ⫻ 10° (azimuth and zenith angles). The correction factor li for portions close to the zenith (i.e., between ␪1 ⫽ 0° and ␪2 ⫽ 10°) is 0.997, while for the portions close to the horizon (i.e., between ␪1 ⫽ 70° and ␪2 ⫽ 80°) it is 0.737. We use an example to evaluate the error in fractional sky cover associated to this effect. Assume that we have an image that has 10% cloudy pixels, that is Ncloudy ⫽ 0.1 ⫻ Ntotal. By applying Eq. (1), we would get f ⫽ 0.1 independent of the position of the cloudy pixels in the image. Now assume that these cloudy pixels are in the portions close to the horizon. In this case, applying Eq. (6) with corrections given by Eq. (7), we obtain f ⫽ 0.087. If the same amount of cloudy pixels are placed close to the zenith, f ⫽ 0.116. Therefore, the relative error induced by neglecting this geometrical correction could be, for this case, about 15%, or an error of slightly greater than 1% in actual fractional sky cover. Although the absolute error may increase with increasing number of cloudy pixels, the relative error decreases when fractional sky cover increases. d. Other sky characteristics: Solar obstruction, cloud brokenness, and cloud patterns 1) SOLAR OBSTRUCTION One sky characteristic difficult to determine from a downward-pointing satellite sensor is solar obstruction. 642 JOURNAL OF ATMOSPHERIC AND OCEANIC TECHNOLOGY We have seen in section 3a that a shadowband is needed to protect the WSC optics and CCD from the sun’s direct radiation. One disadvantage of using a totally opaque shadowband is that it is more difficult to determine the state of solar obstruction. In the case of the upward-pointing SCSC, no shadowband was used, although an opaque disc blocked the sun when images were not being captured. In this case an imageprocessing algorithm was developed to overcome the “blooming” effect of the bright sun on the CCD, the net result being a further loss of FOV due to the area of the image, which was unable to be classified as sky or cloud. An additional image-processing algorithm was developed, which correlated the solar region of the image of interest with that of a previously obtained image on a clear day at a similar SZA (a reference image). The correlation was used to classify the solar region as indicative of solar disk obscured (DO), not obscured (DNO), or partially obscured (DPO). The uncertainty in determining solar obstruction was estimated to be less than ⫾16%, when considering either DNO or (DPO and DO) grouped together. An alternative approach has been used in the design of the shadowband for the TSI. It is partially reflective and attached to the rotating mirror, as the camera is downward-pointing looking onto the mirror. It is possible to determine the relative brightness of pixels along the shadowband and to determine solar obstruction, or sunshine detection as it is referred to by YES. Although the exact method of how the TSI performs sunshine detection has not been revealed by YES, its precursor, the HSI (Long and DeLuisi 1998), uses the brightness of the white metallic area surrounding the mirror as a reference to calculate this parameter. The TSI algorithm looks along the shadowband for an increase in brightness. If the slope of the brightness increase falls above a user-defined threshold, then the sun disk is deemed unobscured by clouds, otherwise it is assumed to be obscured. An example is shown graphically in the right panel of Fig. 2 as the color of the dot in the sunblocking mask. A yellow dot (as in this example) indicates that the sun is not significantly obscured by clouds, whereas a white dot indicates an obscured sun. According to Pfister et al. (2003), an all-sky imager (Allsky 1) that uses the HSI sunshine technique agreed with a collocated TSI in 89% of the cases investigated. Pixels in the sun circle (an approximate 20° radius area centered on the sun position) and horizon area (see Fig. 2) often are interpreted by the processing to be cloudy even when they would not be labeled as such by a human observer. This can occur because of forward scattering from aerosol loading, thin cirrus clouds, or boundary layer haze, which is subvisible elsewhere in VOLUME 23 the image, but tends to “whiten” the affected pixels. As a result of these problems, Pfister et al. (2003) applied a procedure that uses the mean and standard deviation of the cloud fraction over an 11-min period centered on the image of interest to determine whether the sun circle and/or horizon area cloud pixels should be included in the total or not. If both the whole-sky cloud fraction and its variance, and the variance of the sun circle and/or horizon area are small for an 11-min period, then it is very likely that the sun circle and/or horizon area is free of clouds. Pfister et al. (2003) report that this procedure for the sun circle correlates well with the sunshine parameter (i.e., the solar obstruction) for the two imagers analyzed in their paper. Specifically, when the procedure indicates that there are no real clouds in a 20° radius around the sun, the algorithms identify unobscured sun in about 90% of cases. Because different brightness thresholds can be applied to either of the two imagers’ cameras, a mismatch in the sunshine parameter will predominantly occur in situations that cannot be unambiguously classified as either obscured or unobscured sun, for example, situations when the sun disk is partly covered by clouds or when the sun disk is covered by optically thin clouds. This procedure is applied to all-sky image data of at least 1-min resolution, because it depends on the variability through time. As noted in Kassianov et al. (2005), the typical decorrelation time of the sky is about 10 to 15 min. Thus, sky images taken only every 5–10 min do not sample the natural evolution of the sky well enough to adequately track the variability in these problem areas for this “correction” process. In the case of research reported by Sabburg and Long (2004), TSI images were compared to visual sky observations on 5 days (653 images) sampled randomly in the period December 2001 to September 2002. On clear days, the TSI sunshine indicator would incorrectly register solar obstruction near local solar noon when a shadow of the camera housing was cast onto the shadowband. 2) CLOUD BROKENNESS AND CLOUD DISTRIBUTION Besides classifying solar obstruction by cloud across the solar region, algorithms have also been developed for the HSI, SCSC, and TSI sky images to measure cloud properties in other regions of the sky view. One of these properties is a measure of average cloud size and brokenness of the cloud coverage. For example, the cloud retrieval algorithm for the AllSky1 of Pfister et al. (2003) determines an “edge-to-area” ratio, defined as the number of pixels in the image on cloud/clear boundaries divided by the sum of cloudy pixels in the MAY 2006 LONG ET AL. image. A large edge-to-area ratio relates to broken clouds of small diameter, while a small edge-to-area ratio relates to extended clouds. Clear sky and completely covered sky have an edge-to-area ratio defined as zero (Pfister et al. 2003). The SCSC included the parameter CB, the ratio of the perimeter to area of a cloud (similar to the edge-to-area ratio). The perimeter and area measurements were based on the number of pixels classified as cloud, after thresholding the sky image to a binary image. The CB was measured in the range of zero to one (Sabburg and Wong 2000). Sabburg and Long (2004), Sabburg and Wong (2000, 1999), and Pfister et al. (2003) describe algorithms defining the distribution of cloud around the solar region. In the case of the SCSC, this was done in terms of the angle subtended by an arc from the center of the sun to the outer edge of circular sectors of width 2.5°. The algorithm classified which sector contained the greatest area of cloud, of 10 possible concentric sectors, whose angles ranged from 12.5° to 37.5°. In the case of the commercial TSI software, cloud amount is also estimated separately in zenith circle, sun circle, and horizon regions. In an analysis technique written for the TSI, Sabburg and Long (2004) report cloud amounts that were calculated in four separate circular regions concentric about an estimated position of the sun as determined by the TSI processing algorithm. Regions were classified as inner (about 15° radius), middle (about 30° radius), outer (about 45° radius), and extreme (extending beyond the outer region that is within the image area). Errors in cloud amount near to the sun (i.e., in the inner and middle regions) were found to increase from approximately 60° SZA and increasing as the sun approaches the horizon. Causes for this error were previously discussed and can contribute to an overestimation of cloud amount, up to 60% in the inner (sun centered) region to a few percent in the middle region, depending on the aerosol, haze, or ice crystal loading of the atmosphere. A new parameter, defined as cloud uniformity with respect to azimuth (in contrast to sun centered), was also developed for the TSI (Sabburg and Long 2004). The sky image is divided into four quadrants with vertical and horizontal crossbars centered on zenith with the quadrants defined as N to E, N to W, S to W, and S to E. Uniformity was recorded as 1 if cloud amount in the four quadrants of the image was within 20% of the total image cloud amount, otherwise it was listed as 0. 3) CLOUD PATTERNS One of the most difficult research areas of sky-image processing has been that of cloud recognition. Parisi et al. (2004) make reference to some papers relating to 643 this area of research in their overview of sky imagers. In this current paper, we make no attempt to classify cloud patterns (i.e., the parameter that best describes the “type” of cloud or cloud field present) for TSI or WSC images. Cloud pattern classification includes standard cloud types such as cumulus, stratus, and cirrus. Cases of haze, aerosol, and fog classification could also be included under this heading. We do address cloud field properties, such as brokenness, as described previously. In addition, the TSI image processing includes a separation of cloudiness into “thin” and “opaque” classifications, which is based on the amount of blue tint of the clouds in the image. This blue tint occurs when the clouds are optically thin, and thus one can see through them to the background blueness of the clear sky behind the cloud. Although there have been some papers published on analysis of satellite images of the earth view (e.g., Harris and Barrett 1978; Ebert 1987) and overviewed by Parisi et al. (2004), there have only been a few published papers describing limited cloud-type recognition from ground-based sky imagers. For example, by the use of polarizing filters Horvath et al. (2002) have improved algorithms of radiometric cloud detection, particularly promising for very high altitude, thin (i.e., “bluish”) cirrus clouds. Goodall and Hatton (2002) have performed some initial research with both visible and infrared images and have concentrated on the identification of towering cumulus and cumulonimbus clouds. Their results indicate that neural network processing has potential in cloud recognition. For the SCSC, a parameter called “cloud texture” was defined as the standard deviation of the brightness of the cloudy pixels. Brightness was defined as the sum of the values of the three color components (RGB). This gave a value for the variation along the surfaces of the clouds as seen from the ground. From unpublished work by one of the authors of this current paper (Sabburg at the University of Southern Queensland), it is speculated that Lambertian reflection increases for light-textured cloud—for example, cirrus—compared to the specular reflection from a heavier-textured cloud surface—for example, cumulus. Thus it may be possible to use this parameter to assist in the classification of cumulus and cirrus clouds, which is a subject of ongoing research. Further unpublished work by Sabburg, originally developed for use by the Commonwealth Bureau of Meteorology to classify TSI data as stratiform cloud for UV index research, used color, brightness, and pixel transitions in an attempt to classify cloud data as stratus, cumulus, cirrus, or fog. Although the findings for opaque and stratiform (overcast) cloud classification 644 JOURNAL OF ATMOSPHERIC AND OCEANIC TECHNOLOGY VOLUME 23 FIG. 7. (left) A sample gray-scaled HSI image and (right) corresponding gray-scaled cloud decision image taken at 1300 LT 4 Sep 2004 at the Pacific Northwest National Laboratory, Richland, WA. were encouraging, it was not successful for cirrus, stratus, and cumulus clouds. One technique that has been successfully used with CCD cameras for astronomical observations, but not clouds (Buil 1991), is that of Fourier transform or fast Fourier transform (FFT) analysis. The idea is that “signature” frequencies, corresponding to different types of cloud, may be produced from a Fourier transform of a cloudy-sky image [e.g., Garand (1988) applied this idea to satellite cloud images]. Additionally, twodimensional FFT techniques have been applied by one of the authors of this current paper (Calbó at the University of Girona) on terrain topography to investigate the best grid size to be used in mesoscale meteorological modeling (Salvador et al. 1999). This last work indicates that the technique may be quite robust in dealing with any spatial characteristics, including cloud patterns, which will be the subject of further research. 4. Results a. Total sky imager 1) TOTAL SKY IMAGER CORRECTION FOR SUN CIRCLE AND HORIZON AREA CLOUD DETECTION ERRORS The original methodology for correcting the sun circle and horizon area (Fig. 2) cloudiness amount described in Pfister et al. (2003) has been refined and adapted for application to TSI data (Long 2005). In essence, the magnitude and variability of the cloud frac- tion in the sun circle, horizon area, and the remainder of the image (total area minus the sun circle and horizon areas) are used to determine whether or not the cloud pixels in the sun circle and/or horizon area should be included in the total-image sky cover estimate. In the case of the sun circle, it must also be determined whether to count only half of the original cloud pixels (as will be discussed later in this section). The results are smoothed using a running 11-point running mean, that is, if 1-min data are being processed then the amount of adjustment applied is the average over 11 min centered on the point of interest. Figure 7 shows a grayscale sample HSI image (left) and corresponding cloud decision image (right) taken at 1300 local time (LT), 4 September 2004, at the Pacific Northwest National Laboratory located in Richland, Washington. As shown in this relatively extreme example, both the sun circle and horizon areas contain pixels erroneously determined as “cloud” while the sky image shows what an observer would typically label as clear sky. On this day the morning was clear, with cloudiness moving in at about 1320 LT and lasting through about 1520 LT when skies cleared again. More cloudiness then moved slowly in again at around 1700 LT, slowly moving off through about 1840 LT. The sky and cloud decision images show that this day exhibited significant haze, producing the erroneous identification problem. Figure 8 shows the retrieved total-sky cover for the 4 September 2004 daylight period, including the original retrieval, the “first guess” sun circle adjustment (Long MAY 2006 645 LONG ET AL. FIG. 8. Total sky cover retrieval for 4 Sep 2004 at the Pacific Northwest National Laboratory, Richland, WA. The gray line is the original retrieval, the thin black line is the retrieval including the “first guess” adjustment of the sun circle area, and the black line is the final result including all adjustments and smoothing. 2005), and the final adjusted retrieval. The first guess is intended to account for the probability of some error near the sun due to persistent forward scattering for times when the other tests do not subtract the sun circle cloud pixels. There is often some overestimation of cloud amount in the sun circle area, thus the reasoning behind the first-guess adjustment, which in general decreases the sun circle area cloud amount by up to half. As Fig. 8 shows, the adjustment methodology correctly decreased the initial erroneous sky cover values of nearly 20% during clear-sky periods downward in magnitude to near 0%, yet did not decrease the sky cover values for the times when clouds were present. Figure 9 shows relative frequency histograms of various instruments and time periods as noted in the figure caption. In each case, the original retrievals (gray) show a bias away from the “clear” bin (on the left) toward higher values. This result is inconsistent with expectations for these sites, where it is common that the longterm frequency distribution includes about one-third clear sky, one-third overcast, and the remaining onethird distributed in between. This expected distribution is indeed the case when the adjustments detailed here are applied to the retrievals (black) for each case. In the top two plots, the third distribution (striped) is from the available 100° FOV “zenith circle” retrievals (see Fig. 2). This zenith area is far less susceptible to the misidentification problems we are addressing, since the entire horizon area is not included, and (as noted previously) the sun circle problem is generally less for higher sun elevations. As is seen, there is much better agreement with the adjusted distributions than with the origi- nal. Similarly, the third distribution in the bottom plot, produced by the ARM Program using the shortwave flux analysis algorithm (Long and Ackerman 2000; Long and Gaustad 2004), agrees better with the adjusted values than the original. All these results suggest that the adjustment methodology significantly improves the sky-imager retrievals as intended. 2) TOTAL SKY IMAGER SOLAR OBSTRUCTION AND CLOUD DISTRIBUTION STUDIES The TSI located at the campus of the University of Southern Queensland was used to undertake further analysis of the sky characteristics of solar obstruction and cloud uniformity (introduced in section 3d). The analysis of the performance of these characteristics is more extensive than that previously undertaken by Sabburg and Long (2004). The set of images (71 335 in total), captured every 5 min from early morning to late afternoon, is available for the period from June 2003 through December 2004. We chose for study a temporally evenly distributed set of images during this period of up to 10 images per day, resulting in a total of 2427 images covering the SZA range of 4° to 80°. This subset of images was also manually inspected by an independent researcher (with previous experience inspecting SCSC images). Each image was viewed on a computer screen and the researcher recorded the following results in a spreadsheet: (a) for solar obstruction, “0” if the sun was either blocked or not visible due to cloud, otherwise “1.” (b) for cloud uniformity, “0” if cloud was not “uni- 646 JOURNAL OF ATMOSPHERIC AND OCEANIC TECHNOLOGY VOLUME 23 FIG. 9. Sky cover frequency histograms for (a) more than 7 months of data at Pacific Northwest National Laboratory, and (b) for the HSI and (c) TSI deployed during the 3 months of the ARM Cloudiness InterComparison (CIC) field experiment at the Southern Great Plains site. In all plots, gray represents the original retrievals, and black represents the adjusted retrievals. In (a) and (b), striped represents retrievals restricted to a 100° FOV. In (c), striped represents retrievals from the shortwave flux analysis methodology. MAY 2006 647 LONG ET AL. TABLE 3. All available data (SZA, 4° to 80°; TSI cloud fraction, 0%–100%) for (a) DO and (b) uniformity. Algorithm Yes No Undecided Total (a) DO (b) Uniformity Inspection Inspection Yes No Undecided Total Yes No Undecided Total 8 67 0 75 0 2337 0 2337 0 15 0 15 8 2419 0 2427 39 124 0 163 55 2206 0 2261 0 3 0 3 94 2333 0 2427 formly” distributed in the image (i.e., less than 20% of the total cloud in each of four quadrants), otherwise “1.” If the visual inspector was not sure whether to record a “1” or “0,” then a “9” indicated the decision was undecided in either of the two categories. For analysis of results, the data were divided into three groups: all available data, restricted SZA range, and restricted cloud fraction range. The sum of the diagonal values of any one of the matrices shown in Table 3, divided by the total number of images, gives a corresponding indication of the performance of each of the algorithms as 97% and 93% for solar obstruction and cloud uniformity, respectively. On closer inspection the overall performance of 97% was found to be biased due to the exceptional classification (100%) when the disk was not obscured. When the disk was obscured, the algorithm did not perform nearly as well, and neither was the observer able to readily classify the state of the obstruction. This prompted the decision to test if the performance of the algorithm or visual inspection might be affected by the “whitening” phenomenon described in section 3a. As the whitening tends to decrease with decreasing SZA, it was thought that the performance might improve for smaller SZA (higher sun). However, analysis of the smaller SZA data exhibited no improvement in performance. We also investigated whether the success of the comparison between algorithm and inspection improved for separate ranges of cloud fraction. The matrices in Table 4 show the performance of each of the algorithms for cloud fraction less than 50% (99% and 96%), and for a cloud fraction between 51% and 100% (85% and 76%, respectively). These results indicate an increased performance of 3% for both algorithms for cases with less then 50% of the sky-containing clouds. For the mostly cloudy cases, there is a decrease in performance of 11% and 17% for disk obstruction and uniformity, respectively. It could be concluded that the skill of the visual classification of these characteristics decreases with increased cloud fraction. b. Whole sky camera For the WSC, we analyze a set of images taken and processed during one year (November 2001 to October 2002). The only month with a significant number of missing images is August 2002. Images were captured and stored as BMP every 15 min, producing about 13 000 images, of which only about 10 700 taken at SZAs less than 80° were used for the present analysis. For each image, we calculated the fractional sky cover using both the geometrical correction derived in Eq. (6) and the unadjusted ratio of cloudy to total number of pixels [Eq. (1)]. These two values will be named hereafter as fG and fNG, respectively. Using the method described in section 3d, CB was calculated for the WSC images as the number of pixels in the perimeter of cloudy areas divided by the number of cloudy pixels. To investigate the effect of image format, we also computed the corresponding values for a subset of the same images, but stored in JPEG format. Approximately one-third of the images, evenly distributed across all months, were also visually inspected to estimate the corresponding fractional sky cover. This visual inspection was performed by three researchers at the University of Girona (two of the coauthors of this paper and a third colleague). Each inspector looked at close to 1400 images, with a subset of 430 being inspected by all three for cross-comparison and to investigate possible systematic bias of this kind of subjective human estimation of sky cover. The visually determined fractional sky cover will be referred to as fV. To estimate fV we used some visual aids that allowed us to divide the sky dome into 16 sectors, giving a resolution of these estimations of fractional sky cover of 0.0625 (⫽1/16). We also have available about 150 human observations of the sky conditions that were performed from November 2001 to May 2002. Observations were made by the two University of Girona coauthors from the same site where the WSC is installed. Although the observers are not professionally trained, cloud observations were carefully made following WMO recommendations. We recorded fractional sky cover fobs (in 648 JOURNAL OF ATMOSPHERIC AND OCEANIC TECHNOLOGY VOLUME 23 TABLE 4. Data with TSI cloud fraction less than 50% and SZA 4° to 80° (2018 images) for (a) DO and (b) uniformity, and data with TSI cloud fraction 51%–100% and SZA 6° to 80° (409 images) for (c) DO and (d) uniformity. Algorithm Yes No Undecided Total (a) DO (b) Uniformity Inspection Inspection Yes No Undecided Total Yes No Undecided Total 1 13 0 14 0 1997 0 1997 0 7 0 7 1 2017 0 2018 3 44 0 47 38 1930 0 1968 0 3 0 3 41 1977 0 2018 (c) DO (d) Uniformity Inspection Algorithm Yes No Undecided Total Inspection Yes No Undecided Total Yes No Undecided Total 7 54 0 61 0 340 0 340 0 8 0 8 7 402 0 409 36 80 0 116 17 276 0 293 0 0 0 0 53 356 0 409 oktas) and also cloud type and other sky characteristics (e.g., sun obstruction). These observations will be used here to be compared, both with the visual estimations from the images and the computed values. Based on the common set of images, we found that no relevant systematic bias was exhibited when the three trained researchers looked at the same images. Despite the absence of bias, there is some dispersion of values. In 5%–10% of the images, we found differences among two of the estimates of f greater than 0.5. Most of these cases correspond to early morning images, that is, dark images with possible dew on the WSC glass dome. The agreement between estimates is very high for totally cloudless or absolutely overcast skies. For the rest of images, that is, for f in the range 0.05–0.95, the root-mean-square error (rmse) is close to 0.11. This figure can be taken as a measure of the uncertainty when f is determined from WSC images by visual inspection, although for some range of f values, the uncertainty may be larger (see Table 5). The rmse was computed here from the differences between each estimate and the average of the three values. As a consequence of these analyses we decided that, for comparison with automatic estimations, fV would be equal to the average of the three values when available, and equal to the single value when only one researcher had inspected an image. The effect of the geometric correction through Eq. (6) is, as expected, relatively small. We found a determination coefficient r2 ⫽ 0.9998 between fG and fNG. The largest absolute differences are less than 0.04 in fractional sky cover. Corresponding relative errors are always less than 10% (and usually less than 5%), except for almost cloudless skies, when absolute differences of 0.01 may result in relative errors greater than 10%. Despite this minor effect, we have used fG in further analyses. Similarly, f from the JPEG images is almost identical to results from the BMP images in fractional sky cover, with a mean bias deviation (MBD) of 0.003 and rmse of 0.02. Results of the comparison between fG and fV are presented in the box charts of Fig. 10. All differences fG ⫺ fV have been grouped in bins according to fV (top plot). Each bin (except the first and the last ones, which correspond to cloudless and overcast skies, respectively) has a width of 0.10. We can see that the automatic estimation of fractional sky cover is in general TABLE 5. Mean and standard deviation of the visual estimates of f, for several intervals of f, from the set of images analyzed by all three researchers. Interval of f Mean Std dev 0.00 0.00–0.06 0.06–0.12 0.12–0.19 0.19–0.25 0.25–0.31 0.31–0.37 0.37–0.44 0.44–0.50 0.50–0.56 0.56–0.62 0.62–0.69 0.69–0.75 0.75–0.81 0.81–0.87 0.87–0.94 0.94–1.00 1.00 0.00 0.04 0.10 0.16 0.22 0.30 0.36 0.41 0.48 0.56 0.61 0.68 0.73 0.79 0.85 0.92 0.97 1.00 0.00 0.03 0.05 0.07 0.08 0.09 0.14 0.07 0.13 0.21 0.16 0.19 0.16 0.15 0.13 0.07 0.03 0.00 MAY 2006 LONG ET AL. FIG. 10. Differences between automatic estimation of fractional sky cover fG and visual estimation fV: (upper) depending on fractional sky cover, and (lower) depending on SZA. Boxes show the median and the percentiles 25 and 75. Additional error bars represent the percentiles 10 and 90. lower than the human visual estimation. Most medians correspond to negative differences, with absolute values always less than 0.20 and in general less than 0.10. The dispersion of differences is larger for broken-cloud conditions ( fV in the range 0.35–0.85). This behavior is likely due to an overestimation of the human estimate, which consists of counting large sectors of the sky that can be “patched” with small clear areas as cloudy, while the automatic estimation is based upon pixel counts only. Automatic and manual estimations are virtually identical as far as overcast conditions are concerned. For cloudless skies, the automatic estimate hardly ever results in fG ⫽ 0.00. This is due to circumsolar areas that are considered cloudy by the automatic method, given that these areas appear white when there is some 649 amount of haze or aerosols, as mentioned previously. For the whole dataset, MBD between fG and fV is ⫺0.001, and rmse is 0.21. Figure 10 (bottom) shows the same differences fG ⫺ fV as in the top plot, but versus SZA. The medians of the differences are practically 0 for all SZAs, which is a good characteristic of the image processing. It should be noted that about two-thirds of the data in the top plot resides in the first two and last bins (i.e., the nearly clear and overcast bins), which are about evenly distributed by SZA in the bottom plot. Dispersion is somewhat larger for smaller SZAs. There are two possible reasons for this behavior. First, there are fewer images taken at these lower SZA values, which correspond to noontime of summer months. Second, these same noontime summer data are when aerosol optical depths at Girona are usually higher than in other hours and seasons (González et al. 1998). Thus, the effect of the “white” circumsolar area under high aerosol conditions is enhanced at smaller SZA, resulting in an overestimate of f. This also may be related to the fact that we are using a single threshold (R/B ⫽ 0.6) for all images and suggests that a slightly higher value of this ratio should be used for summer conditions in Girona. In comparing fG and fobs, the best agreement is found for overcast skies, followed by cloudless or almost cloudless (less than 1 okta) skies. In the latter case, as expected, fG tends to be greater than fobs, because of the already explained difficulty of obtaining fG ⫽ 0 by using our automatic retrieval. In all other cases (2 oktas ⱕ fobs ⱕ 7 oktas) fG is systematically biased toward lower values or, conversely, the human observations systematically indicate higher cloud amounts. The MBD between fG and fobs is ⫺0.12, and the rmse is 0.28. The maximum differences correspond to cases when the human observation has reported cirriform clouds: from the 150 observations there are seven cases with fG ⫺ fobs ⬍ ⫺0.5. In these seven cases either Ci or Cs were reported. The number of direct observations of the sky is not large enough to derive robust conclusions from the commented differences. However, this comparison seems to confirm the tendency that has already been detected when comparing fG with fV. In summary, the automatic estimate derived from WSC images generally results in lower fractional sky cover values than the human estimates from either direct observations or visual inspection of corresponding sky images. The most frequent value of CB ranges from 0.10 to 0.15, with most CB values less than 0.35. This is true for sky conditions corresponding to fG in the range 0.05– 0.95. Obviously, when the sky is virtually cloudless ( fG ⬍ 0.05) or almost overcast ( fG ⬎ 0.95), CB tends to be 0. Logically, most frequent CB values are higher for 650 JOURNAL OF ATMOSPHERIC AND OCEANIC TECHNOLOGY VOLUME 23 FIG. 11. Frequency distributions of CB values for four different ranges of fG as noted in the figure legend. scattered cloudy conditions and lower when fractional sky cover is small or large. For example, for fG in the range 0.25–0.45, typical CB is 0.3, while for fG ⬎ 0.65, CB is less than 0.1 in general. The major importance of CB, however, is the dispersion of values for a given value of fractional sky cover. Different CB values indicate different sky conditions: smaller CB means compact clouds, while larger CB means patchy clouds. Figure 11 shows the frequency distributions of CB for four different ranges of fG. We can see that, for fG between 0.35 and 0.45, there is maximum dispersion of CB values, indicating that these clouds may correspond either to a few clouds covering a part of the sky or a larger number of broken cloudy areas likely occupying the whole sky dome. Figure 11 also confirms that dispersion of CB values is smaller when fG is greater. The CB values computed from JPEG images tend to be smaller (by a factor of 2) than CB values from BMP images. This is due to the more physically representative “smoothing” of the JPEG format (discussed below). However, the relative frequency distribution of CB values from JPEG images is quite similar to the distribution obtained from BMP images that is shown in Fig. 11. While it is true that a BMP image does better capture each individual CCD element value, commercial CCD arrays such as we are using have element-to-element sensitivity differences that affect the clear/cloud classification. It is not “normal” for one isolated pixel to be “cloudy” when all of its surrounding pixels are not. Using an element-by-element map, such as a BMP image, often results in isolated pixels erroneously being classified as cloud. While this results in only a small error in total sky cover, it can have a significant effect on a parameter such as CB as noted above. JPEG compression, which by its nature is a slightly “smoothed” rendering at typical default JPEG compression settings (75–80), tends to compensate for the CCD element-toelement sensitivity differences giving sky cover retrievals that much better reflect the way nature behaves in the sky. 5. Summary In this paper we have shown that the application of automatic digital image-processing techniques on sky images is a useful method to complement, or even replace, traditional human observations of sky cover, and there is likely potential for inferring cloud type. Although some uncertainty exists in fractional sky cover retrievals from sky images, previous work has shown this uncertainty is no greater than that attached to human observations for the commercially available sky imager and processing technique (i.e., for the TSI) discussed here. Even for the WSC imager, the uncertainty is still acceptable and comparable to human observational uncertainty. We note that cloud cover has been traditionally recorded as eighths (oktas) or tenths of the sky dome by human observers. This means that an uncertainty of at least 0.125 or 0.10, respectively, is to be expected. Unlike human observations, current sky condition descriptions from digital images do not include cloud typing in the traditional way (i.e., using cloud genera). However, other equally interesting (for radia- MAY 2006 651 LONG ET AL. tion studies, e.g.) sky characteristics can be implied, such as cloud brokenness and distinction between optically thin and thick clouds. The main advantages of sky imagers compared to human observations are threefold. First, sky imagers can provide an almost continuous observation of the sky. Classically, cloud observations are made every 3 h, or on an hourly basis at some meteorological stations. Second, sky imagers can provide long-term sky condition information at relatively low cost, compared to the cost of human observers. Finally, where human observations of clouds are subjective, decreasing their precision, observation of clouds by automatic devices such as sky imagers is objective and highly reproducible. One of the imagers presented in this paper was designed and built by researchers at the University of Girona. This imager (WSC) has been continuously taking sky images since the Northern Hemisphere summer of 2001. One year of such images has been analyzed by using a simple process that consists of initial masking of parts of the image and using a single threshold to distinguish between cloudless and cloudy pixels. When computing the fractional sky cover, an expression that accounts for the differences between the actual solid angles and the corresponding image areas has been considered. The effect of this correction is minor, but nevertheless the correction has been applied because it does not present particular difficulties in processing the images. With this imager and simpler processing methodology, fractional sky cover can be estimated with an uncertainty of about 0.2. More specifically, imagerderived sky cover tends to be greater than the corresponding human observations for amounts less than 0.2, but less than the human observations for amounts ranging between 0.2 and 0.8. The two values virtually always agree for overcast conditions. We see two directions for future research stemming from the current work: improving the hardware and improving the image processing. One possibility for device improvement is mounting a fish-eye camera on a solar tracker and shading the lens with a shading sphere, instead of a shadow strip or shadowband. With this approach, the area of the sky obscured could be reduced. However, it is also realized that the smaller the “dome” or mirror surface used, the greater the portion of the sky image adversely affected by obstructions such as rain- or dewdrops. Regarding the image processing, we will investigate further parameters such as CB, and how these parameters relate with classical cloud types. In addition, suggestions made in this paper about the use of Fourier analyses techniques applied to ground-based sky images will be further explored. Acknowledgments. Dr. Long acknowledges the support of the Climate Change Research Division of the U.S. Department of Energy as part of the ARM Program. Dr. Sabburg would like to thank Nathan Downs for his dedicated work in coding the specialized USQ TSI algorithms and evaluating their uncertainties, as well as Rosalie Sabburg (Environmental Sciences Graduate), for visually inspecting the subset of USQ TSI images. Dr. Calbó would like to thank Magda Llach (UdG), for visually inspecting her set of WSC images. We gratefully thank the anonymous reviewers, whose efforts lead to improvement of this paper. REFERENCES Buil, C., 1991: CCD Astronomy—Construction and Use of an Astronomical CCD Camera. Willmann-Bell, 321 pp. Calbó, J., D. Pagès, and J. A. González, 2005: Empirical studies of cloud effects on UV radiation: A review. Rev. Geophys., 43, RG2002, doi:10.1029/2004RG000155. Ebert, E., 1987: A pattern recognition technique for distinguishing surface and cloud types in the polar regions. J. Climate Appl. 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https://openalex.org/W2146023959	https://pure.mpg.de/pubman/item/item_2019154_4/component/file_2019153/22606-119059-1-PB.pdf	English	null	Testing variational estimation of process parameters and initial conditions of an earth system model	Tellus. Series A, Dynamic meteorology and oceanography	2,014	cc-by	12,992	Corresponding author. email: Simon.Blessing@FastOpt.com #Deceased ABSTRACT We present a variational assimilation system around a coarse resolution Earth System Model (ESM) and apply it for estimating initial conditions and parameters of the model. The system is based on derivative information that is efficiently provided by the ESM’s adjoint, which has been generated through automatic differentiation of the model’s source code. In our variational approach, the length of the feasible assimilation window is limited by the size of the domain in control space over which the approximation by the derivative is valid. This validity domain is reduced by non-smooth process representations. We show that in this respect the ocean component is less critical than the atmospheric component. We demonstrate how the feasible assimilation window can be extended to several weeks by modifying the implementation of specific process representations and by switching off processes such as precipitation. Keywords: data assimilation, climate modelling, coupled oceanatmosphere model, earth system model, automatic differentiation, adjoint model Tellus A 2014. # 2014 S. Blessing et al. This is an Open Access article distributed under the terms of the Creative Commons CC-BY 4.0 License (http:// creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license. 1 Citation: Tellus A 2014, 66, 22606, http://dx.doi.org/10.3402/tellusa.v66.22606 By SIMON BLESSING1, THOMAS KAMINSKI1, FRANK LUNKEIT2, ION MATEI2, RALF GIERING1, ARMIN KO¨ HL2, MARKO SCHOLZE2,3, P. HERRMANN4#, KLAUS FRAEDRICH2 and DETLEF STAMMER2, 1FastOpt, Lerchenstraße 28a, DE-22767 Hamburg, Germany; 2CEN, University of Hamburg, Bundesstraße 53, DE-20146 Hamburg, Germany; 3Lund University, So¨lvegatan 12, SE-223 62 Lund, Sweden; 4Max-Planck-Institut fu¨r Meteorologie, Bundesstraße 53, DE-20146 Hamburg, Germany By SIMON BLESSING1*, THOMAS KAMINSKI1, FRANK LUNKEIT2, ION MATEI2, RALF GIERING1, ARMIN KO¨ HL2, MARKO SCHOLZE2,3, P. HERRMANN4#, KLAUS FRAEDRICH2 and DETLEF STAMMER2, 1FastOpt, Lerchenstraße 28a, DE-22767 Hamburg, Germany; 2CEN, University of Hamburg, Bundesstraße 53, DE-20146 Hamburg, Germany; 3Lund University, So¨lvegatan 12, SE-223 62 Lund, Sweden; 4Max-Planck-Institut fu¨r Meteorologie, Bundesstraße 53, DE-20146 Hamburg, Germany (Manuscript received 14 August 2013; in final form 17 January 2014) (Manuscript received 14 August 2013; in final form 17 January 2014) PUBLISHED BY THE INTERNATIONAL METEOROLOGICAL INSTITUTE IN STOCKHOLM SERIES A DYNAMIC METEOROLOGY AND OCEANOGRAPHY PUBLISHED BY THE INTERNATIONAL METEOROLOGICAL INSTITUTE IN STOCKHOLM SERIES A DYNAMIC METEOROLOGY AND OCEANOGRAPHY 1. Introduction It is built around the MITgcm (Marshall et al., 1997a, 1997b) and infers a combination of initial and boundary conditions of the ocean circulation. Meanwhile, multiple versions of the system are being applied by several research groups around the world in different setups (e.g. Hoteit et al., 2005; Ko¨ hl and Stammer, 2008). As another example, the adjoint (Kauker et al., 2009) of the Arctic coupled sea-ice ocean model NAOSIM is employed to initialise seasonal predictions of the Arctic ice conditions (Kauker et al., 2010). A more fundamental challenge results from the non- linearity of the climate system: The usefulness of derivative code depends on the capability of the linearisation around a point to represent the model in the point’s neighbourhood. This capability is closely connected with the concept of predictability, which Lorenz (1963) analysed for a non- linear three-dimensional system that possesses a strange attractor. Lea et al. (2000) use this system to demonstrate that the usefulness of the linearisation of the long-term mean of the state variables around the system’s parameters decreases with increasing integration period. Ko¨ hl and Willebrand (2002) analyse how this affects the parameter estimation from the long-term mean state via a gradient method for the same model as well as for a high-resolution quasi-geostrophic model of the oceanic circulation. In this estimation context, the poor linearisability of the long-term mean shows up in the form of multiple local minima in the modeldata misfit. Ko¨ hl and Willebrand (2002) as well as Thuburn (2005) extend the adjoint approach by a statistical concept to enhance the usefulness of the gradient informa- tion. Pires et al. (1996) using the Lorenz model and Tanguay et al. (1995) using a b-plane model address the linearisation problem in the context of four-dimensional variational data assimilation, estimating initial conditions that minimise the modeldata misfit. Pires et al. (1996) and Swanson et al. (1998) present a quasi-static variational assimilation ap- proach that tracks the absolute cost function minimum through successive increments of the assimilation window. In the adjoint-based assimilation system around their ESM, Sugiura et al. (2008) are improving linearisability through the simulation of time-averaged fields and an approximate adjoint with an artificial damping term following an initial calibration of seven parameters through a Green’s functions approach. Abarbanel et al. (2010) also suggest a variable damping term, and present an analysis of its effect on their cost function. 1. Introduction tion, simplified process representations, and/or reduced sets of uncertain (tunable) parameters. For example, Jones et al. (2005) employ FAMOUS, a reduced resolution version of its parent general circulation model HadCM3 to demonstrate the systematic tuning of eight process parameters. This subset of the full parameter space, which for the atmosphere component alone has about 100 dimensions (Murphy et al., 2004), had to be kept small for computational reasons. This is because even a parameter space of as few as eight dimensions can only be efficiently searched for an optimal parameter set by a gradient algorithm. Such gradient algorithms minimise the modeldata misfit quantified by a cost function through the use of the cost function’s gradient. Jones et al. (2005) had to restrict the dimension of the control space because they approximated the gradient (i.e. sensitivity) informa- tion in the optimisation procedure by inaccurate finite difference calculations of multiple model runs (depending on the chosen perturbation size), at a computational cost proportional to the number of tunable process parameters. State-of-the-art climate predictions rely on numerical models of the earth system. One of the major sources of uncertainty in these predictions is the correct representation and parameterisation of the processes underlying the climate system (see e.g. Cubasch et al., 2001). A further source is the uncertainty in the initial state, that is, the state of the climate system at the beginning of the integration. Syste- matic use of observational information has the potential to reduce both types of uncertainty. Due to their high complex- ity, state-of-the-art earth system models (ESMs) are extre- mely demanding in terms of computer time. This complicates the systematic estimation of process parameters (calibration) and of the initial state (initialisation) from observations. These systematic approaches can, thus, typically only be pursued for models with reduced spatio-temporal resolu- Computing parameter sensitivities with the adjoint avoids any restriction on the dimension of the parameter (page number not for citation purpose) (page number not for citation purpose) S. BLESSING ET AL. 2 2 and initial state space. This is because the associated computational cost is independent of this dimension, as will be explained in Section 2.2 below. This concept has been demonstrated for several components of the earth system. To the atmospheric component, the adjoint approach is being routinely applied at operational centres for numerical weather prediction (NWP) for forecast initialisation (see e.g. Rabier et al., 2000). 1. Introduction Adjoint-based calibration has been demonstrated (e.g. Blessing et al., 2004; Kaminski et al., 2007) for the Portable University Model of the Atmosphere (PUMA, Fraedrich et al., 2005c). nature, imposed by the model’s code size and complex- ity. For many of the above-listed component models (PUMA, MITgcm, NAOSIM, BETHY, JSBACH, JULES, ORCHIDEE), the derivative code has been generated by an automatic differentiation tool (Transformation of Algo- rithms in Fortran (TAF), Giering and Kaminski, 1998). Sugiura et al. (2008) pioneered assimilation into an ESM by coupling the adjoints of their component models. This approach is tedious, error prone, and inflexible as it requires hand coding the coupling on the derivative code level. The alternative approach, which consists of automatic differ- entiation of the entire ESM, has not been pursued yet. The present study demonstrates, for the first time, the feasibility of this coupled model differentiation, using an ESM consist- ing of the Planet Simulator (PlaSim, Fraedrich et al., 2005a, 2005b; Fraedrich, 2012) coupled to MITgcm (Marshall et al., 1997a, 1997b). and initial state space. This is because the associated computational cost is independent of this dimension, as will be explained in Section 2.2 below. This concept has been demonstrated for several components of the earth system. To the atmospheric component, the adjoint approach is being routinely applied at operational centres for numerical weather prediction (NWP) for forecast initialisation (see e.g. Rabier et al., 2000). Adjoint-based calibration has been demonstrated (e.g. Blessing et al., 2004; Kaminski et al., 2007) for the Portable University Model of the Atmosphere (PUMA, Fraedrich et al., 2005c). For the ocean component of the earth system, an adjoint- based assimilation system has been operated for more than a decade (Stammer et al., 2002, 2003). It is built around the MITgcm (Marshall et al., 1997a, 1997b) and infers a combination of initial and boundary conditions of the ocean circulation. Meanwhile, multiple versions of the system are being applied by several research groups around the world in different setups (e.g. Hoteit et al., 2005; Ko¨ hl and Stammer, 2008). As another example, the adjoint (Kauker et al., 2009) of the Arctic coupled sea-ice ocean model NAOSIM is employed to initialise seasonal predictions of the Arctic ice conditions (Kauker et al., 2010). For the ocean component of the earth system, an adjoint- based assimilation system has been operated for more than a decade (Stammer et al., 2002, 2003). TESTING VARIATIONAL ESTIMATION Configurations marked ‘slow’ use a 20-minute time-step in the atmosphere, and in one case (Exp. 4 of Table 3 described in Section 3), even a 10-minute time-step. A useful diagnostic for the performance of an adjoint- based assimilation system is the length of the feasible assimilation window, that is, the assimilation window over which the system can successfully operate. For a coarse resolution version of PUMA, the atmospheric component of our ESM, Kaminski et al. (2007) demonstrated feasible assimilation windows of up to 100 d for parameter estima- tion. Here we use the same diagnostic to study the perfor- mance of the assimilation system around our ESM. A number of modifications were made to PlaSim in order to enhance its performance in a variational assimila- tion system (see Appendix). Two configurations, called ‘standard’ and ‘minimal’ are used. ‘Standard’ uses most of PlaSim’s components except for the terrestrial biosphere model, while in ‘minimal’ also the hydrological cycle is excluded, that is, evaporation, precipitation, and runoff. Moreover, in the moisture-free ‘minimal’ atmosphere there is no cloud-radiative feedback and the soil moisture is set to climatology. Configurations marked ‘w/o ocean’ replace the ocean with climatological sea surface temperature (SST). Table 1 gives an overview for quick reference. We further use the tags soft to mark experiments which do use smooth replacements for some occurrences of the if, where, min, max, abs, etc. statements, which proved problematic in initial tests, and hard for those which do not (see Appendix for details). The layout of the remainder of this paper is as follows. Section 2 will present the components of the assimilation system, Section 3 will describe the experimental setup, and Section 4 will present the results. Section 5 will provide a discussion and Section 6 a summary and conclusions. 1Available via http://www.cen.uni-hamburg.de/en/research/cen- models/cesam.html. TESTING VARIATIONAL ESTIMATION 3 provided by Lorenc and Payne (2007), together with a sketch of a seamless four-dimensional variational assimilation approach, which models probability density functions for the uncertain, small-scale processes. cloud cover, precipitation, runoff, soil temperature and wetness, surface fluxes, a thermodynamic sea-ice model, and a terrestrial biosphere component (SIMBA). The MITgcm is a state-of-the-art finite volume model of the general oceanic circulation, including a model of sea-ice dynamics and rheology (Zhang et al., 1998). In NWP, it is common to run the assimilation with ‘simplified physics’, that is, to remove a set of particularly non-linear processes or replace them by less complex and smoother formulations (Rabier et al., 2000). What is feasible for the short assimilation windows typical for NWP can be problematic on longer time scales, where it may result in considerable biases in the simulated state of the system. For the terrestrial biosphere component, it has been shown (Knorr et al., 2010; Kaminski et al., 2012) that the per- formance in the above-mentioned CCDAS is considerably improved by reformulation of some crucial process formu- lations (e.g. of leaf phenology). Formulations that rely on step functions or non-differentiabilities were replaced by formulations that resulted in smooth dependency of the simulation on initial conditions and process param- eters. For the phenology this was achieved by adopting a statistical concept (Knorr et al., 2010) as opposed to a concept that, simply speaking, simultaneously removes all leaves within a given grid cell. The current study transfers this reformulation concept to our ESM. In the coupling, sea surface temperature and salinity are computed by the ocean model and used by the atmospheric model. In turn, the atmospheric model passes back heat flux, precipitation minus evaporation, runoff, wind stress, and, optionally, short wave radiative heat flux, atmospheric sur- face pressure, and snow and ice mass. Of the optional quan- tities, we use only short wave radiative heat flux since the sea-ice component of the MITgcm is deactivated in the pres- ent study. Instead, the thermodynamic ice model of PlaSim is used. In the current setup, the models run in turns, and the exchanged quantities are interpolated between the grids. For all experiments, a resolution of 48 in the ocean and 5.68 (T21) in the atmosphere and land surface components is used. A time-step of 8 hours is used in the ocean and 48 minutes in the atmosphere. 1. Introduction A summary of the linearisation topic is For the terrestrial biosphere component, this approach is demonstrated by the Carbon Cycle Data Assimilation System (CCDAS, http://ccdas.org, Rayner et al., 2005; Scholze et al., 2007; Kaminski et al., 2012, 2013), which performs a combined parameter and initial state estimation in the terrestrial biosphere model BETHY (Knorr and Heimann, 2001). CCDAS also features uncertainty pro- pagation, based on second derivative information. The CCDAS concept is being transferred (see e.g. Luke, 2011; Kuppel et al., 2012; Kaminski et al., 2013; Schu¨ rmann et al., 2013) to several further terrestrial biosphere models (JSBACH, JULES, ORCHIDEE), all of which are compo- nent models in ESMs that contribute climate projections to the IPCC’s 5th assessment report. The construction of an analogous assimilation system around an entire ESM is clearly desirable. Such a system could allow, for example, the initialisation of climate model predictions in a way consistent with model dynamics. Another application could be the use of paleo records as constraints on the process parameters of the underlying ESM. Furthermore, the impact of all process parameters and the initial state on the model’s climate sensitivity could be rigorously assessed in a single adjoint run. First steps into this direction were taken by Lee et al. (2000) and Galanti et al. (2003) who used ocean models (in the first case a beta plane model, and in the latter the MOM3 general circulation model) coupled to a simple statistical atmospheric compo- nent, derived through a singular value decomposition. One of the challenges associated with the set-up of an assimilation system around an entire ESM is of a technical 2.2. Assimilation 2.2. Assimilation We use the observational information to constrain a vec- tor of control variables, which can be a combination of initial and boundary conditions as well as parameters in the process formulations of the model. Our experiments will investigate several choices of control vectors summa- rised in Table 2. P10 is a control vector of process param- eters from PlaSim, controlling the time scale for Rayleigh friction in the uppermost two atmospheric layers, the diffusion time scales for divergence, vorticity, and tempera- ture, the point of mean long wave radiation transmissivity in a layer, and four degrees of freedom of diffusion and sur- face fluxes. I2 controls a globally uniform perturbation of initial conditions of atmospheric surface pressure and temperature at all levels. I4 is as I2, but additionally includes global-scale perturbations of salinity and temperature of the ocean. Finally, I3D controls a gridpoint-wise perturbation of atmospheric vorticity, divergence, surface pressure, and temperature, as well as of oceanic salinity and temperature. Figure 1 shows a schematic illustration of a cost func- tion that includes a step function. The red curve displays the observational term (for perfect model and observations) with the step function while the green curve displays the effect of smoothing the step. In this case, the smoothing is not strong enough to avoid a secondary minimum. Adding a strong enough prior term (dark blue) removes the secondary minimum (magenta). All of our experiments (see Section 3), with the exception of one, use pseudo observations produced from known true values of the control variables without added noise. In this context there are three options for the prior term: Our assimilation system implements a probabilistic inver- sion concept (see Tarantola, 2005) that describes the state of information on a specific physical quantity by a prob- ability density function (PDF). The prior information on the control variables is quantified by a PDF in control space and the observational information by a PDF in the space of observations, at all sampling times and locations. Their respective means are denoted by xprior and d and their respective covariance matrices by Cprior and Cobs, where Cobs accounts for uncertainties in the observations as well as uncertainties from errors in simulating their counterpart (model error). 2.1. The model Table 1. Model conﬁgurations Configuration Atm. time-step (min.) Atm. hydr. cycle Coupled with MITgcm std w ocean 48 yes yes minimal w ocean 48 no yes std w/o ocean 48 yes no minimal w/o ocean 48 no no slow w/o ocean 10 no no slow w ocean 20 no yes Table 1. Model conﬁgurations Table 1. Model conﬁgurations The ESM introduced here is the CESAM1 (CEN Earth System Assimilation Model). It consists of the PlaSim (Fraedrich et al., 2005a, 2005b; Fraedrich, 2012) coupled to the MITgcm (Marshall et al., 1997a, 1997b). The relevant components of the PlaSim include the spectral PUMA (Fraedrich et al., 2005c), including schemes for radiation, 1Available via http://www.cen.uni-hamburg.de/en/research/cen- models/cesam.html. S. BLESSING ET AL. 4 where M(x) denotes the model operated as a mapping of the parameters onto simulated counterparts of the observa- tions. In the non-linear case, we approximate the posterior PDF by a Gaussian with mean value xpost, which is also termed maximum a posteriori probability (MAP) estimate. Without the prior term, it is termed maximum likelihood estimate (MLE). The first term of eq. (1) quantifies the modeldata misfit (observational term) and the second term the prior information. In NWP the latter term is called background term. 2.2. Assimilation If the prior and observational PDFs were Gaussian and the model linear, the posterior PDF would be Gaussian, too, and completely characterised by its mean xpost and its covariance matrix Cpost. Further, xpost would minimise the following cost function: i. discard the prior term ii. use true values as prior xprior as is illustrated in Fig. 1. ii. use true values as prior xprior as is illustrated in Fig. 1. iii. use different values than the true values as prior xprior xprior Options (i) and (ii) allow us to assess the progress of the iterative minimisation of J(x) through the difference between the current and the true values of the control variables. For a successful minimisation, this difference, for example, expressed as a Euclidean norm, should con- verge to zero. By contrast, for Option (iii) we would expect the prior term to shift the minimum from the true value towards the prior. This is why we discard this option. We note, however, that Option (iii) is the usual choice for assimilation of real data, and is particularly important in underdetermined setups. The effect of Options (ii) and (iii) is to smooth the cost function, and thus mask potential problems in the observational term. Figure 1 schematically illustrates the smoothing effect for Option (ii). For Option (iii), the effect of the prior term will depend on its loca- tion relative to the two minima in the observational term. For our experiments, we choose Option (i) in order to make a clear assessment of the properties of the observational term. We demonstrate, however, the effect of including a prior term (Option (ii)) for two of our experiments, which use the P10 control vector (described in Section 3 and Table 2) with standard deviations set to 100% of the respective parameter values and zero off-diagonal elements in the uncertainty covariance matrix Cprior. We also use the prior uncertainty to scale the control vector in all our JðxÞ ¼ 1 2 ½ðMðxÞ dÞTC1 obs:ðMðxÞ dÞ þ ðx xpriorÞTC1 priorðx xpriorÞ; (1) (1) Table 2. Control vectors Name Atmosphere Dim. Ocean Dim. P10 10 process parameters 10 I2 scalar pert. for ps, T 2 0 I4 scalar pert. for ps, T 2 scalar pert. for S, T 2 I3D spatially explicit: z, D, ps, T 63 488 spatially explicit: S, T 61 942 Table 2. TESTING VARIATIONAL ESTIMATION TESTING VARIATIONAL ESTIMATION 5 5 cost function control parameter observational term obs. term with soft step prior term obs. plus prior term with soft step obs. plus prior term Fig. 1. Schematic illustration of a cost function that includes a step function, including the effects of smoothing and prior (background) term. cost function control parameter Fig. 1. Schematic illustration of a cost function that includes a step function, including the effects of smoothing and prior (background) term. experiments, that is, the control vector is quantified in multiples of the prior uncertainty. tively) was generated through TAF. The TLM requires the CPU time of about 2.3 model runs to provide a single gradient component and the function value, while the ADM requires the CPU time of about four model runs to provide the entire gradient and the function value. This includes an efficient two-level-check-pointing scheme (Griewank, 1992) to allow long integrations. The model’s MPI paralle- lisation capabilities were preserved in the derivative code without degradation of the above performance ratios. We note that repeated invocation of TAF can be used to generate code for evaluation of higher-order derivatives. For exam- ple, Kaminski et al. (2003) describe the generation of code for evaluation of the Hessian. The assimilation consists of an iterative minimisation of J through variation of x by a quasi-Newton algorithm (Fletcher and Powell, 1963). This procedure determines the search direction through the gradient of J with respect to x. This gradient of the cost function with respect to the control variables is provided by automatic differentiation (Griewank, 1989) of the source code through TAF (Giering and Kaminski, 1998). The automatic differentiation procedure decomposes the code that evaluates the entire function J into simple elementary functions such as ‘’ or ‘sin’ for which the derivative (local Jacobian matrix) is known. By applying the chain rule of calculus to the sequence of local Jacobians, the derivative of the composite function can then be evaluated accurately up to rounding error. This multiple matrix product can be evaluated in arbitrary order. The tangent linear model (TLM) uses the same order as the evaluation of the function, while the adjoint model (ADM) uses the reverse order. Both yield (up to rounding error) identical results for the gradient of J(x) of eq. (1), but the memory and CPU time requirements differ. 2.2. Assimilation Control vectors Name Atmosphere Dim. Ocean Dim. P10 10 process parameters 10 I2 scalar pert. for ps, T 2 0 I4 scalar pert. for ps, T 2 scalar pert. for S, T 2 I3D spatially explicit: z, D, ps, T 63 488 spatially explicit: S, T 61 942 Scalar pert. for atmospheric surface pressure (ps) is applied to the co- efficient m0, n1 of the spherical harmonic in spectral representa- tion. z denotes vorticity, D divergence, S salinity, and T temperature. Table 2. Control vectors Scalar pert. for atmospheric surface pressure (ps) is applied to the co- efficient m0, n1 of the spherical harmonic in spectral representa- tion. z denotes vorticity, D divergence, S salinity, and T temperature. TESTING VARIATIONAL ESTIMATION While the requirements for the gradient calculations using one TLM run per control variable are proportional to the number of control variables, the requirements using the ADM are proportional to the number of dependent variables but virtually independent of the number of control variables. An efficient TLM and ADM pair (comprising 174 000 and 387 000 lines of Fortran code excluding comments, respec- We note that TAF relies on a number of global analyses, that is, analyses of the entire function code. For example, an activity analysis traces all variables on the path from control variables to the cost function value. This analysis also covers the interfaces of the component models with the coupler. Treating the differentiation of the model com- ponents and the coupler separately, as demonstrated by Sugiura et al. (2008) for their ESM, would have required the user to perform this activity analysis and assure the correct coupling on the level of the generated component derivative codes. Even though there was pre-existing deri- vative code for some of the model components (Marotzke et al., 1999; Blessing, 2000; Blessing et al., 2008; Rivie` re et al., 2009), we apply our coupled model differentiation approach. This means we apply TAF to generate the deriv- ative of J with respect to the parameters x to the entire coupled model at once. Thus, TAF automatically generates S. BLESSING ET AL. 6 experiment with the ERA-40 data is derived by optimis- ing the atmospheric initial conditions in a pre-assimilation over 200 iterations. This procedure uses as additional term in eq. (1), a surface pressure tendency penalty to suppress gravity waves as given in eq. 2.4 of Zou et al. (1993), summed over all time steps during the first 6 hours of a 1-d assimilation window, while the observational constraint was constructed as the time-averaged data over the full assimilation window. All but the aforementioned experi- ment will be conducted as identical twin experiments that assimilate pseudo data. This means we use default values of the control vector to generate pseudo data. Next, we start the iterative assimilation procedure from a perturbed control vector. 2.3. Data sets for assimilation ‘Identical twin’ experiments use pseudo-data in the assim- ilation, which were generated with the model itself from a prescribed control vector, thus guaranteeing full consistency of data and model, and allowing us to know the ‘true’ control vector. For one other experiment, data interpolated from ERA-40 simulations (Uppala et al., 2005) are used in the atmosphere. In either case, data are provided at all grid points and levels of the respective subsystem, with the exception of atmospheric temperature at the upper- most level. For the atmosphere we are using vorticity, temperature, and surface pressure, while in the ocean these are temperature and salinity. As in Ko¨ hl and Willebrand (2002), all data are time-averaged over the assimilation window and no noise is added. Consequently, the cost function is evaluated at the end of the assimilation window. Given the spatial resolution of T21 and 10 levels in the atmosphere and 48 and 15 levels in the ocean, this amounts to 40 960 time-averaged observations from the atmosphere and (restricting to wet points) 61 942 from the ocean, totalling 102 902. Now, in the most favourable case of a linear model, we can expect a solution of this type of inverse problem to take as many iterations as there are components in the control vector (Powell, 1964). Since for an ESM one can usually only afford an iteration number of a few tens or hundreds, we can only expect setups with low dimensional control vectors to converge. For the setups with high-dimensional control vector, that is, I3D, we will only perform 20 iterations. In this context, we call an experiment successful, if reductions of the cost function and of the Euclidean distance of the control vector to the default values are achieved at the same time. In an apparently underdeter- mined setup such as I3D (with 125 430 control variables constrained by 102 902 observations) the improvement of the control vector is of particular importance. For Cobs of eq. (1), off-diagonal elements where set to zero, that is, we assume uncorrelated uncertainty. The diagonal elements are the squares of the following standard deviations: In the atmosphere we use 3 K for temperature, 5 hPa for surface pressure, and 2105 1/s for vorticity. Our ocean uncertainties vary in space with standard deviations of 0.43.1 K for temperature and 0.150.8 PSU for salinity with the higher values towards the surface. 2.3. Data sets for assimilation These numbers are a rough guess of the actual uncertainties and effectively determine the relative weight given to the individual observation. Within the iterative minimisation, the trajectory of the control vector through the control space is highly depen- dent on the initial parameter vector. This means that two minimisations starting from neighbouring control vectors typically explore quite different regions in control space even if they converge to the same minimum. For an example, see Fig. 4 in Clerici et al. (2010). To assess the robustness of our experimental results, we carry out each experiment as a small ensemble with four members, each of which starts from a different point in control space. For the identical twin experiments with the I4 control vector the first member starts with the following perturbations of the control vector: in atmosphere and ocean 0.1 K for temperature, 1 per mil of the atmospheric surface pressure, 0.1 PSU for salinity. For the other members, the same magnitudes are used, but with varied signs. For example in the ‘min w ocean’ configuration this uniform initial TESTING VARIATIONAL ESTIMATION For the identical twin experiments with short control vectors, that is, P10, I2, I4 as defined in Table 2, we call an experiment successful if we can accurately (Euclidean distance to default reduced by at least five orders of magnitude) recover the default parameter values through assimilation of the data, with a strongly (by more than five orders of magnitude) reduced gradient of the cost function. a single derivative code for all coupled model components, including the coupler. No further hand-coding is required, that is, for the reasons given, safer, more flexible and sustainable. In the context of this study, it allowed us to generate derivative code versions for a variety of com- binations of model configurations, control vectors, and observational data sets. TESTING VARIATIONAL ESTIMATION An ensemble size of four is low, but appears to be sufficient for a first assessment. state perturbation yields, after a 26 d integration, a per- turbation of about 1.5 K in the lowest atmospheric layer (standard deviation), with maximum values of 18 K and 10 hPa. For the P10 control vector, a 10% perturbation of each component is used. The I3D control vector uses a globally uniform perturbation of the same magnitude as in the I4 case, but sign and magnitude are varied to generate the other ensemble members. An ensemble size of four is low, but appears to be sufficient for a first assessment. the atmosphere (configuration ‘min w ocean’, Exp. 3). The simplification of the atmosphere can be regarded as a step towards the setup of the Kaminski et al. (2007) study, where the atmosphere is even reduced to its dynamical core. To approach the 100-d assimilation window of the Kaminski et al. study, we test a configuration ‘slow w/o ocean’ which removes the ocean, simplifies the atmosphere, and reduces its time step. The reduced time step is motivated by the study of Zhu and Kamachi (2000), who report stability problems for the linearisation of certain numerical time integration schemes. The reduced time step can thus be regarded as a way to render the cost function more regular. The config- uration ‘slow w/o ocean’ works robustly for an assimilation window of 56 d (Exp. 4, Fig. 3). 3. Experimental setup Our experiments are designed to verify the correctness of the derivatives and to identify potential problems under a variety of situations. They present the first steps towards an assimilation system in a coupled model environment. We will examine several combinations of model configura- tions and control vectors. For each of the model configurations, we generate a consistent snapshot of the model state (restart file) recorded at the end of a 10-yr integration. The restart file for the TESTING VARIATIONAL ESTIMATION TESTING VARIATIONAL ESTIMATION 1 std w ocean P10 soft ID-twin 1 1 2 std w/o ocean P10 soft ID-twin 1 0 3 min w ocean P10 soft ID-twin 1 2 4 slow w/o ocean P10 soft ID-twin 56 4 5 slow w/o ocean P10 soft ERA-40 1 4 6 std w ocean I4 soft ID-twin 1 4 7 std w ocean I4 hard ID-twin 1 3 8 std w ocean I4 soft ID-twin 3 0 9 std w/o ocean I2 soft ID-twin 3 0 10 min w ocean I4 soft ID-twin 26 3 11 min w ocean I4 hard ID-twin 26 3 12 slow w ocean I4 soft ID-twin 26 0 13 std w ocean I3D hard ID-twin 1 3 14 min w ocean I3D hard ID-twin 1 4 15 min w ocean I3D hard ID-twin 26 3 16 std w ocean I3D hard ID-twin 26 0 17 min w ocean I3D soft ID-twin 26 3 Exp. No. Configuration Ctrl. Smoothness Observations Ass. Wdw. (d) Succ. Mbr. 1 std w ocean P10 soft ID-twin 1 1 2 std w/o ocean P10 soft ID-twin 1 0 3 min w ocean P10 soft ID-twin 1 2 4 slow w/o ocean P10 soft ID-twin 56 4 5 slow w/o ocean P10 soft ERA-40 1 4 6 std w ocean I4 soft ID-twin 1 4 7 std w ocean I4 hard ID-twin 1 3 8 std w ocean I4 soft ID-twin 3 0 9 std w/o ocean I2 soft ID-twin 3 0 10 min w ocean I4 soft ID-twin 26 3 11 min w ocean I4 hard ID-twin 26 3 12 slow w ocean I4 soft ID-twin 26 0 13 std w ocean I3D hard ID-twin 1 3 14 min w ocean I3D hard ID-twin 1 4 15 min w ocean I3D hard ID-twin 26 3 16 std w ocean I3D hard ID-twin 26 0 17 min w ocean I3D soft ID-twin 26 3 state perturbation yields, after a 26 d integration, a per- turbation of about 1.5 K in the lowest atmospheric layer (standard deviation), with maximum values of 18 K and 10 hPa. For the P10 control vector, a 10% perturbation of each component is used. The I3D control vector uses a globally uniform perturbation of the same magnitude as in the I4 case, but sign and magnitude are varied to generate the other ensemble members. TESTING VARIATIONAL ESTIMATION 1 std w ocean P10 soft ID-twin 1 1 2 std w/o ocean P10 soft ID-twin 1 0 3 min w ocean P10 soft ID-twin 1 2 4 slow w/o ocean P10 soft ID-twin 56 4 5 slow w/o ocean P10 soft ERA-40 1 4 6 std w ocean I4 soft ID-twin 1 4 7 std w ocean I4 hard ID-twin 1 3 8 std w ocean I4 soft ID-twin 3 0 9 std w/o ocean I2 soft ID-twin 3 0 10 min w ocean I4 soft ID-twin 26 3 11 min w ocean I4 hard ID-twin 26 3 12 slow w ocean I4 soft ID-twin 26 0 13 std w ocean I3D hard ID-twin 1 3 14 min w ocean I3D hard ID-twin 1 4 15 min w ocean I3D hard ID-twin 26 3 16 std w ocean I3D hard ID-twin 26 0 17 min w ocean I3D soft ID-twin 26 3 state perturbation yields, after a 26 d integration, a per- turbation of about 1.5 K in the lowest atmospheric layer (standard deviation), with maximum values of 18 K and 10 hPa. For the P10 control vector, a 10% perturbation of each component is used. The I3D control vector uses a globally uniform perturbation of the same magnitude as in the I4 case, but sign and magnitude are varied to generate the other ensemble members. An ensemble size of four is low, but appears to be sufficient for a first assessment. 4. Results First we address model parameter estimation that is the the atmosphere (configuration ‘min w ocean’, Exp. 3). The simplification of the atmosphere can be regarded as a step towards the setup of the Kaminski et al. (2007) study, where the atmosphere is even reduced to its dynamical core. To approach the 100-d assimilation window of the Kaminski et al. study, we test a configuration ‘slow w/o ocean’ which removes the ocean, simplifies the atmosphere, and reduces its time step. The reduced time step is motivated by the study of Zhu and Kamachi (2000), who report stability problems for the linearisation of certain numerical time integration schemes. The reduced time step can thus be regarded as a way to render the cost function more regular. The config- uration ‘slow w/o ocean’ works robustly for an assimilation Exp. No. Configuration Ctrl. Smoothness Observations Ass. Wdw. (d) Succ. Mbr. TESTING VARIATIONAL ESTIMATION 7 Table 3. Experiments. Column 1 indicates the experiment number, column 2 the conﬁguration from the list in Table 1, column 3 the control vector from the list in Table 2, column 4 the level of smoothing applied to the atmospheric component (see Section 2.1), column 5 the observational data set (see Section 2.3), column 6 the length of the assimilation window, column 7 the number of successful members out of our four member ensemble Table 3. Experiments. Column 1 indicates the experiment number, column 2 the conﬁguration from the list in Table 1, column 3 the control vector from the list in Table 2, column 4 the level of smoothing applied to the atmospheric component (see Section 2.1), column 5 the observational data set (see Section 2.3), column 6 the length of the assimilation window, column 7 the number of successful members out of our four member ensemble state perturbation yields, after a 26 d integration, a per- turbation of about 1.5 K in the lowest atmospheric layer (standard deviation), with maximum values of 18 K and 10 hPa. For the P10 control vector, a 10% perturbation of each component is used. The I3D control vector uses a globally uniform perturbation of the same magnitude as in the I4 case, but sign and magnitude are varied to generate the other ensemble members. An ensemble size of four is low, but appears to be sufficient for a first assessment. 4. Results First we address model parameter estimation, that is, the control vector P10, in a set of identical twin experiments. Table 3 summarises our experimental results. Exp. 1 shows that in the most complex configuration ‘std w ocean’, we cannot even reliably recover our parameter vector over a 1-d assimilation window. Three out of four ensemble members fail to find a minimum. The minimisations get stuck at edges in the cost function. This is because, with a given minimal step size along a local downhill direction that is pointing towards an upward jump, the optimisation algorithm cannot achieve any further decrease of the cost function. Figure 2 illustrates this situation, where the stopping point is on the right hand side of the jump, and except for the jump point the cost function has ascending slope, that is, the downhill direction points towards the left. In our model such jumps can typically be traced back to if-statements in the convective precipitation. TESTING VARIATIONAL ESTIMATION Removing the ocean does not help (Exp. 2). What helps is the simplification of the atmosphere (configuration ‘min w ocean’, Exp. 3). The simplification of the atmosphere can be regarded as a step towards the setup of the Kaminski et al. (2007) study, where the atmosphere is even reduced to its dynamical core. To approach the 100-d assimilation window of the Kaminski et al. study, we test a configuration ‘slow w/o ocean’ which removes the ocean, simplifies the atmosphere, and reduces its time step. The reduced time step is motivated by the study of Zhu and Kamachi (2000), who report stability problems for the linearisation of certain numerical time integration schemes. The reduced time step can thus be regarded as a way to render the cost function more regular. The config- uration ‘slow w/o ocean’ works robustly for an assimilation window of 56 d (Exp. 4, Fig. 3). We note that repeating, as a test, Exp. 1 with a prior contribution still yields one successful member while repeat- ing Exp. 3 with a prior term does increase the number of successful members from two to four. This behaviour confirms our expectation from Fig. 1: While a prior term cannot avoid step functions (probably induced by atmo- spheric processes in the ‘std’ configuration) it can help to avoid secondary minima in the smoother atmospheric ‘min’ configuration. Exp. 5 tests the Exp. 4 setup with ERA data instead of pseudo data. Over an assimilation window of 1 d all four members converge to very proximate minima. Figure 4 shows the convergence for one of the members, which reduces the gradient norm by more than five orders of magnitude. The value of the cost function is reduced by 1%, and the parameter vector is considerably changed. 5 the observational data set (see Section 2.3), column 6 the length of the assimilation window, column 7 the number of successful members out of our four member ensemble Exp. No. Configuration Ctrl. Smoothness Observations Ass. Wdw. (d) Succ. Mbr. 4. Results First we address model parameter estimation, that is, the control vector P10, in a set of identical twin experiments. Table 3 summarises our experimental results. Exp. 1 shows that in the most complex configuration ‘std w ocean’, we cannot even reliably recover our parameter vector over a 1-d assimilation window. Three out of four ensemble members fail to find a minimum. The minimisations get stuck at edges in the cost function. This is because, with a given minimal step size along a local downhill direction that is pointing towards an upward jump, the optimisation algorithm cannot achieve any further decrease of the cost function. Figure 2 illustrates this situation, where the stopping point is on the right hand side of the jump, and except for the jump point the cost function has ascending slope, that is, the downhill direction points towards the left. In our model such jumps can typically be traced back to if-statements in the convective precipitation. Removing the ocean does not help (Exp. 2). What helps is the simplification of We note that repeating, as a test, Exp. 1 with a prior contribution still yields one successful member while repeat- ing Exp. 3 with a prior term does increase the number of successful members from two to four. This behaviour confirms our expectation from Fig. 1: While a prior term cannot avoid step functions (probably induced by atmo- spheric processes in the ‘std’ configuration) it can help to avoid secondary minima in the smoother atmospheric ‘min’ configuration. Exp. 5 tests the Exp. 4 setup with ERA data instead of pseudo data. Over an assimilation window of 1 d all four members converge to very proximate minima. Figure 4 shows the convergence for one of the members, which reduces the gradient norm by more than five orders of magnitude. The value of the cost function is reduced by 1%, and the parameter vector is considerably changed. S. BLESSING ET AL. 8 0.02105 0.0211 0.02115 0.0212 0.02125 0.0213 0.02135 0.0214 –1e–07 –8e–08 –6e–08 –4e–08 –2e–08 0e+00 cost function section of control space Fig. 2. Cost function over a section of control space at the stopping point of one of the unsuccessful members of Exp. 1. Except for the x-value of the jump, the curve has a very small positive derivative (about 0.041 left, and 0.033 right of the jump), that is, an ascending slope. 4. Results The units of the x-axis are relative to the stopping point. 0.02105 0.0211 0.02115 0.0212 0.02125 0.0213 0.02135 0.0214 –1e–07 –8e–08 –6e–08 –4e–08 –2e–08 0e+00 cost function section of control space Cost function over a section of control space at the stopping point of one of the unsuccessful members of Exp 1 Excep Fig. 2. Cost function over a section of control space at the stopping point of one of the unsuccessful members of Exp. 1. Except for the x-value of the jump, the curve has a very small positive derivative (about 0.041 left, and 0.033 right of the jump), that is, an ascending slope. The units of the x-axis are relative to the stopping point. The procedure has apparently found a minimum which is not necessarily a global one, but it is also possible that the model in the minimal configuration just cannot match the ERA-data any better. We note that the value of x2, that is, twice that of the cost function at the mini- mum (Tarantola, 2005) of about 7414 is about a factor of 5.5 smaller than expected when assimilating 40 960 (:5.57417) observations (see Section 2.3) to estimate 10 unknown parameters (see Table 2) without the use of prior information. One could fix this by scaling down our data uncertainties by a factor of ﬃﬃﬃﬃﬃﬃﬃ 5:5 p (see e.g. Me´ nard and Chang, 2000). We do not do this here because in the 1e–10 1e–08 1e–06 0.0001 0.01 1 100 10000 0 10 20 30 40 50 60 iteration # \|\|gradient\|\| cost func. \|par 1\| \|par 2\| \|par 3\| \|par 4\| \|par 5\| \|par 6\| \|par 7\| \|par 8\| \|par 9\| \|par 10\| Fig. 3. Convergence of the minimisation for control vector P10, conﬁguration ‘slow w/o ocean’, assimilation of pseudo observations, and a 56-d assimilation window (Exp. 4): Cost function (solid red, ‘’), norm of its gradient (green dashed, ‘’), and absolute difference of components of control vector to true value over iteration number (par 110, see legend). 1e–10 1e–08 1e–06 0.0001 0.01 1 100 10000 0 10 20 30 40 50 60 iteration # \|\|gradient\|\| cost func. \|par 1\| \|par 2\| \|par 3\| \|par 4\| \|par 5\| \|par 6\| \|par 7\| \|par 8\| \|par 9\| \|par 10\| Fig. 3. Convergence of the minimisation for control vector P10, conﬁguration ‘slow w/o ocean’, assimilation of pseudo observations, and a 56-d assimilation window (Exp. 4. Results 4): Cost function (solid red, ‘’), norm of its gradient (green dashed, ‘’), and absolute difference of components of control vector to true value over iteration number (par 110, see legend). TESTING VARIATIONAL ESTIMATION 9 1e–04 1e–03 1e–02 1e–01 1e+00 1e+01 1e+02 \|\|gradient\|\| 3700 3710 3720 3730 3740 3750 cost func. 0 1 2 3 4 5 6 0 50 100 150 200 250 iteration # \|\|parameter\|\| Fig. 4. Convergence of the minimisation for control vector P10, conﬁguration ‘slow w/o ocean’, assimilation of ERA observations, and a 1-d assimilation window (Exp. 5): Norm of its gradient (top), cost function (centre), and absolute difference of the components of the control vector to the default value (labelled ‘true’ value in ID-twin experiments, bottom) over iteration number. Fig. 4. Convergence of the minimisation for control vector P10, conﬁguration ‘slow w/o ocean’, assimilation of ERA observations, and a 1-d assimilation window (Exp. 5): Norm of its gradient (top), cost function (centre), and absolute difference of the components of the control vector to the default value (labelled ‘true’ value in ID-twin experiments, bottom) over iteration number. For a 3-d assimilation window, the assimilation does not work anymore (Exp. 8). Removing the ocean (Exp. 9) does not help. We can, however, achieve considerable extensions of the assimilation window if we simplify the atmospheric component. Configuration ‘min w ocean’ is mostly success- ful for an assimilation window of 26 d with (Exp. 10) and without (Exp. 11, Fig. 6) soft switches. Interestingly, reduc- ing the time step deteriorates the estimation of initial con- ditions (Exp. 12), even though it improved the estimation For a 3-d assimilation window, the assimilation does not work anymore (Exp. 8). Removing the ocean (Exp. 9) does not help. We can, however, achieve considerable extensions of the assimilation window if we simplify the atmospheric component. Configuration ‘min w ocean’ is mostly success- ful for an assimilation window of 26 d with (Exp. 10) and without (Exp. 11, Fig. 6) soft switches. Interestingly, reduc- ing the time step deteriorates the estimation of initial con- ditions (Exp. 12), even though it improved the estimation absence of a prior term a uniform scalar has no impact on the minimisation. Figure 5 shows that the estimated parameter vector achieves a slight improvement of the predictive skill beyond the assimilation window. Next, we present experiments where we estimate initial conditions (control vectors I2, I4, and I3D). 4. Results We start with the most complex configuration, ‘std w ocean’, and the I4 control vector, which works for assimilation windows of 1 d with (Exp. 6) and mostly without (Exp. 7) soft switches. 1.4 1.6 1.8 2 2.2 2.4 2.6 0 0.5 1 1.5 2 RMS-error of atmospheric temperatures [K] integration time [d] prior-data post-data Fig. 5. RMS of temperature difference during and after assimilation window for Exp. 5. 1.4 1.6 1.8 2 2.2 2.4 2.6 0 0.5 1 1.5 2 RMS-error of atmospheric temperatures [K] integration time [d] prior-data post-data Fig. 5. RMS of temperature difference during and after assimilation window for Exp. 5. 10 S. BLESSING ET AL. 1e–06 1e–04 1e–02 1e+00 1e+02 1e+04 \|\|gradient\|\| 1e–20 1e–15 1e–10 1e–05 1e+00 1e+05 cost func. 1e–12 1e–10 1e–08 1e–06 1e–04 1e–02 1e+00 0 5 10 15 20 iteration # \|par 1\| \|par 2\| \|par 3\| \|par 4\| Fig. 6. As Fig. 4 but for convergence of the minimisation for control vector I4, conﬁguration ‘min w ocean’, assimilation of pseudo observations, and a 26-d assimilation window (Exp. 11). Fig. 6. As Fig. 4 but for convergence of the minimisation for control vector I4, conﬁguration ‘min w ocean’, assimilation of pseudo observations, and a 26-d assimilation window (Exp. 11). of parameters. Fig. 7 shows the cost function over a section of the control space from the true value to the first guess of the first member of Exp. 12. At this large scale the cost function looks smooth. Note also the high curvature (expressed as the second derivative) of the cost function at the minimum of about 100 000, compared to a curva- ture of the prior term (not used in the experiment) of 1. This indicates a strong constraint by the observations. The discontinuities which hamper the minimisation are of the type shown in Fig. 2 and only visible at much finer scales. As mentioned, we use the low dimensional control vectors because they have the potential to converge within an affordable number of iterations. For initialisation of climate predictions, however, we want to correct the 3D structure of the initial field. Our final set of experiments 0 200 400 600 800 1000 0e+00 5e–02 1e–01 2e–01 2e–01 2e–01 cost function section of control space obs. term ﬁtted parabola 0 200 400 600 800 1000 0e+00 5e–02 1e–01 2e–01 2e–01 2e–01 cost function section of control space obs. TESTING VARIATIONAL ESTIMATION TESTING VARIATIONAL ESTIMATION 11 ocean model operate on longer time scales than those in the atmospheric model. In particular, switching off fast atmo- spheric processes such as precipitation increases the feasible assimilation window. The non-linear behaviour of these processes is aggravated by their numerical implementation, which often incorporates non-differentiable statements. A step function, for example, produces discontinuities in the cost function, which may provide an obstacle for gradient-based minimisation. In this study, the replacement of some of these formulations by differentiable approxima- tions in the soft experiments has been limited to just a few parts in PlaSim. Hence, we expect future studies to reveal the full potential that lies in the reformulation of such processes in a differentiable way, possibly using statistical concepts as demonstrated by (Knorr et al., 2010) and (Kaminski et al., 2012). tests this type of control vector (I3D) and limits the number of iterations of our gradient algorithm to 20. Recall that in this context we call an experiment successful, if reductions of the cost function and of the Euclidean distance of the control vector to the default values are achieved at the same time. Over 1 d, our most complex configuration ‘std w ocean’ (Exp. 13) has three successful ensemble members (9, 13, and 14% reduction of norm of parameter difference to truth and cost function reductions of 32, 20, and 19%), while one member got stuck without reduction in the norm of the parameter difference to truth nor in the cost function. By contrast, over the same assimilation window in configuration ‘min w ocean’ (Exp. 14) all four ensemble members are successful, with 11, 12, 15, and 15% reduction of norm of parameter difference to truth and cost function reductions of 34, 20, 30, and 20%. In this latter configura- tion, three ensemble members were also successful for an assimilation window of 26 d (Exp. 15, Fig. 8) with 2, 5, and 10% reduction of norm of parameter difference to truth and cost function reductions of 10, 17, and 20%, while the same assimilation fails for the configuration ‘std w ocean’ (Exp. 16). Running the configuration ‘min w ocean’ with soft switches (Exp. 17) again yields three successful ensemble members with comparable results (parameter vector: 5, 7, and 10% reduction; cost function: 25, 14, and 28% reduction). The size of the time-step in dynamical models is typically a trade-off between performance and simulation quality. TESTING VARIATIONAL ESTIMATION A long time-step results in a fast model integration, while a short time step typically reduces discretisation error and thus enhances the quality of the simulation. Exceeding a certain threshold (imposed by the CFL criterion) even results in an unstable integration. In our experiments, a reduction of the atmospheric time step improves the performance for parameter estimation (Exp. 4) but dete- riorates it for the estimation of the initial state (Exp. 12). An obvious qualitative difference between parameter and initial state estimation is that model parameters directly influence the cost function at each time-step throughout the integration, while the influence of the initial state is indirect, because it has to be propagated from time step to time step through the integration of the dynamical system. 4. Results term ﬁtted parabola Fig. 7. Cost function over a section of control space from the true value (origin) to the ﬁrst guess (marked with vertical line) of the ﬁrst of four (unsuccessful) members of Exp. 12 (control vector I4, ‘slow w ocean’; solid line, ‘’) and a parabola ﬁtted at the known minimum (dashed line; second deriv. is about 100 000). Fig. 7. Cost function over a section of control space from the true value (origin) to the ﬁrst guess (marked with vertical line) of the ﬁrst of four (unsuccessful) members of Exp. 12 (control vector I4, ‘slow w ocean’; solid line, ‘’) and a parabola ﬁtted at the known minimum (dashed line; second deriv. is about 100 000). 5. Discussion Running the uncoupled atmospheric model does not yield results superior to the coupled one, as we see in Exp. 2/1 and Exp. 9/8. This reflects the fact that the processes in the 1e+01 1e+02 1e+03 1e+04 \|\|gradient\|\| 4.5e+02 5.0e+02 5.5e+02 6.0e+02 cost func. 38 39 40 41 42 43 0 5 10 15 20 iteration # \|\|parameter\|\| Fig. 8. As Fig. 4 but for convergence of the minimisation for control vector I3D, conﬁguration ‘min w ocean’, assimilation of pseudo observations, and a 26-d assimilation window (Exp. 15). Fig. 8. As Fig. 4 but for convergence of the minimisation for control vector I3D, conﬁguration ‘min w ocean’, assimilation of pseudo observations, and a 26-d assimilation window (Exp. 15). S. BLESSING ET AL. 12 This propagation has the potential to dampen or compli- cate the structure of the sensitivity of the cost function to an initial state change. Our experiments may show the balance of two mechanisms with opposite effect. On one hand a reduced time step enhances stability of the line- arised model (Zhu and Kamachi, 2000), but on the other hand it increases the number of time steps required to cover a given assimilation window. It may be that the first mech- anism is dominant for parameter estimation while the second mechanism is dominant for the estimation of initial conditions. To confirm these findings further research is required, including theoretical studies with simple models. This propagation has the potential to dampen or compli- cate the structure of the sensitivity of the cost function to an initial state change. Our experiments may show the balance of two mechanisms with opposite effect. On one hand a reduced time step enhances stability of the line- arised model (Zhu and Kamachi, 2000), but on the other hand it increases the number of time steps required to cover a given assimilation window. It may be that the first mech- anism is dominant for parameter estimation while the second mechanism is dominant for the estimation of initial conditions. To confirm these findings further research is required, including theoretical studies with simple models. (eq. 1) in our experiments (except for a demonstration) in order to make a clearer assessment of the constraints on the coupled model provided by the observational term. Through its parabolic contribution to the cost function, a prior term would have stabilised the inverse problem. 5. Discussion This would have clearly facilitated the minimisation and possibly would have masked convergence problems im- posed by the observational term. In that respect we can regard our assessment as conservative. We find that the performance of the coupled model in the assimilation system is highly dependent on the selec- tion of atmospheric processes and their implementation. A reduced atmospheric configuration with a number of processes deactivated shows significantly better perfor- mance than the standard configuration, while inclusion or exclusion of the dynamical ocean component has only a minor effect. Reducing the atmospheric time-step helps the estimation of process parameters but complicates the estimation of initial conditions. We note that the absolute performance of the system is likely to change with the resolution of the model. For example, we would expect a degraded performance for enhanced resolution of the ocean or atmosphere component or both. Nevertheless, the above findings should hold over a range of resolutions, because the responsible mechanisms are resolution-independent. Assimilation of ERA-data in Exp. 5 certainly is more challenging than the identical twin experiments. Even though we used an assimilation procedure to prepare the initial state for the experiment, it is obvious from the RMS error growth in Fig. 5 that the model trajectory quickly diverges from the data. Still it is possible to improve this situation slightly by the parameter estimation. The difficulty lies in a combination of four factors: First, the atmospheric configuration is simplified. Second, the model resolution is coarse compared to the data source. Third, despite the pre-assimilation procedure the initial state is still sub-optimal. Fourth, the P10 control space is small. We can eliminate the first two of these factors by repeating the same procedure with the ERA data replaced by observations generated by the model from initial conditions of a different year. In this case the cost function reduction is stronger by a factor of more than 10. This indicates the potential for a more realistic model and higher resolution. The performance in the reduced configuration is much better when estimating parameters by assimilating pseudo data generated by the model itself instead of by assimilating ERA data, in spite of careful preparation of the initial conditions. Part of this difference may be attributed to the coarse resolution and a too large degree of simplification in the reduced configuration with a limited number of control variables. 5. Discussion More work is required to improve the balance between realistic process representations and good perfor- mance in the assimilation system. The efficient handling of longer control vectors was demonstrated in the present study. Another perspective to further extend the feasible assimilation window is the combined use of a reduced and full configuration in a variational assimilation system, where the reduced configuration is used to provide an approximate gradient. 8. Appendix Given the limited assimilation window, we do not expect a strong detrimental effect for an assimilation. However, simulations including atmospheric moisture require a closed hydrological cycle, for example, rain, which is currently implemented in a form far from smooth. (3) The PlaSim does its time stepping in spectral space and has to deal with spurious negative moisture stem- ming from the Fourier-transform. The original model uses a redistribution algorithm which fills up negative moisture at affected grid cells, taking it from a certain domain. Out of vertical column containing the affected grid cell, latitude band, and global domain, it chooses the smallest domain that contains enough moisture. Switching this off had a positive effect on the smoothness of the cost function at the expense of formal moisture conservation. Given the limited assimilation window, we do not expect a strong detrimental effect for an assimilation. However, simulations including atmospheric moisture require a closed hydrological cycle, for example, rain, which is currently implemented in a form far from smooth. (3) The PlaSim does its time stepping in spectral space and has to deal with spurious negative moisture stem- ming from the Fourier-transform. The original model uses a redistribution algorithm which fills up negative moisture at affected grid cells, taking it from a certain domain. Out of vertical column containing the affected grid cell, latitude band, and global domain, it chooses the smallest domain that contains enough moisture. Switching this off had a positive effect on the smoothness of the cost function at the expense of formal moisture conservation. Given the limited assimilation window, we do not expect a strong detrimental effect for an assimilation. However, simulations including atmospheric moisture require a closed hydrological cycle, for example, rain, which is currently implemented in a form far from smooth. Fraedrich, K., Jansen, H., Kirk, E., Luksch, U. and Lunkeit, F. 2005a. The planet simulator: towards a user friendly model. Meteorol. Z. 14, 299304. Fraedrich, K., Jansen, H., Kirk, E. and Lunkeit, F. 2005b. The planet simulator: green planet and desert world. Meteorol. Z. 14(3), 305314. Fraedrich, K., Kirk, E., Luksch, U. and Lunkeit, F. 2005c. The portable university model of the atmosphere (PUMA): storm track dynamics and low frequency variability. Meteorol. Z. 14, 735745. Galanti, E., Tziperman, E., Harrison, M., Rosati, A. and Sirkes, Z. 2003. A study of ENSO prediction using a hybrid coupled model and the adjoint method for data assimilation. Mon. TESTING VARIATIONAL ESTIMATION 13 (4) In the PlaSim some of the min, max, and abs statements were replaced by smooth approximations. (4) In the PlaSim some of the min, max, and abs statements were replaced by smooth approximations. References Abarbanel, H., Kostuk, M. and Whartenby, W. 2010. Data assimilation with regularized nonlinear instabilities. Q. J. Roy. Meteorol. Soc. 136(648), 769783. DOI: 10.1002/qj.600. Blessing, S. 2000. Development and applications of an adjoint GCM. Master’s Thesis University of Hamburg Germany Abarbanel, H., Kostuk, M. and Whartenby, W. 2010. Data assimilation with regularized nonlinear instabilities. Q. J. Roy. Meteorol. Soc. 136(648), 769783. DOI: 10.1002/qj.600. Meteorol. Soc. 136(648), 769783. DOI: 10.1002/qj.600. Blessing, S. 2000. Development and applications of an adjoint GCM. Blessing, S. 2000. Development and applications of an adjoint GCM. Master’s Thesis. University of Hamburg, Germany. Blessing, S., Fraedrich, K. and Lunkeit, F. 2004, Climate diagnostics by adjoint modelling: a feasibility study. In: The KIHZ Project: Towards a Synthesis of Holocene Proxy Data and Climate Models (eds. H. Fischer, T. Kumke, G. Lohmann, G. Flo¨ ser, H., H. von Storch and co-authors). Springer, Heidelberg, pp. 383396. Blessing, S., Fraedrich, K. and Lunkeit, F. 2004, Climate diagnostics by adjoint modelling: a feasibility study. In: The KIHZ Project: Towards a Synthesis of Holocene Proxy Data and Climate Models (eds. H. Fischer, T. Kumke, G. Lohmann, G. Flo¨ ser, H., 7. Acknowledgements This work was supported by the European Community within the 6th Framework Programme for Research and Technological Development under contract no. 212643 (THOR) to Hamburg University and supported in part also through the DFG funded CLISAP excellence initiative. The presented research is building on work on an assimila- tion system developed around only PlaSim that was funded by NERC through its QUEST programme. M. Scholze contributed through that phase and acknowledges support through a CLISAP fellowship. P. Herrmann was supported through a Max-Planck Fellow Grant to D. Stammer. The authors wish to thank E. Kirk for his advice throughout this study and valuable comments on the manuscript. This work was supported by the European Community within the 6th Framework Programme for Research and Technological Development under contract no. 212643 (THOR) to Hamburg University and supported in part also through the DFG funded CLISAP excellence initiative. We note that the smoothing effect of the above modifica- tions is limited, because, unlike Knorr et al. (2010), in this initial study we refrained from redevelopment of entire process representations (such as convection) in smooth form. The presented research is building on work on an assimila- tion system developed around only PlaSim that was funded by NERC through its QUEST programme. M. Scholze contributed through that phase and acknowledges support through a CLISAP fellowship. P. Herrmann was supported through a Max-Planck Fellow Grant to D. Stammer. The authors wish to thank E. Kirk for his advice throughout this study and valuable comments on the manuscript. 6. Summary and conclusions We demonstrated a coupled model differentiation approach that applies, for the first time, automatic differentiation to an entire ESM at once. The generated derivative code is efficient and easy to maintain and to adapt to changes in the ESM code. No hand-coding is required at the deriva- tive level. We further constructed a variational assimilation system around the ESM and demonstrated the assimilation of pseudo observations as well as of an atmospheric data set based on ERA, and addressed estimation of process parameters and initial conditions. For both applications, using pseudo and ERA data, we quantify the performance of the assimilation system by the length of the feasible assimilation window. The study presented a first step towards a flexible variational assimilation system for initialisation of predic- tions and calibration of the ESM against observations. The system was demonstrated in an idealised set up. Obvious next steps are an increase of spatio-temporal resolution, extension/improvement of the process representations in a differentiable form, and the simultaneous use of observa- tions of the entire climate system. A further obvious appli- cation is sensitivity studies based on the tangent and adjoint ESM. The TAF compliance of the system assures fast updates after any modification of the ESM code. The focus of this study lies on the behaviour of the adjoint ESM in a standard assimilation environment, rather than on the construction of a sophisticated assimilation system, for example, with split in inner and outer optimisation loops (see e.g. Rabier et al., 2000). We also refrained from using a prior (or background) term in the cost function 8. Appendix Based on a set of initial tests with PlaSim, we identified several spots in the process implementations that produced a non-smooth shape in the cost function. For these spots we modified the implementation. Even though these modifica- tions are specific to the model at hand, it is instructive to give a few examples. Blessing, S., Greatbatch, R., Fraedrich, K. and Lunkeit, F. 2008. Interpreting the atmospheric circulation trend during the last half of the twentieth century: application of an adjoint model. J. Clim. 21, 46294646. 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https://openalex.org/W3134736764	https://figshare.com/articles/journal_contribution/A_state-dependent_damping_method_to_reduce_collision_force_and_its_variability/23480681/2/files/41216415.pdf	English	null	A State-Dependent Damping Method to Reduce Collision Force and Its Variability	IEEE robotics & automation letters	2,021	cc-by	9,444	Published in IEEE Robotics and Automation Letters Link to external publisher version https://doi.org/10.1109/LRA.2021.3062307 Licence Licence This work is made available under the CC BY 4.0 licence and should only be used in accordance with that licence. For more information on the specific terms, consult the repository record for this item. Document Version Published version Citation for this work (American Psychological Association 7th edition) Hamid, E., Herzig, N., Abad Guaman, S. A., & Nanayakkara, T. (2021). A state-dependent damping method to reduce collision force and its variability (Version 2). University of Sussex. https://hdl.handle.net/10779/uos.23480681.v2 Citation for this work (American Psychological Association 7th edition) Hamid, E., Herzig, N., Abad Guaman, S. A., & Nanayakkara, T. (2021). A state-de reduce collision force and its variability (Version 2). University of Sussex. https://hdl.handle.net/10779/uos.23480681.v2 A state-dependent damping method to reduce collision force and its variability Elham Hamid, Nicolas Herzig, Sara Adela Abad Guaman, Thrishantha Nanayakkara A State-Dependent Damping Method to Reduce Collision Force and Its Variability E. Hamid1, N. Herzig2, S-A. Abad3, and T. Nanayakkara4, Senior Member, IEEE, [3]. Furthermore, it is known that the knee joint not only manages to decrease the magnitude but also the variability of the force as well as the motion variability doing a similar task at a similar ground condition [4]. However, it is a complex computational problem to adjust the internal impedance of a robotic joint to decrease the variability of one quantity without compromising that of another quantity [5]. Abstract—This paper investigates the effect of biologically inspired angle-dependent damping proﬁle in a robotic joint primarily on the magnitude and the variability of the peak collision force. Joints such as the knee that experience collision forces are known to have an angle-dependent damping proﬁle. In this paper, we have quantiﬁed and compared three damping proﬁles. Our numerical and experimental results show that the proposed hyperbolic angle-dependent damping proﬁle can minimize both the magnitude and the variability of the peak collision force (average magnitude and variability reduction of ≈26% and ≈47% compared to the peak constant damping proﬁle). Very often, the variability of the force across the collision between the robot and the environment cause uncertainty about the state variables of the robotic joint. We show that by increasing the slope of the proposed hyperbolic angle-dependent damping proﬁle we can also reduce the variability and the magnitude of post-collision peak displacement and peak velocity compared to those of constant damping proﬁle. This was achieved while reducing the root mean square of power consumed by the robotic joint. The dynamics of systems that involve periodic impulsive collisions with the environment cause sudden jumps in the state-space, which varies at each step on the same terrain [6], [7]. Previous research on a simple passive dynamic walker known as the rimless wheel has revealed that the distribution of the coefﬁcient of restitution and the coefﬁcient of friction and their interactions play a signiﬁcant role in the state variability [8]. Therefore, any solution that would reduce the variability of states upon collisions will help to reduce the chances of failure. The complexity in robot-environment interaction that in- volves dynamics that are punctuated by collisions is a major challenge for closed-loop control systems. This paper was recommended for publication by Editor Clement Gosselin upon evaluation of the Associate Editor and Reviewers’ comments. This work was supported in part by the Engineering and Physical Sciences Research Council (EPSRC) MOTION grant (EP/N03211X/2 and EP/N03208X/1), and EPSRC RoboPatient grant (EP/T00603X/1). Copyright and reuse: This work was downloaded from Sussex Research Open (SRO). This document is made available in line with publisher policy and may differ from the published version. Please cite the published version where possible. Copyright and all moral rights to the version of the paper presented here belong to the individual author(s) and/or other copyright owners unless otherwise stated. For more information on this work, SRO or to report an issue, you can contact the repository administrators at sro@sussex.ac.uk. Discover more of the University’s research at https://sussex.figshare.com/ This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/LRA.2021.3062307, IEEE Robotics and Automation Letters This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/LRA.2021.3062307, I and Automation Letters n accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation inform and Automation Letters 1 IEEE ROBOTICS AND AUTOMATION LETTERS. PREPRINT VERSION. FEBRUARY, 2021 A State-Dependent Damping Method to Reduce Collision Force and Its Variability Several studies have investigated fast collision detection and reactive behaviour mechanisms [9]–[11] or different numerical methods of pre- dicting the consequence of variability on the stability [12], [13] or search for impedance parameters that can reduce the variability [14]. However, in the studies mentioned above, intensive computational algorithms for real-time control were used. Index Terms—Actuation and Joint Mechanisms, Compliance and Impedance Control 1Elham Hamid is with Centre for Robotics Research, Department of Informatics, King’s College London, London WC2R 2LS, UK. elham.hamid@kcl.ac.uk 2 Nicolas Herzig is with the School of Engineering and Informatics, University of Sussex, Brighton, BN1 9QT, UK. nh366@sussex.ac.uk 3 Sara-Adela Abad is with the Department of Mechanical Engineering, UCL, London, WC1E 7JE, UK; and with the Institute for Applied Sustainabil- ity Research, Av. Granados E13-55 e Isla Marchena, No.44, Quito, 170503, Ecuador. s.abad-guaman@ucl.ac.uk Manuscript received: 10, 13, 2020; Revised 1, 10, 2021; Accepted 2, 6, 2021. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ 2 Nicolas Herzig is with the School of Engineering and Informatics, University of Sussex, Brighton, BN1 9QT, UK. nh366@sussex.ac.uk 3 3 Sara-Adela Abad is with the Department of Mechanical Engineering, UCL, London, WC1E 7JE, UK; and with the Institute for Applied Sustainabil- ity Research, Av. Granados E13-55 e Isla Marchena, No.44, Quito, 170503, Ecuador. s.abad-guaman@ucl.ac.uk I. INTRODUCTION FEBRUARY, 2021 2 2 Small Rack Aluminum Plate Force Sensor Environment Motor Robotic Joint Motor 𝑚! 𝑐!+ 𝑐"! 𝑘! 𝑚# 𝑘# 𝑐#+ 𝑐"# 𝜏 ! 𝑙$ Rack Pinion Pinion Linear bearing Linear bearing Fig. 1. Experimental setup: The force sensor measures the collision force at the point of contact between the robot and the aluminium plate representing the environment. To replicate the impact between a robot with the environ- ment, the motor representing the environment was set to move the aluminum plate sinusoidally. The angle of rotation of the robotic joint motor, represents the robot joint angle. Measurements Line fitting Hyperbolic shape Fig. 2. The damping proﬁle of the knee joint against the knee joint angle. This proﬁle is based on the viscosity values and their corresponding joint angles, provided in [17], at a given constant torque value. Small Rack Aluminum Plate Force Sensor Environment Motor Robotic Joint Motor 𝑚! 𝑐!+ 𝑐"! 𝑘! 𝑚# 𝑘# 𝑐#+ 𝑐"# 𝜏 ! 𝑙$ Rack Pinion Pinion Linear bearing Linear bearing Linear bearing Linear bearing Aluminum Plate Fig. 2. The damping proﬁle of the knee joint against the knee joint angle. This proﬁle is based on the viscosity values and their corresponding joint angles, provided in [17], at a given constant torque value. a controller. We do not intend to exactly mimic the biology of the knee joint, but we intend to take inspiration from its control over the impedance properties. We approximate the angle-dependent damping proﬁle found by Zhang et al. [17] to a hyperbolic function of the joint angle (ch(θr)) given by: Fig. 1. Experimental setup: The force sensor measures the collision force at the point of contact between the robot and the aluminium plate representing the environment. To replicate the impact between a robot with the environ- ment, the motor representing the environment was set to move the aluminum plate sinusoidally. The angle of rotation of the robotic joint motor, represents the robot joint angle. ch(θr) = c0 + (cmax −c0)(1 −e−η\|θr\|) (1) (1) where θr is the angle of the robotic joint. Zhang et al. [17] also showed that at the relaxed state and zero degree ﬂexion, the knee joint damping coefﬁcient is not zero. Saying that c0 = 0.2 Ns/rad represents the minimum damping coefﬁcient of a robotic joint. cmax = 5 Ns/rad refers to the maximum damping coefﬁcient of the robotic joint. II. ANGLE-DEPENDENT DAMPING CONTROL Zhang et al. [17] found out that the internal impedance of a human’s knee joint is a nonlinear function of the joint angle. Furthermore, Morland et al. [19], showed that an angle-dependent damping control could provide better control in legged locomotion using a rimless wheel. However, a quantiﬁcation of the angle-depended damping proﬁle remains to be performed. In the contact phase of the feet with the environment, using the impedance control (controlling the stiffness, damping and inertia), the knee joint manages the impact during the strike, and it provides stability by absorbing and controlling the collision force [16]. By taking the viscosity values from [17] for a given constant torque value and plotting them against their joint angles, the damping proﬁle of the knee joint (shown in Fig. 2) was obtained. During the strike, the damping proﬁle can be approximated as a hyperbolic function because the knee joint angle varies in a small range between 10◦and 25◦on average [20]. Active impedance control for the knee joint is obtained by muscular control of co-contracting antagonistic muscle pairs which can control the joint torques in humans and animals [21]. There are two ways of taking inspiration from the knee joint, one is to design the cam proﬁle of the knee, and the other is to incorporate its properties into I. INTRODUCTION These parameters are set to be constants for both simulation and experiment. (cmax −c0) is to prevent the damping coefﬁcient of the joint exceeding the minimum and maximum damping coefﬁcients of the joint. (1 −e−η\|θr\|) provides hyperbolic shape of the damping. Where η is a scaling factor in the angle-dependent damping function determining its slope (η ∈[0, 0.5, 1, 1.5]). In other words, increasing η rises the damping coefﬁcient ch. The values are chosen based on the limitation of the experimental setup. Furthermore, since the proposed function is angle- dependent, a periodic damping proﬁle would be achieved for periodic angle variation. a robot. We provide analytical and experimental validations of this generic method. The results show that it minimised both the variability and magnitude of the peak collision force as well as that of the post-collision peak velocity and peak displacement without complicated computations. Interestingly, this was achieved while minimising the root mean square of power. To isolate the effect of the hyperbolic angle-dependent damping proﬁle on a robotic joint, we keep a constant propor- tional gain and conduct experiments in the horizontal plane to only study the normal force. I. INTRODUCTION M M INIMISING collision force and its variability is im- portant for many robotic applications such as legged locomotion and collaborative robots (cobots). There are several methods for collision avoidance [1], [2]. However, there are robotic applications where contact is part of the task, such as legged locomotion, object passing, physical examination, etc. In such tasks, predictability depends on both the collision force and its variability. It is believed that for most animals, one of the top priorities is to minimise the peak collision force to reduce the risk of injury and permanent joint damages Based on laws governing interaction dynamics, body in dynamic association with the environment should adjust its internal impedance to preserve a stable dynamic coupling with the environment [15]. The knee joint is an example that is vital for efﬁciency and stability of legged locomotion in human and animals [5], [16]. However, its variable impedance (stiffness, damping, and inertia) [17] and its computational role in walking remain to be understood. In this paper, we focus on implementing a simple active impedance control method that can better emulate the mor- phological impedance proﬁle of the knee joint to be imple- mented in any robotic joint and to provide efﬁcient and stable behaviour. Indeed angle-dependent stiffness is also important, however studying the combined effect of angle-dependent stiffness and angle-dependent damping is beyond the scope of this paper. Furthermore, it is known that the damping proﬁle can intrinsically stabilise the system [18]. This study focuses not only on the magnitude but also on the variability of the collision force, post-collision peak displacement and velocity. We propose a hyperbolic angle-dependent damping control framework that can be easily implemented in each motor of 1Elham Hamid is with Centre for Robotics Research, Department of Informatics, King’s College London, London WC2R 2LS, UK. elham.hamid@kcl.ac.uk 2 Nicolas Herzig is with the School of Engineering and Informatics, University of Sussex, Brighton, BN1 9QT, UK. nh366@sussex.ac.uk 3 4Thrishantha Nanayakkara is with Dyson School of Design Engineering, Imperial College London, London SW7 2DB, UK. t.nanayakkara@imperial.ac.uk 4Thrishantha Nanayakkara is with Dyson School of Design Engineering, Imperial College London, London SW7 2DB, UK. t.nanayakkara@imperial.ac.uk 4Thrishantha Nanayakkara is with Dyson School of Design Engineering, Imperial College London, London SW7 2DB, UK. t.nanayakkara@imperial.ac.uk Digital Object Identiﬁer (DOI): see top of this page. Digital Object Identiﬁer (DOI): see top of this page. IEEE ROBOTICS AND AUTOMATION LETTERS. PREPRINT VERSION. III. EXPERIMENTAL SETUP To represent the during collision phase and before and after collision phase of a robotic joint with the environment, we developed an experimental setup shown in Fig. 1. A PD controlled back-drivable MAXON motor (EC-max 40 mm, 120 W, brush-less, geared at 1 : 81 gearing ratio, motor part number 283873) was used to provide active impedance control of the robotic joint as a function of its angle θr. This motor was attached to an 8 cm diameter pinion. The proportional (Kp = 8) gain was set to be constant, and the derivative (Kd) gain was controlled as a function of θr. To represent the environment (any object that comes into contact with the robot), an aluminium plate attached to a small horizontal rack was set to be in contact with another 8 cm diameter pinion. The pinion was attached to another MAXON motor (EC 60 mm, 400 W, brush-less, part number 167131) with high PD gains (Kp = 30000 and Kd = 3772). Therefore, its position and velocity will not be affected when colliding article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/LRA.2021.3062307, IEEE Robotics and Automation Letters E.HAMID et al.: A STATE-DEPENDENT DAMPING METHOD TO REDUCE COLLISION FORCE AND ITS VARIABILITY 3 ed for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/LRA.2021.3062307, IEEE Robotics and Automation Letters E.HAMID et al.: A STATE-DEPENDENT DAMPING METHOD TO REDUCE COLLISION FORCE AND ITS VARIABILITY 3 with the robot. Each MAXON motor was connected to an EPOS 5/50. The collision force was measured by a 50 N Load Cell attached to the 3D printed part connected to the motor representing the robotic joint. where θr and ˙θr are the angular displacement and velocity of the motor representing the robotic joint. Parameter Je is deﬁned as Je = Igm + Ip + mer2. Where Iem = 8.01e−5 kgm2, Ip = 6.4e−4 kgm2 and me = 12.4 kg are moment of inertia of the motor representing the environment and the moment of inertia of the pinion, as well as the total mass that the motor representing the environment must move (the aluminium plate and the small rack attached to it). B. Before and After Collision Phase For before and after the collision where there is no contact between the robot and the environmental object, the physical system’s information matrix (Ai) is given by: Ai =   0 1 0 0 −kr Jr −ch(θr)−cir Jr 0 0 0 0 0 1 0 0 −ke Je −ce−cie Je   (6) III. EXPERIMENTAL SETUP r = 4 cm refers to the radius of motor’s pinion. Parameter Jr is deﬁned as Jr = Irm + Ip + mrr2. Where Irm = 1.01e−5 kgm2 refers to the moment of inertia of the motor representing the robotic joint. The inertia of the link of the robot in contact with the environment is negligible in comparison to the inertia of the environment. The total mass of the robotic joint’s motor has to move is mr = 8.2 kg. As explained in section II, ch(θr) represents the hyperbolic angle-dependent damping of the robotic joint controlled by (1). Parameter kr = 8 N/rad, is the stiffness coefﬁcient of the robotic joint motor. cir = 0.8 Ns/rad and cie = 0.4 Ns/rad are the coefﬁcient of friction/damping of the robotic joint motor and the environmental object motor. The torque applied to the robot during the normal collision between the robot and the environment, τc(θr) is given by: This paper intends to show the effect of the hyperbolic angle-dependent damping proﬁle on a robotic joint. Since the torque contributed by gravity also changes with the joint angle, for clarity, the gravity effect of the robot was removed. Furthermore, to only study the normal force at the point of contact, both motors were constrained to move horizontally. A. During Collision Phase (6) The model represents an elastic collision, perpendicular to the plane of impact, with a spring and a damper in parallel acting between the two objects that come into contact (robot and environment). Two phases were considered: when the robot and the environmental object are independent, and when they are coupled during impact. The conservation of momentum is applied to the two objects during the transition from one phase to another. When the robotic joint comes into contact with the environmental object, the system’s dynamics changes to a double-mass-spring-damper system. Then the equations of motion of the system at the contact phase can be used to ﬁnd the collision force. The A matrix for during collision phase (Ac) is given by: IV. ANALYTICAL MODEL In this work, the motor representing the environmental ob- ject follows a sinusoidal movement. As shown in the schematic design of the experimental setup (1), τs is the torque for the desired sinusoidal motion of the environmental object and is given by: τs = keθ∗ e + (ce + cie) ˙θ∗ e (2) (2) where ke = 90 N/rad, ce = 9 Ns/rad, θe and θ∗ e = l0 + A sin(2πft) are the constant stiffness, damping, and the actual and desired joint angle of the motor representing the environment. l0 = 4.7 rad, A = 4.97 rad, f = 1 Hz, and t s are the initial distance of the environmental object from the robot, amplitude, frequency and time. ˙θe and ˙θ∗e are the actual and desired velocity of the motor representing the environment. The aim of this research is to study the effect of a hyperbolic angle-dependent damping control of a robotic joint actuator on the collision force denoted Fc. Simulations were conducted for two phases of relative movement between the environmental object and the robot; before and after collision phase and during collision phase. The state space representation of the system is in the form of ˙x = Ax + Bτs. The state vector is described by: x = h θr ˙θr θe ˙θe iT and B = [0 0 0 1/Je]T . Where the A matrix that holds the physical system’s information for either during collision phase or before and after collision phase. τc(θr) = −(ch(θr) + cir) ( ˙θr −˙θe) −kr(θr −θe) (4) (4) where the collision force Fc applied to the robot at the point of contact is Fc = τc(θr)/r. Observing the torque coming from the PD controller of the robotic joint motor and angle of the robotic joint, we can compute the power P of the robotic joint regulating the impedance, P = τr(θr) ˙θr. Where τr(θr) is the torque coming from the PD controller of the robotic joint actuator given by: τr(θr) = (ch(θr) + cir) ˙θr + krθr (5) (5) This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ V. COEFFICIENT OF VARIATION ANALYSIS In robotic applications that involve dynamics that are punc- tuated by impulsive collisions with the environment, pre- dictability and consequently stability depends on minimizing both the magnitude and variability of the peak collision force as well as the state variability (displacement and velocity). Therefore, In this research when comparing our proposed method with other two constant damping proﬁles, the sum- mation of the coefﬁcient of variation of the collision force CvF , angular peak velocity Cvv and peak displacement Cvd of the robotic joint should be calculated and compared. Ac =   0 1 0 0 −kr Jr −ch(θr)−cir Jr kr Jr ch(θr)+cir Jr 0 0 0 1 kr Je ch(θr)+cir Je −kr−ke Je −ch(θr)−cir−ce−cie Je   (3) CvF = σFc µFc , Cvv = σ ˙θr µ ˙θr , Cvd = σθr µθr (7) Cvr = CvF + Cvv + Cvd (8) (7) (3) (8) This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/LRA.2021.3062307, IEEE Robotics and Automation Letters This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/LRA.2021.3062307, IEEE Robotic and Automation Letters IEEE ROBOTICS AND AUTOMATION LETTERS. PREPRINT VERSION. FEBRUARY, 2021 4 c! c" * * * * * * * * Fig. 3. An example of the damping proﬁle found from the experiment on the hyperbolic angle-dependent damping function for different values of η. cp is the maximum damping found at the maximum angle during the hyperbolic angle-dependent damping proﬁle experiment and cm is the damping value found at the middle of the hyperbolic angle-dependent damping proﬁle. c! c" * * * * * * * * For the third experiment, rather than looking for the max- imum damping values cp, the damping values were chosen at the middle of the hyperbolic angle-dependent damping proﬁle cm = cp+c0 2 . Therefore, for each value of η, the damping of the robotic joint PD controller was set to the constant damping values (cr = cm) shown in Table I. VI. EXPERIMENTAL PROCEDURE The ODE113 function of MATLAB was used to solve the differential equations (6 and 3) derived previously. Then, the simulated results were plotted. Fig. 4(a) presents the simulation results for the peak collision force. It can be observed that by implementing our proposed hyperbolic angle- dependent damping proﬁle ch no matter what the value of η the peak collision force will stay constant around its minimum value. However, for the other two constant damping proﬁles (cp and cm) not only the peak collision force values were higher but they had a rising trend when increasing the value of η. Three sets of experiments were carried out. The ﬁrst set of experiments was to test the effect of the proposed hyperbolic angle-dependent damping proﬁle on the behaviour of a robotic joint. Then to evaluate the efﬁciency of our proposed function, the results from the ﬁrst set of experiments were compared with two sets of different constant damping proﬁles (second and third set of experiments). During the ﬁrst set of experiments, the damping of the robotic joint PD controller was set to be the proposed hy- perbolic angle-dependent damping (cr = ch(θr)), found from (1). This was repeated for 10 trials, and the average of these 10 damping proﬁles was calculated. Fig. 3, presents a sample of a hyperbolic angle-dependent damping proﬁle for all values of η. In order to see the effect of increasing damping coefﬁcient on the magnitude and variability of the collision force as well as the state variables, this process was repeated for every value of η ∈[0, 0.5, 1, 1.5]. The post-collision peak velocity and displacement are shown in Fig. 4(b) and (c) respectively. It can be observed that for all three damping proﬁles, both the peak velocity and peak displacement decrease as the value of η increase. However, cm shows higher peak velocity and displacement than our pro- posed damping proﬁle ch and peak constant damping proﬁle cp. Another factor to compare the performance of our proposed damping proﬁle ch with the other two constant damping proﬁles cp and cm was to calculate the root mean square of the power that was consumed to control the robotic joint. Fig. 4(d), shows that the proposed hyperbolic angle-dependent ch provides lower root mean square of the power and cp the highest. VI. EXPERIMENTAL PROCEDURE Table II presents the percentage of reduction when comparing the proposed hyperbolic angle-dependent damping proﬁle ch with the medium constant damping proﬁle cm (α = ch−cm cm %); as well as comparing the proposed hyperbolic angle-dependent damping proﬁle ch with the peak constant damping proﬁle cp (β = ch−cp cp %). For the second experiment, the damping value reached at the maximum angle during each trial of the ﬁrst experiment were found. After that, the average of the 10 maximum damping values cp for each value of η was calculated and shown in Table I. Then for every value of η, the damping of the robotic joint PD controller was set to be these constant maximum damping values (cr = cp). These are the average of the applied damping values in the ﬁrst set of experiments that control the displacement where the robotic joint was at its maximum joint angle. The results from the hyperbolic angle-dependent damping proﬁle were compared with those for cp. Therefore, for each value of η, the corresponding constant damping value of cp was chosen and tested for 10 trials. With this experiment, we aim to compare the hyperbolic angle-dependent damping strategy with high constant damping strategy. V. COEFFICIENT OF VARIATION ANALYSIS In addition, it can be observed that cm and cp increases as higher value of η is chosen (Table I). Basically, these three sets of experiments aimed to see the effect of our proposed angle- dependent damping function on the behaviour of a robotic joint in comparison to other constant damping. Note that, the environment-robotic joint strike was modelled as an inelastic collision. Fig. 3. An example of the damping proﬁle found from the experiment on the hyperbolic angle-dependent damping function for different values of η. cp is the maximum damping found at the maximum angle during the hyperbolic angle-dependent damping proﬁle experiment and cm is the damping value found at the middle of the hyperbolic angle-dependent damping proﬁle. TABLE I CONSTANT DAMPING PARAMETERS FOR THE EXPERIMENT η cp(Ns/rad) cm(Ns/rad) 0 0.2 0.2 0.5 1 0.6 1 1.6 0.9 1.5 2.18 1.19 TABLE I CONSTANT DAMPING PARAMETERS FOR THE EXPERIMENT η cp(Ns/rad) cm(Ns/rad) 0 0.2 0.2 0.5 1 0.6 1 1.6 0.9 1.5 2.18 1.19 where, σ and µ represent the standard deviation and the average of the state variables respectively. The aim is to ﬁnd the damping proﬁle that gives the minimum coefﬁcient of variation of the robotic joint Cvr. argmin ∀(Cr)∈R [Cvr] (9) argmin ∀(Cr)∈R [Cvr] (9) (9) This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ TABLE II SIMULATION RESULTS: THE ANGLE-DEPENDENT DAMPING PROFILE VERSUS CONSTANT DAMPING PROFILES. THE NEGATIVE AND POSITIVE SIGNS REPRESENT THE REDUCTION AND INCREASE OF THE PARAMETERS RESPECTIVELY. Magnitude of Magnitude of Magnitude of RMS of Power Peak Collision Force Peak Velocity Peak Displacement η α% β% α% β% α% β% α% β% 0.5 -41.984 -58.819 -23.974 1.706 -5.466 -1.330 -7.150 -28.265 1 -55.961 -71.291 -28.165 3.201 -0.622 0.826 -7.475 -35.987 1.5 -62.609 -76.466 -33.773 1.094 -3.028 -0.652 -2.878 -36.754 cm and cp) when the value of η increases. The average peak displacement and peak velocity for cm were higher than the other two damping proﬁles. Interestingly, experiments show approximately equal average peak displacement for both ch and cp. The experimental results in Fig. 6(d) shows the root mean square of the power that was consumed by the robotic joint implementing different damping proﬁles. The root mean square of the power consumption for our proposed damping proﬁle ch was smaller than other constant damping proﬁles cm and ch. It can also be seen that the root mean square of the power consumption for both cm and ch are approximately equal at η = 1. Fig. 5. An example of the force sensor reading for ch at η = 0 and the corresponding relative angular velocity between the two actuators. Fig. 5. An example of the force sensor reading for ch at η = 0 and the corresponding relative angular velocity between the two actuators. Fig. 7 presents the variation of peak collision force, post- collision peak velocity, as well as the peak displacement. The variability was measured by calculating the standard deviation of the values. Fig. 7(a) shows the variation of peak collision force. The experimental results for the proposed damping proﬁle ch, for all values of η, showed a higher negative trend on the variability. However, a gradual rise was mainly seen for cm and cp, except for cm at η = 1. Fig. 7(b) presents the variability of the post-collision peak velocity. It can be observed that for every value of η, ch provides a smaller variability of the post-collision peak velocity compared to cm and cp. Fig. 7(c) presents the variability of the peak dis- placement for each value of η and all three damping proﬁles. It can be observed that the lowest (0.012 for 3 signiﬁcant decimal places) and highest variability (0.043) belong to the proposed damping proﬁle ch and the peak constant damping proﬁle cp respectively. In general, the standard deviation of the peak displacement is lower for our proposed damping proﬁle ch. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ TABLE II TABLE II SIMULATION RESULTS: THE ANGLE-DEPENDENT DAMPING PROFILE VERSUS CONSTANT DAMPING PROFILES. THE NEGATIVE AND POSITIVE SIGNS REPRESENT THE REDUCTION AND INCREASE OF THE PARAMETERS RESPECTIVELY. B. Experimental results Fig. 5 presents an example of the raw collision force data as well as the relative angular velocity between the robotic cle has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/LRA.2021.3062307, IEEE Robotics and Automation Letters has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citati and Automation Letters AMID et al.: A STATE-DEPENDENT DAMPING METHOD TO REDUCE COLLISION FORCE AND ITS VARIABILITY E.HAMID et al.: A STATE-DEPENDENT DAMPING METHOD TO REDUCE COLLISION FORCE AND ITS VARIABILITY 5 5 (a) (b) (c) (d) Fig. 4. Simulation results: (a) The peak collision force (b) the post-collision peak displacement, (c) the post-collision peak velocity, and (d) the root mean square of power for all three types of damping (ch refers to our proposed hyperbolic angle-dependent damping proﬁle, cp and cm refer to the maximum and medium constant damping proﬁles found from the ﬁrst set of experiments) for every value of η. Which η represent the slope of the damping proﬁle. (a) (c) (d) (d) (c) (d) Fig. 4. Simulation results: (a) The peak collision force (b) the post-collision peak displacement, (c) the post-collision peak velocity, and (d) the root mean square of power for all three types of damping (ch refers to our proposed hyperbolic angle-dependent damping proﬁle, cp and cm refer to the maximum and medium constant damping proﬁles found from the ﬁrst set of experiments) for every value of η. Which η represent the slope of the damping proﬁle. TABLE II SIMULATION RESULTS: THE ANGLE-DEPENDENT DAMPING PROFILE VERSUS CONSTANT DAMPING PROFILES. THE NEGATIVE AND POSITIVE SIGNS REPRESENT THE REDUCTION AND INCREASE OF THE PARAMETERS RESPECTIVELY. Table III presents the percentage of reduction of joint actuator and the environmental actuator. As it can be observed, the ﬁrst peak of collision force causes a rise in the relative angular velocity due to the compliance of the robot actuator. This is followed by force and velocity oscillations due to the PD controllers of both actuators involved in the collision. As seen in Fig. 6(a), the proposed hyperbolic angle- dependent damping proﬁle ch resulted in the smallest average peak collision forces across all values of η. In addition, it was found that increasing the damping coefﬁcient by increasing the values of η will not affect the peak collision force. Indeed, the peak collision forces were found to be similar ≈11 N. On the other hand, average peak collision force increased signiﬁcantly for cm and cp, ≈14 N and ≈16 N respectively, compared to that for ch. According to experimental results in Fig. 6(b) and (c), the peak displacement and post-collision peak velocity are decreasing for all three damping proﬁles (ch, joint actuator and the environmental actuator. As it can be observed, the ﬁrst peak of collision force causes a rise in the relative angular velocity due to the compliance of the robot actuator. This is followed by force and velocity oscillations due to the PD controllers of both actuators involved in the collision. As seen in Fig. 6(a), the proposed hyperbolic angle- dependent damping proﬁle ch resulted in the smallest average peak collision forces across all values of η. In addition, it was found that increasing the damping coefﬁcient by increasing the values of η will not affect the peak collision force. Indeed, the peak collision forces were found to be similar ≈11 N. On the other hand, average peak collision force increased signiﬁcantly for cm and cp, ≈14 N and ≈16 N respectively, compared to that for ch. According to experimental results in Fig. 6(b) and (c), the peak displacement and post-collision peak velocity are decreasing for all three damping proﬁles (ch, This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/LRA.2021.3062307, IEEE Robotics and Automation Letters IEEE ROBOTICS AND AUTOMATION LETTERS. PREPRINT VERSION. FEBRUARY, 2021 6 (a) (b) (c) (d) Fig. 6. TABLE III PERIMENTAL RESULTS: THE ANGLE-DEPENDENT DAMPING PROFILE VERSUS CONSTANT DAMPING PROFILES. Magnitude of Variability of Magnitude of Variability of Magnitude of Variability of RMS of Power Peak Collision Force Peak Collision Force Peak Displacement Peak Displacement Peak Velocity Peak Velocity η α% β% α% β% α% β% α% β% α% β% α% β% α% β% 0.5 -19.942 -22.768 -38.907 -23.202 13.083 -0.361 -34.002 -46.057 -5.167 -0.319 -19.360 -40.250 -14.273 -18.563 1 -23.092 -28.955 -35.085 -49.715 -14.899 0.945 -37.947 -52.440 -3.481 -0.974 -45.420 -53.591 0.779 -5.555 1.5 -22.984 -27.448 -63.776 -67.122 -16.064 1.395 -35.704 64.603 -6.012 -0.335 -10.109 -24.311 -7.953 -15.165 Fig. 8. Experimental results: The coefﬁcient of variation of the robotic joint Cvr for all three types of damping (ch, cp and cm) and for every value of η. proﬁles. It can be observed that our proposed hyperbolic angle- dependent damping proﬁle ch provides the lowest Coefﬁcient of variation in comparison to the other two constant damping proﬁles. Table IV present the percentage of reduction of the coefﬁcient of variation of the robotic joint calculated for all three damping proﬁles. TABLE II SIMULATION RESULTS: THE ANGLE-DEPENDENT DAMPING PROFILE VERSUS CONSTANT DAMPING PROFILES. THE NEGATIVE AND POSITIVE SIGNS REPRESENT THE REDUCTION AND INCREASE OF THE PARAMETERS RESPECTIVELY. Experimental results: (a) The average peak collision force (b) the average post-collision peak displacement, (c) the average post-collision peak velocity, and (d) the root mean square of power for all three types of damping (ch, cp and cm) for every value of η. Which η represent the slope of the damping proﬁle. The solid vertical lines indicate the error bar across the 10 trials for every value of η. (a) (b) (b) (c) (a) (d) (b) (c) (d) Fig. 6. Experimental results: (a) The average peak collision force (b) the average post-collision peak displacement, (c) the average post-collision peak velocity, and (d) the root mean square of power for all three types of damping (ch, cp and cm) for every value of η. Which η represent the slope of the damping proﬁle. The solid vertical lines indicate the error bar across the 10 trials for every value of η. (a) (b) (c) Fig. 7. Experimental results: The standard deviation of (a) the peak collision force, (b) the post-collision peak velocity, and (c) the post-collision peak displacement for all three types of damping (ch, cp and cm) for every value of η which represent the slope of the damping proﬁle. (b) (c) (a) (b) Fig. 7. Experimental results: The standard deviation of (a) the peak collision force, (b) the post-collision peak velocity, and (c) the post-collision peak displacement for all three types of damping (ch, cp and cm) for every value of η which represent the slope of the damping proﬁle. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ TABLE III TABLE III EXPERIMENTAL RESULTS: THE ANGLE-DEPENDENT DAMPING PROFILE VERSUS CONSTANT DAMPING PROFILES. Coefﬁcient of Variation of the Robotic Joint Cvr η α% β% 0.5 -22.238 -20.740 1 -22.334 -38.426 1.5 -39.395 -35.915 In contrast, we propose a simple approach that can be implemented in a control algorithm or a hardware mechanism of a robotic joint without any need for sensor reading for a closed-loop control, quick reaction or sophisticated numerical calculation or control policies for predictions. This approach minimises both the magnitude and variability of collision force as well as the above stated important factors underpinning predictability and show similar trends. The differences in exact magnitudes can come from i) Implementing high constant values of damping (cm and cp) at all times, especially at the time of the collision. However, in the case of hyperbolic angle-dependent damping (ch), the damping is around its minimum value (c0) at the time of the collision. ii) Unmodiﬁed sources of damping such as gear friction and the coefﬁcient of restitution, can cause the difference between the experimental and simulation magnitudes. iii) the response time of the motor representing the PD controller of the robotic joint. In general, it can be noted that a high constant damping proﬁle is not necessary to minimise the magnitude of the peak displacement and post- collision peak velocity of the robotic joint. As it was observed, high constant damping proﬁle would minimise the magnitude of peak displacement and velocity, but it maximises their variability as well as the magnitude and variability of the peak collision force and the root mean square of the power. Our proposed hyperbolic angle-dependent damping ch can achieve the same state reduction as the highest constant damping proﬁle, cp. In this study, we have also introduced a function that not only takes into account the magnitude but also the variability of peak collision force, peak post-collision velocity and displacement where the results were plotted in Fig. 8. The results conﬁrms that our proposed hyperbolic function provides the smallest values of the Cvr across all values of η. This results in similar changes in the variability of other state variables such as post-collision peak velocity, and peak displacement due to collision. In particular, the variability of states depends on the reduction of the magnitude of the peak collision force and its variability. High variability in state transitions (post-collision peak velocity and peak dis- placement) push the system to failure states [7]. In addition to the improvements mentioned above, smaller variability of the post-collision peak velocity was achieved, as illustrated in Fig. 7(b). VIII. DISCUSSION Two factors are vital for systems that involve punctuated dynamics due to collisions; decreasing the magnitude of the peak collision forces and their variability [7], [8]. Furthermore, decreasing the variability of the peak collision force increases predictability and consequently, the stability of the robotic joint. Fig. 8. Experimental results: The coefﬁcient of variation of the robotic joint Cvr for all three types of damping (ch, cp and cm) and for every value of η. magnitude and variability comparing the proposed hyperbolic angle-dependent damping proﬁle ch with the medium constant damping proﬁle cm (α%); as well as comparing the proposed hyperbolic angle-dependent damping proﬁle ch with the peak constant damping proﬁle cp (β%). Fig. 8 compare the coefﬁ- cient of variation of the robotic joint Cvr for all three damping In this work, we proposed a biologically inspired hyperbolic angle-dependent damping proﬁle that can be implemented in any robotic joint using a pure mechanical design, impedance control in a servo motor, or a mixed of the two. It was found that the proposed hyperbolic damping proﬁle can signiﬁcantly icle has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/LRA.2021.3062307, IEEE Robotics and Automation Letters ticle has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/LRA.2021.3062307, IEEE Robotics and Automation Letters This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/LRA.2021.3062307, IEEE Robotics and Automation Letters E.HAMID et al.: A STATE-DEPENDENT DAMPING METHOD TO REDUCE COLLISION FORCE AND ITS VARIABILITY 7 has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Cita and Automation Letters E.HAMID et al.: A STATE-DEPENDENT DAMPING METHOD TO REDUCE COLLISION FORCE AND ITS VARIAB 7 Coefﬁcient of Variation of the Robotic Joint Cvr η α% β% 0.5 -22.238 -20.740 1 -22.334 -38.426 1.5 -39.395 -35.915 Similarly, less variability was found for peak displacement (except for η = 1.5), shown in Fig. 7(c). This conﬁrms the effect of minimizing the peak collision force’s magnitude and variability on those of other state variables. A signiﬁcant reduction of the magnitude of the peak dis- placement for hyperbolic angle-dependent damping proﬁle ch and peak damping constant cp was observed (Fig. 4(b) and Fig. 6(b)); interestingly, their magnitudes were similar for all values of η. This means that our proposed damping proﬁle ch could minimise the peak displacement as much as the maximum damping proﬁle cp while minimising the magnitude and variability of the peak collision force and the root mean square of the power consumed by the robotic joint. This is due to the fact that both ch and cp provide similar high damping value at the peak displacement, which prevents the system from moving further. However, ch only applies high damping where the angle is high and this made our control system more efﬁcient by achieving smaller root mean square of power compared to cm and cp, Fig. 4(d) and Fig. 6(d). Note that this angle-dependent damping proﬁle was implemented based on the relative robotic joint angle from the inception of a collision. This can be implemented in a control algorithm in an industrial application using force or current sensor to detect a collision to consider the relative angle. In future work, we will study the impact of such an implementation on the control strategy efﬁciency. In case of larger collisions causing higher angle change, the safer method is to turn the motors off (fully compliant). There have been several studies trying to provide stabil- ity. One of the proposed methods is based on environment reaction force feedback control. However, this method raises challenges in terms of placing several sensors at the point of contact [10], [11] for the closed-loop control. Authors in [9] focused on applying a reaction force by searching policy for optimum gains for a feedback controller. Other methods involve predicting the consequence of variability on the sta- bility of the system [12], [13]. All the methods mentioned above make the system vulnerable to external perturbations as fast collision detection, numerical calculation, control and reactive behaviour mechanisms are needed. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ TABLE IV EXPERIMENTAL RESULTS: THE ANGLE-DEPENDENT DAMPING PROFILE VERSUS CONSTANT DAMPING PROFILES. reduce both the magnitude and the variability of the peak collision force. It is interesting to note that the collision force stayed around its minimum value irrespective of the value of η, the value of the damping coefﬁcient (Fig. 4(a) and Fig. 6(a)). It is suggesting that the exact rate of change of damping is not so critical as far as it starts low and rises with the joint angle. Consequently, the hyperbolic shape allows the spring to absorb the initial impact force without disturbance from the damper. The damping effect comes at a later stage after storing some collision energy in the spring. However, this is not the case for the other two constant damping proﬁles as they present a positive trend in the magnitude of the peak collision force as the value of η increases. Moreover, it can be noted that the proposed method can make the system more predictable across all values of η, Fig. 7(a). Therefore, a higher value of η (damping coefﬁcient) can be chosen to bring the robotic joint to the desired velocity and displacement value without increasing the peak collision force and its variability. Coefﬁcient of Variation of the Robotic Joint Cvr η α% β% 0.5 -22.238 -20.740 1 -22.334 -38.426 1.5 -39.395 -35.915 Coefﬁcient of Variation of the Robotic Joint Cvr η α% β% 0.5 -22.238 -20.740 1 -22.334 -38.426 1.5 -39.395 -35.915 show similar trends. The differences in exact magnitudes can come from i) Implementing high constant values of damping (cm and cp) at all times, especially at the time of the collision. However, in the case of hyperbolic angle-dependent damping (ch), the damping is around its minimum value (c0) at the time of the collision. ii) Unmodiﬁed sources of damping such as gear friction and the coefﬁcient of restitution, can cause the difference between the experimental and simulation magnitudes. iii) the response time of the motor representing the PD controller of the robotic joint. In general, it can be noted that a high constant damping proﬁle is not necessary to minimise the magnitude of the peak displacement and post- collision peak velocity of the robotic joint. As it was observed, high constant damping proﬁle would minimise the magnitude of peak displacement and velocity, but it maximises their variability as well as the magnitude and variability of the peak collision force and the root mean square of the power. Our proposed hyperbolic angle-dependent damping ch can achieve the same state reduction as the highest constant damping proﬁle, cp. In this study, we have also introduced a function that not only takes into account the magnitude but also the variability of peak collision force, peak post-collision velocity and displacement where the results were plotted in Fig. 8. The results conﬁrms that our proposed hyperbolic function provides the smallest values of the Cvr across all values of η. There have been several studies trying to provide stabil- ity. One of the proposed methods is based on environment reaction force feedback control. However, this method raises challenges in terms of placing several sensors at the point of contact [10], [11] for the closed-loop control. Authors in [9] focused on applying a reaction force by searching policy for optimum gains for a feedback controller. Other methods involve predicting the consequence of variability on the sta- bility of the system [12], [13]. All the methods mentioned above make the system vulnerable to external perturbations as fast collision detection, numerical calculation, control and reactive behaviour mechanisms are needed. IX. CONCLUSION [4] J. W. Smith, J. C. Christensen, R. L. Marcus, and P. C. LaStayo, “Muscle force and movement variability before and after total knee arthroplasty: A review,” World Journal of Orthopedics, vol. 5, no. 2, p. 69, 2014. This paper, for the ﬁrst time, provides experimental and analytical evidence for the ability of an angle-dependent hyperbolic damping proﬁle in a robotic joint to reduce the peak collision force and its variability. Bio inspiration was taken from the human knee joint that is known to have an angle-dependent impedance proﬁle. Three sets of experiments and simulations were conducted to compare the performance of the proposed hyperbolic angle-dependent damping proﬁle with two other constant damping proﬁles. The results provide important design guidelines for variable impedance joints in robots to improve stability by reducing the collision force as well as its variability in closed-loop control. [5] F. Bianchi, G. Bartoli, K. Shoar, M. R. A. Fernandez, V. Pereno, J. Zir- jakova, A. Jiang, and T. Nanayakkara, “Adaptive internal impedance control for stable walking on uncertain visco-elastic terrains,” in 2012 IEEE/RSJ International Conference on Intelligent Robots and Systems, pp. 2465–2470, IEEE, 2012. [6] M. Garcia, A. Chatterjee, A. Ruina, and M. Coleman, “The simplest walking model: stability, complexity, and scaling,” J Biomech Eng, 1998. [7] K. Byl and R. Tedrake, “Metastable walking on stochastically rough terrain,” Proceedings of robotics: science and systems IV, pp. 6490– 6495, 2008. [8] T. Nanayakkara, K. Byl, H. Liu, X. Song, and T. Villabona, “Dominant sources of variability in passive walking,” in 2012 IEEE International Conference on Robotics and Automation, pp. 1003–1010, IEEE, 2012. ¨ ¨ [9] Y. S¸ahin, F. M. Botsalı, M. Kalyoncu, M. Tinkir, ¨U. ¨Onen, N. Yılmaz, ¨O. K. Baykan, and A. C¸ akan, “Force feedback control of lower extremity exoskeleton assisting of load carrying human,” in Applied Mechanics and Materials, vol. 598, pp. 546–550, Trans Tech Publ, 2014. Table III shows that the proposed hyperbolic angle- dependent damping proﬁle ch achieves peak force reduction of ≈−22.006% compared to the medium constant damping pro- ﬁle cm and ≈−26.390% compared to peak constant damping proﬁle cp. The corresponding average variability reduction of the peak collision force was ≈−45.922% and ≈−46.680%. Also, the proposed damping proﬁle had led to a reduction of peak displacement and post-collision peak velocity, which was, at some extent, similar to when the maximum constant damping proﬁle cp was implemented. IX. CONCLUSION The proposed hyper- bolic angle-dependent damping proﬁle ch achieved peak post- collision displacement reduction of ≈−14.682% compared to the medium constant damping proﬁle cm and ≈−0.659% compared to peak constant damping proﬁle cp. The corre- sponding average variability reduction of peak post-collision displacement were ≈−35.884% and ≈−11.298%. [10] D. Owaki, L. Morikawa, and A. Ishiguro, “Listen to body’s message: Quadruped robot that fully exploits physical interaction between legs,” in 2012 IEEE/RSJ International Conference on Intelligent Robots and Systems, pp. 1950–1955, IEEE, 2012. [11] C. D. Remy, K. Bufﬁnton, and R. Siegwart, “Stability analysis of passive dynamic walking of quadrupeds,” The International Journal of Robotics Research, vol. 29, no. 9, pp. 1173–1185, 2010. [12] J. Schmitt, M. Garcia, R. Razo, P. Holmes, and R. J. Full, “Dynamics and stability of legged locomotion in the horizontal plane: a test case using insects,” Biological cybernetics, vol. 86, no. 5, pp. 343–353, 2002. [13] I. Wijesundera, M. N. Halgamuge, A. Nirmalathas, and T. Nanayakkara, “Predicting the mean ﬁrst passage time (mfpt) to reach any state for a passive dynamic walker with steady state variability,” PloS one, vol. 13, no. 11, p. e0207665, 2018. [14] V. Pereno, K. Shoar, G. Bartoli, F. Bianchi, and T. Nanayakkara, “Stable walking on variable visco-elastic terrains using meta-parameters for passive state migration,” in 2013 IEEE/RSJ International Conference on Intelligent Robots and Systems, pp. 3126–3131, IEEE, 2013. Table III also shows that the proposed hyperbolic angle- dependent damping proﬁle ch achieves average post-collision peak velocity reduction of ≈−4.886% compared to the medium constant damping proﬁle cm and ≈−0.542% com- pared to peak constant damping proﬁle cp. The corresponding average variability reduction of the post-collision peak velocity was ≈−24.963% and ≈−39.384%. Furthermore, Table III shows that the proposed hyperbolic angle-dependent damping proﬁle ch achieves a reduction of root mean square of power up to ≈−7.149% compared to the medium constant damp- ing proﬁle cm and ≈−13.094% compared to peak constant damping proﬁle cp. [15] N. Hogan, “Impedance control: An approach to manipulation: Part i- theory,” Journal of dynamic systems, measurement, and control, vol. 107, no. 1, pp. 1–7, 1984. pp [16] S. Masouros, A. Bull, and A. Amis, “i) biomechanics of the knee joint,” Orthopaedics and Trauma, vol. 24, pp. 84–91, 2010. [17] L.-Q. Zhang, G. Nuber, J. Butler, M. Bowen, and W. Z. Coefﬁcient of Variation of the Robotic Joint Cvr η α% β% 0.5 -22.238 -20.740 1 -22.334 -38.426 1.5 -39.395 -35.915 In contrast, we propose a simple approach that can be implemented in a control algorithm or a hardware mechanism of a robotic joint without any need for sensor reading for a closed-loop control, quick reaction or sophisticated numerical calculation or control policies for predictions. This approach minimises both the magnitude and variability of collision force as well as the above stated important factors underpinning predictability and stability. This method can be a good candidate for a multi-joint scenario because the angle-dependent damping will be im- plemented at a joint level. The movement of each joint due to collision will depend on the conﬁguration of the robot Furthermore, our proposed damping proﬁle ch and cp pro- vided similar magnitudes of the post-collision peak velocity; Fig. 4(c) and Fig. 6(c). The experimental and simulation results This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/LRA.2021.3062307, IEEE Robotics and Automation Letters This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/LRA.2021.3062307, IEEE Robotics and Automation Letters 8 IEEE ROBOTICS AND AUTOMATION LETTERS. PREPRINT VERSION. FEBRUARY, 2021 at the point of collision with an external object. The prove of stability has not been addressed in this paper. However, reasoning on the passivity of the system we can deduct that the system is stable. The proposed control algorithm is tested for a limited range of collisions. It would be interesting to test harder collisions in the future to ﬁnd the range that the proposed method is useful. REFERENCES [1] E. Ohashi, T. Aiko, T. Tsuji, H. Nishi, and K. Ohnishi, “Collision avoid- ance method of humanoid robot with arm force,” IEEE Transactions on Industrial Electronics, vol. 54, no. 3, pp. 1632–1641, 2007. [2] M. P. Polverini, A. M. Zanchettin, and P. Rocco, “Real-time collision avoidance in human-robot interaction based on kinetostatic safety ﬁeld,” in 2014 IEEE/RSJ International Conference on Intelligent Robots and Systems, pp. 4136–4141, IEEE, 2014. [3] Y. Blum, H. R. Vejdani, A. V. Birn-Jeffery, C. M. Hubicki, J. W. Hurst, and M. A. Daley, “Swing-leg trajectory of running guinea fowl suggests task-level priority of force regulation rather than disturbance rejection,” PloS one, vol. 9, no. 6, p. e100399, 2014. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ IX. CONCLUSION Rymer, “In vivo human knee joint dynamic properties as functions of muscle contraction and joint position,” Journal of biomechanics, vol. 31, no. 1, pp. 71–76, 1997. [18] A. Radulescu, M. Howard, D. J. Braun, and S. Vijayakumar, “Ex- ploiting variable physical damping in rapid movement tasks,” in 2012 IEEE/ASME International Conference on Advanced Intelligent Mecha- tronics (AIM), pp. 141–148, IEEE, 2012. [19] M. F. E. Morland, K. Althoefer, and T. Nanayakkara, “Novel method to form adaptive internal impedance proﬁles in walkers,” in 2015 37th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC), pp. 7764–7767, IEEE, 2015. The percentage of reduction of the coefﬁcient of varia- tion of the robotic joint Cvr value as seen in Table IV of up to ≈−27.989% and ≈−31.693% was achieved when comparing the proposed hyperbolic angle-dependent damping proﬁle ch with medium constant damping proﬁle cm and the maximum constant damping proﬁle cp respectively. Simulation results based on a spring-variable damper-mass model has conﬁrmed the experimental data. and Biology Society (EMBC), pp. 7764–7767, IEEE, 2015 [20] M. R. Shorten and D. S. Winslow, “Spectral analysis of impact shock during running,” Journal of Applied Biomechanics, vol. 8, no. 4, pp. 288– 304, 1992. [21] N. Hogan, “Adaptive control of mechanical impedance by co-activation of antagonist muscles,” IEEE Transactions on automatic control, vol. 29, no. 8, pp. 681–690, 1984.
https://openalex.org/W4382122858	https://revistaseletronicas.pucrs.br/index.php/teo/article/download/44037/27973	Portuguese	null	O aggiornamento da teologia matrimonial na Exortação Amoris Laetitia	Teocomunicação	2,023	cc-by	10,579	O aggiornamento da teologia matrimonial na Exortação Amoris Laetitia The aggiornamento of the matrimonial theology in the Exhortation Amoris Laetitia El aggiornamento de la teología matrimonial en la Exhortación Amoris Laetitia Resumo: A família e o matrimônio sempre foram preocupações para a Igreja. Diante da crise hodierna em que vive o matrimônio, busca-se responder como a teologia pode ajudar os casais cristãos a viverem a alegria do amor. Na doutrina matrimonial católica foi predominante a ênfase na procriação, mas, a partir do Concílio Vaticano II, a fi nalidade unitiva do matrimônio também foi valorizada. A Exortação pós-Sinodal Amoris Laetitia encontra-se em continuidade com a perspectiva do Vaticano II. Assim, neste trabalho, busca-se primeiro estudar o desenvolvimento da teologia matrimonial e o acento dado na procriação; depois, analisa-se a teologia do matrimônio presente na Amoris Laetitia, para, por fi m, à luz do documento, indicar caminhos para os cônjuges católicos descobrirem e viverem na alegria do amor. Jonas Emerim Velho1 id / 6 6 Jonas Emerim Velho1 orcid.org/0000-0002-9764-160X jonas.velho@yahoo.com Jonas Emerim Velho1 orcid.org/0000-0002-9764-160X jonas.velho@yahoo.com Recebido em: 20 nov. 2022. Aprovado em: 10 fev. 2023. Publicado em: 8 maio. 2023. Palavras-chave: matrimônio; Amoris Laetitia; alegria. Abstract: The family and marriage have always been concerns for the Church. Faced with the current crisis in which marriage lives, the aim is to answer how theology can help Christian couples to live the joy of love. In Catholic matrimonial doctrine, the emphasis on procreation was predominant, but since the Second Vatican Council, the unitive purpose of marriage is also valued. The Post-Synodal Exhortation Amoris Laetitia is in continuity with the perspective of Vatican II. Thus, in this work, we fi rst seek to study the development of matrimonial theology and the emphasis given to procreation; then, the theology of marriage present in Amoris Laetitia is analyzed, to fi nally, in the light of the document, indicate ways for catholics spouses to discover and live in the joy of love. Keywords: marriage; Amoris Laetitia; joy. Resumen: La familia y el matrimonio han sido siempre preocupaciones de la Iglesia. Ante la crisis actual que vive el matrimonio cristiano, buscamos respon- der cómo la teología puede ayudar a las parejas cristianas a vivir la alegría del amor. En la doctrina matrimonial católica predominaba el énfasis en la procrea- ción, pero desde el Concilio Vaticano II también se valora la fi nalidad unitiva del matrimonio. O aggiornamento da teologia matrimonial na Exortação Amoris Laetitia La Exhortación postsinodal Amoris Laetitia está en continuidad con la perspectiva del Concilio Vaticano II. Así, en este trabajo, buscamos primero estudiar el desarrollo de la teología matrimonial y el énfasis dado a la procre- ación; luego, se analiza la teología del matrimonio presente en Amoris Laetitia, para fi nalmente, a la luz del documento, indicar caminos para que los esposos cristianos descubran y vivan la alegría del amor. Palabras clave: matrimonio; Amoris Laetitia; felicidad. OPEN ACCESS OPEN ACCESS http://dx.doi.org/10.15448/0103-314X.2023.1.44037 Teocomunicação, Porto Alegre, v. 53, n. 1, p. 1-14, jan.-dez. 2023 e-ISSN: 1980-6736 \| ISSN-L: 0103-314X 1 Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brasil. Recebido em: 20 nov. 2022. Aprovado em: 10 fev. 2023. Publicado em: 8 maio. 2023. instituídos por Jesus Cristo, Verbo encarnado (DENZINGER; HÜNERMANN, 2007, n. 1801). Nesse sentido, Müller afirma: instituídos por Jesus Cristo, Verbo encarnado (DENZINGER; HÜNERMANN, 2007, n. 1801). Nesse sentido, Müller afirma: predominou na teologia matrimonial católica durante séculos, acentuando, no amor conjugal, a finalidade da procriação. Também uma visão mais jurídico-canônica prevaleceu sobre uma fundamentação mais bíblica e existencial. Con- tudo, a partir do aggiornamento proposto pelo Concílio Vaticano II, a teologia matrimonial passou a ser traduzida com linguagem mais histórica e concreta, em uma antropologia integral, na qual a finalidade unitiva do matrimônio está ao lado da procriativa. Por matrimônio cristão compreende-se uma comunhão de vida total, exclusiva e pessoal entre um homem batizado e mulher batizada, realizada em liberdade, na qual se reflete a aliança de Cristo com sua Igreja, e mediante a qual o matrimônio se torna sinal eficaz da comunicação da graça divina (MÜLLER, 2015, p. 531). Com dupla finalidade da união e da procriação, santifica homem e mulher tornando-os participan- tes da vida e do amor divinos. No entanto, sabe-se que nem sempre a compreensão dessa dupla finalidade foi tão clara, pois, durante séculos se entendeu a predominância do fim procriativo sobre o unitivo no matrimônio, o que teve con- sequências para o ensino da fé e da moral cristã. Para estudar o desenvolvimento da teologia do matrimônio, faz-se necessário passar por algumas etapas que marcam sua estrutura- ção: a tradição judaica no Antigo Testamento, a plenitude da revelação no Novo Testamento, a influência filosófica grega na antropologia cristã e a nova perspectiva mais personalista. Essa última influenciará o Concílio Vaticano II, que marca o início de um caminho de assimilação de uma antropologia integral na doutrina matrimonial católica. Com dupla finalidade da união e da procriação, santifica homem e mulher tornando-os participan- tes da vida e do amor divinos. No entanto, sabe-se que nem sempre a compreensão dessa dupla finalidade foi tão clara, pois, durante séculos se entendeu a predominância do fim procriativo sobre o unitivo no matrimônio, o que teve con- sequências para o ensino da fé e da moral cristã. O Papa Francisco na Exortação Amoris Laeti- tia, último documento pontifício sobre a família, afirma que a alegria do amor na família deve ser também o júbilo da Igreja (AL 1). O documento insere-se em uma lógica de misericórdia pastoral, onde, apesar de suas fragilidades, o casal cristão é chamado a descobrir e viver na alegria do amor. 1.1 No Antigo Testamento Em muitas culturas o casamento é um ato reli- gioso. Em geral, ele é entendido como realidade que lança pontes entre mundos diferentes, esta- belece ordem, serve ao bem do clã e por isso é celebrado culticamente. A hierogamia (casamento sagrado) era realizada em culto de união sexual com a divindade, mediante um representante, como sacerdotes ou mulheres do templo (NO- CKE, 2012, p. 326). “A associação da sexualidade ao sagrado foi um atributo assumido por muitas culturas, onde o corpo assume elementos que reverenciam o sagrado como instância reguladora da vida humana” (ALMEIDA; CASTRO; SANTOS, 2020, p. 567). Para isso, em primeiro lugar, estuda-se a cons- trução da teologia do matrimônio ao longo da história, com a ênfase dada na finalidade da procriação. Em segundo lugar, busca-se com- preender aspectos centrais na teologia matri- monial presente na Exortação Amoris Laetitia. E, em terceiro, à luz do documento, encontram-se caminhos para auxiliar os casais cristãos a vive- rem na alegria do amor, apesar de seus limites. instituídos por Jesus Cristo, Verbo encarnado (DENZINGER; HÜNERMANN, 2007, n. 1801). Nesse sentido, Müller afirma: Assim, procura-se, nesta pesquisa, analisar a teologia matrimonial presente na Exortação e as orientações que dela decorrem para os cônjuges cristãos viverem na alegria do amor. Para estudar o desenvolvimento da teologia do matrimônio, faz-se necessário passar por algumas etapas que marcam sua estrutura- ção: a tradição judaica no Antigo Testamento, a plenitude da revelação no Novo Testamento, a influência filosófica grega na antropologia cristã e a nova perspectiva mais personalista. Essa última influenciará o Concílio Vaticano II, que marca o início de um caminho de assimilação de uma antropologia integral na doutrina matrimonial católica. A realidade em que se vive o matrimônio na atualidade é marcada por divórcios, novas núp- cias, uma antropologia dualista e uma cultura do provisório que atinge também as relações humanas. Diante disso, constata-se a relevância do tema abordado, na tentativa de auxiliar a pas- toral matrimonial da Igreja Católica no acompa- nhamento de casais cristãos, que são chamados a viver a alegria do amor, mesmo em meio a situações variadas e complexas. Introdução A teologia do matrimônio cristão desenvolveu-se ao longo da histó- ria do cristianismo, com base na Revelação contida nas Escrituras e na compreensão antropológica. A perspectiva agostiniana do casamento Artigo está licenciado sob forma de uma licença Creative Commons Atribuição 4.0 Internacional. 1 Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brasil. 2/14 Teocomunicação, Porto Alegre, v. 53, n. 1, p. 1-14, jan.-dez. 2023 \| e-44037 instituídos por Jesus Cristo, Verbo encarnado (DENZINGER; HÜNERMANN, 2007, n. 1801). Nesse sentido, Müller afirma: instituídos por Jesus Cristo, Verbo encarnado (DENZINGER; HÜNERMANN, 2007, n. 1801). Nesse sentido, Müller afirma: 1 A teologia do matrimônio cristão ao longo da história O matrimônio é entendido, na doutrina cristã, como um dos sete sacramentos da nova aliança, Na tradição de Israel, no entanto, casar-se tem Jonas Emerim Velho O aggiornamento da teologia matrimonial na Exortação Amoris Laetitia 3/14 Jonas Emerim Velho O aggiornamento da teologia matrimonial na Exortação Amoris Laetitia 3/14 po (Gn 2, 24)” (MÜLLER, 2015, p. 533). A união do homem e da mulher ultrapassa os outros afetos humanos, pois o homem deve deixar seus pais para unir-se à sua mulher. “O casamento remete ao primeiro instante da criação, ele pertence à vocação do homem. Jesus não terá de instituí-lo, só o confirmará, sublinhando, com os mesmos termos, sua origem divina” (SESBOÜÉ, 2021, p. 54). O poema sacerdotal é caracterizado pela cria- ção do ser humano à imagem e semelhança de Deus (Gn 1, 26). “Homem e mulher, em sua comunhão pessoal, são os dons e as tarefas da fecundidade, da dominação da terra e da respon- sabilidade sobre o mundo. Essa comunhão en- contra-se sob a bênção de Deus e sob a palavra da promessa (Gn 1, 27s.)” (MÜLLER, 2015, p. 533). Em escritos mais recentes do Antigo Testa- mento, como o livro de Tobias, a bênção de Deus sobre o amor entre homem e mulher reflete-se em uma vida conjugal que busca glorificar a Deus (Tb 8, 4-9). po (Gn 2, 24)” (MÜLLER, 2015, p. 533). A união do homem e da mulher ultrapassa os outros afetos humanos, pois o homem deve deixar seus pais para unir-se à sua mulher. “O casamento remete ao primeiro instante da criação, ele pertence à vocação do homem. Jesus não terá de instituí-lo, só o confirmará, sublinhando, com os mesmos termos, sua origem divina” (SESBOÜÉ, 2021, p. 54). caráter profano. No Antigo Testamento encon- tram-se festas e costumes para celebrá-lo (Tb 8, 19s.), contudo, sem a sanção de um ato religioso. Adverte-se contra a prostituição cúltica (Dt 23, 18s.). Está em primeiro plano a preocupa- ção com a descendência, também se fala de carinho e erotismo (sobretudo no Cântico dos Cânticos, mas também em Gn 24, 67; 29, 20; 1Sm 18, 20. 28). A divisão do ser humano em dois sexos não é expressão de dicotomia cós- mica, e, sim, dádiva do Criador (Gn 1, 27; 5, 2), da mesma maneira como a elementar força de atração mútua (Gn 2, 21-34). 1 A teologia do matrimônio cristão ao longo da história Em contrapartida, o domínio unilateral do homem sobre a mulher é consequência do pecado na concepção javista (Gn 3, 16) (NOCKE, 2012, p. 326). O poema sacerdotal é caracterizado pela cria- ção do ser humano à imagem e semelhança de Deus (Gn 1, 26). “Homem e mulher, em sua comunhão pessoal, são os dons e as tarefas da fecundidade, da dominação da terra e da respon- sabilidade sobre o mundo. Essa comunhão en- contra-se sob a bênção de Deus e sob a palavra da promessa (Gn 1, 27s.)” (MÜLLER, 2015, p. 533). A literatura profética recorre ao matrimônio como imagem para descrever a relação de Deus com Israel, ressaltando a misericórdia do marido (Javé), em detrimento da ingratidão e infidelida- de da esposa (Israel). Exemplo disso é o profeta Oseias, “que por ordem de Deus, se casa com uma prostituta (Os 1, 2-9) e adúltera (Os 3, 1-5), para expor aos olhos de Israel sua infidelidade para com seu Deus, mas também para anunciar o amor francamente paradoxo de Deus” (NOCKE, 2012, p. 327). A riqueza antropológica do matri- mônio e as suas implicações na vida das pessoas (amor, intimidade, fidelidade e outros) são ima- gens expressivas de que os autores sagrados se serviram para conseguirem dizer de forma eficaz a aliança de amor de Deus para com o seu povo (SCHILLEBEECKX, 1969, p. 38, 39). Em escritos mais recentes do Antigo Testa- mento, como o livro de Tobias, a bênção de Deus sobre o amor entre homem e mulher reflete-se em uma vida conjugal que busca glorificar a Deus (Tb 8, 4-9). 1.2 No Novo Testamento À luz do evento Cristo, nota-se que o matrimô- nio está inserido no processo histórico-salvífico da redenção humana, e mostra-se novamente o propósito original do matrimônio, conforme o capítulo 19 do evangelho de Mateus, ao apre- sentar Jesus questionado sobre a possibilidade do divórcio. Conforme a lei mosaica em vigor, o marido poderia despedir sua esposa com uma carta de divórcio (Dt 24, 1-2). No entanto, Jesus condena essa prática sendo uma concessão de Moisés à “dureza do coração” dos homens, pois “no prin- cípio não era assim” (Mt 19, 8). Jesus retorna ao plano original de Deus a homem e mulher, antes do pecado, no qual a fidelidade incondicional faz parte da ordem criacional do matrimônio, que proíbe, em princípio, despedir um parceiro. “As comunidades neotestamentárias procuram viver de acordo com o apelo de Jesus e conciliá-lo simultaneamente com os problemas emergentes concretos” (NOCKE, 2012, p. 327). Porém, é no relato veterotestamentário da criação que o matrimônio é abordado em um horizonte mais amplo, da relação entre homem e mulher criada sem o pecado, e depois perturbada por ele. É o que se percebe nos poemas javista e sacerdotal da criação no livro do Gênesis. No poema javista da criação, mais antigo que o primeiro, destaca-se a relação pessoal e equiva- lente entre homem e mulher. Somente na mulher formada a partir de sua costela, Adão reconhece uma correspondente (Gn 2, 18). “O homem, que reconhece na mulher a natureza humana comum e a igualdade (“carne de minha carne”), deixa sua família de origem e une-se à sua mulher, de modo que ambos se tornam “uma só carne”, ou seja, uma comunhão de vida, de amor e de cor- No Novo Testamento, encontra-se uma teolo- gia paulina sobre uma noção conjugal baseada Teocomunicação, Porto Alegre, v. 53, n. 1, p. 1-14, jan.-dez. 2023 \| e-44037 4/14 na relação esponsal de Cristo-Igreja como forma de exortar os casais das novas comunidades cris- tãs a enfrentarem os problemas da vida familiar (PERES, 2020, p. 22). São Paulo, ao falar-nos do matrimônio, versa sobre o plano da relação de amor que Cristo nutre pela Igreja e que por ela se entregou na cruz. 1.3 Ênfase na procriação Os teólogos cristãos dos primeiros séculos tinham de caminhar entre delimitar a depra- vação sexual e a dificuldade com as correntes de pensamento dualista, que desprezavam o corpo e o prazer, considerando a matéria como realidade má, e somente a alma como realidade boa, como os estóicos e os maniqueus. Nesse contexto, “Os Padres da Igreja defendiam unani- memente a bondade natural do matrimônio e sua importância para a salvação e a vida na graça” (MÜLLER, 2015, p. 535). A Carta aos Efésios é evocada como funda- mento para o matrimônio cristão. As regras para a vida familiar colocadas pelo autor no capítulo 5, dizem que as mulheres devem se sujeitar aos maridos e, os maridos, devem amar suas mulheres. Para o amor dos maridos, se dá uma fundamentação teológica: a figura do amor de Cristo à Igreja: ele se “entregou por ela” (Ef 5, 25). Assim, o que é dito em Gn 2, 24 a respeito de homem e mulher (que “o homem se unirá à sua mulher e os dois serão uma só carne”) é agora remetido a Cristo e à Igreja. Os pensadores cristãos defendiam o matri- mônio como instituído por Deus e eticamente permitido, mas também se influenciaram pelo dualismo antropológico. Nesse contexto encon- tra-se Agostinho. “Exemplo típico é o pensamento de Agostinho. Pessoalmente experimentado e enojado por aventuras sexuais, depois marcado pelo maniqueísmo, por fim engajado na contro- vérsia com o monge cristão Pelágio e sua ênfase antimaniqueísta do livre-arbítrio” (NOCKE, 2012, p. 329). Exegetas católicos veem aqui mais do que uma mera comparação, e até mais do que somente uma relação entre protótipo e seguimento. O protótipo de Cristo não é apenas um exemplo que é seguido na imagem do matrimônio terre- no, e, sim, essa imagem, o matrimônio terreno e sua efetivação prática, também, é constituído, em sua essência, pelo modelo de Cristo. A imagem, o matrimônio terreno, recebe, assume e representa o protótipo. [...] No matrimônio terreno é preservada essencialmente a relação de Cristo com a Igreja. Por isso, seria possível interpretar o matrimônio sacramentalmente a partir de Ef 5, 21-33 (NOCKE, 2012, p. 328). Agostinho, sabendo que a Escritura afirma a bondade da união do homem e da mulher, e ao mesmo tempo influenciado pelo estoicismo e maniqueísmo, considera impossível a prática da sexualidade sem o pecado, isso devido ao pecado original e à concupiscência. 1.2 No Novo Testamento Neste desejo de entregar-se em vista do bem de quem se ama, no caso de Cristo pela humanidade pecadora, agora os casais são interpelados a viver essa mesma atitude na união de ambos e o façam “no Senhor” (1Cor7,39) (KASPER, 2014, p. 26). a que o próprio Cristo vive por sua esposa, a Igreja. O matrimônio natural só encontrará sua plena realização no sacramento, no qual participa do mistério de amor entre Cristo e a Igreja, do desígnio divino de salvação. Dizendo que ele é sacramento, “expressamos que ele é sinal eficaz do Mistério santo, ou seja, do desígnio do Pai” (SCOLA, 2003, p. 339). O matrimônio se torna “bom” por meio dos três “bens” (bona), que compensam as carências: fidelidade (fides), prole (proles) e sacramento (sacramentum). Fidelidade significa que não haja relações fora do matrimônio com alguém outro; prole, que a criança seja aceita com cari- nho e educada responsavelmente; sacramento, por fim, que não haja separação do matrimônio (NOCKE, 2012, p. 329). por fim, que não haja separação do matrimônio (NOCKE, 2012, p. 329). temporânea, de inspiração cristã católica, que enfatiza a pessoa e seu valor absoluto, bem como os laços de solidariedade entre pessoas. Segundo o pensador francês Emmanuel Mounier, Na perspectiva agostiniana, o matrimônio é compreendido com a finalidade de criar um espaço ordenado e delimitado para o “controle” da sexualidade humana, perversa em si mesma, devido ao pecado. “Agostinho associou a queda e o mal à condição frágil do corpo e da sexuali- dade. Logo, o prazer sexual, enquanto realidade indissociável do ato de amor, deveria apenas estar a serviço da procriação e não para a obtenção da intimidade” (ALMEIDA; CASTRO; SANTOS, 2020, p. 568). Somente nele, o desejo sexual atinge seu objetivo legítimo, a procriação da espécie humana. “O matrimônio proporciona, assim, o ordenamento do desejo sexual, da procriação e da estabilidade social” (FONTANA, 2018, p. 90). Além disso, na sua obra Dos bens conjugais e a santa virgindade onde confronta-se com o monge Joviniano, o bispo de Hipona defende a igual dignidade entre o matrimônio e o celibato, “Agostinho respondia afirmando que o celibato é superior ao matrimônio” (DICCIONARIO..., 2001, p. 871, tradução nossa).2 O personalismo consiste precisamente nisso: numa oposição ao individualismo. Enquanto este último mantém o homem centrado sobre si mesmo, a primeira preocupação do persona- lismo é descentrá-lo para colocá-lo nas largas perspectivas abertas pela pessoa (2004, p. 45). Nesse sentido, essa filosofia busca um equi- líbrio entre o coletivismo (que tende a ver na pessoa somente uma unidade numérica) e o individualismo (que tende a enfraquecer os laços de solidariedade entre as pessoas), afirmando a centralidade e a dignidade da pessoa humana. O personalismo quer dizer, com isso, que a pessoa é um absoluto em comparação com qualquer outra realidade material ou social e com qualquer outra pessoa humana. [...] Nenhuma outra pessoa, nenhuma outra co- letividade, nenhum organismo pode utilizá-la legitimamente como um meio (AMORIM, 2010, p. 78). Nessa corrente tem lugar central a pessoa e a sua realização em comunidade de pessoas. A Constituição Pastoral Gaudium et Spes (47-52) trata do matrimônio como preocupação para a Igreja na atualidade, e o apresenta com influência da antropologia personalista. 2 Do original: Agustín respondía afirmando que el celibato es superior al matrimonio. 1.3 Ênfase na procriação Assim, desenvolve sua teoria dos três bens do matrimônio, que terá grande predominância na teologia e moral matrimonial nos séculos seguintes. Nessa relação de amor entre homem e mulher, o autor da Carta aos Efésios chama de mistério profundo (mysterion/sacramentum) (5, 32). Nes- se sentido interpreta-se que no “amor do ho- mem e da mulher, através do qual o matrimônio existe, tem sua origem, por conseguinte, nesta autodoação de Jesus pela Igreja, representa-a simbolicamente e é interiormente plenificado por esta doação de Cristo (Ef 5, 21.33; 2Cor 11,2; Ap 19, 7)” (MÜLLER, 2015, p. 533). Assim, a forma do amor esponsal não poderá ser outra, senão Jonas Emerim Velho O aggiornamento da teologia matrimonial na Exortação Amoris Laetitia Jonas Emerim Velho 5/14 2 O matrimônio na Exortação Amoris Laetitia A Exortação Apostólica Amoris Laetitia, a mais longa da história da Igreja, traz presente conclu- sões do Sínodo dos Bispos sobre a família, de suas duas Assembleias Sinodais: a extraordinária, em outubro de 2014, e a ordinária, em outubro de 2015. Convém recordar que a Exortação não modifica a doutrina da Igreja sobre o matrimônio e a família, mas a interpreta com outra perspectiva. A pessoa, deste modo, era considerada como um ser inacabado, que tem a tarefa de ir cons- tituindo-se, o que acontece na relação com Deus, com os outros e com o mundo. [...] con- centrava-se mais no amor presente na relação entre os cônjuges, buscando captar as exigên- cias do amor na situação de amadurecimento em que as pessoas envolvidas se encontravam (ALMEIDA; CASTRO; SANTOS, 2020, p. 571). Francisco deixa claro que não quer em nada romper com a tradição nem modificar a doutri- na estabelecida sobre o matrimônio. [...] O que ele pretende é manter a mesma orientação, segundo o espírito do Concílio Vaticano II, em- bora ousando aprofundar mais nas tentativas de soluções pastorais do que os anteriores documentos papais sobre a família, dentro dos limites da doutrina católica (KASPER, 2019, p. 12). O magistério pontifício pós-conciliar conti- nuou preocupado no desenvolvimento de uma visão da família e do matrimônio em um âmbito personalista, embora ainda em uma linguagem jurídico-canônica. A Encíclica Humane Vitae, de Paulo VI, apesar de toda polêmica por seu tema controverso, aborda o amor conjugal com dois fins, unitivo e procriativo, como inseparáveis. No entanto, “A teologia desse documento foi considerada por alguns, como um retorno à compreensão pré-conciliar, onde a norma está acima do sujeito, e o acento à procriação em detrimento da finalidade também unitiva do matrimônio” (VELHO, 2021, p. 74). João Paulo II, na Exortação pós-sinodal Familiaris Consortio, na terceira parte, valoriza o tema da comunhão con- jugal, em continuidade com o Vaticano II. Porém, “o documento se inscreve na linha da Humanae Vitae, na medida em que continua adotando a lei natural como categoria de leitura da moral matrimonial” (PASSOS, 2018, p. 114). E a Encíclica Deus Caritas Est, de Bento XVI, valoriza o amor erótico, enaltecendo a beleza do amor conjugal no matrimônio. “O Papa Bento XVI retomou esta discussão em sua primeira encíclica Deus Caritas Est, apresentando o amor humano como eros e ágape” (DCE 7). personalista A doutrina católica matrimonial pré-conciliar, alicerçada no paradigma agostiniano, preocu- pa-se em evidenciar o caráter divino e eterno do pacto conjugal, e a vivência da sexualidade sendo ordenada em vista da prole. Contudo, “No século XIX, surgiu um ideal de matrimônio (o ideal romântico), que via o matrimônio de modo menos objetivo, sob o aspecto da procriação e da garantia econômica, e mais sob o aspecto ‘pessoal’, como união de amor” (NOCKE, 2012, p. 333). Assim, a teologia do Concílio Vaticano II trouxe, ou pelo menos introduziu, uma nova compreensão desse sacramento, a partir da perspectiva da antropologia personalista, que “estimulou uma compreensão mais pessoal do matrimônio” (MÜLLER, 2015, p. 539). Esta perspectiva mais personalista será as- sumida no Concílio, e de modo particular, na Constituição Pastoral Gaudium et Spes. Segundo esta, tanto a abertura à procriação quanto a exigência da plena fidelidade e indis- solubilidade do matrimônio, têm como raiz e fundamento a íntima união de vida e amor das pessoas dos cônjuges (n. 48). A união é, antes de tudo, uma realidade pessoal, um fato de amor. O ato conjugal, sem perder seu consti- tutivo sentido de orientação à transmissão da vida, é o contexto onde a linguagem desse mesmo amor é capaz de melhor explicitá-lo e aprofundá-lo (ALMEIDA; CASTRO; SANTOS, 2020, p. 571). O Vaticano II compreendeu o matrimônio como comunidade de vida e amor, assumida no pacto conjugal e expressa nos atos próprios dos cônjuges. Nesse sentido, os bens e fins do O personalismo é uma corrente filosófica con- 6/14 Teocomunicação, Porto Alegre, v. 53, n. 1, p. 1-14, jan.-dez. 2023 \| e-44037 Quanto mais os dois se encontram [...], tanto mais se realiza a verdadeira natureza do amor em geral” (LIMA, SANCHES, 2017, p. 542). matrimônio estão fundados em um encontro amoroso. “A categoria filosófica do personalismo foi fundamental para reintegrar toda a afetividade e os sentimentos num dinamismo de amor, con- duzidos por valores éticos” (ALMEIDA; CASTRO; SANTOS, 2020, p. 571). A influência personalista se faz perceber na noção de pessoa que funda- menta a renovação conciliar: 3 Do original: Che cosa sono per Papa Francesco il gaudium, la laetitia? Il termine “gioia” (nelle sue varie declinazioni: alegría, gozo…) è uno dei più ricorrenti del vocabolario bergogliano. [...] Di quale gioia sta parlando qui Papa Francesco? Essa è un frutto dello Spirito Santo, che sgorga dal cuore di Cristo risorto. Solo l’incontro con il Signore può dare questa gioia, non una decisione etica o l’adesione a una idea. 2 O matrimônio na Exortação Amoris Laetitia Para ele, “eros e ágape [...] nunca se deixam separar completamente um do outro. A Amoris Laetitia é um clamor por uma mu- dança de comportamento e de ação frente à realidade antropológica. Ela é fonte de discer- nimento pastoral, sob a ótica da misericórdia, a partir das condições históricas das pessoas no mundo atual (FUMAGALLI, 2019, p. 17). No título da Exortação aparece um dos temas recorrentes no pontificado de Francisco: a alegria. Nesse documento a ênfase é dada na alegria do amor. Já na sua primeira Exortação Apóstolica o Papa afirma: “A alegria do Evangelho enche o coração e a vida inteira daqueles que se encontram com Jesus” (EG 1). Nesse sentido, Spadaro aprofunda: O que significa a palavra o gaudium, a laetitia, para o Papa Francisco? O termo “gioia” (nas suas várias declinações: alegria, prazer...) é um dos mais recorrentes do vocabulário bergo- gliano. [...] De que alegria está falando o Papa Francisco? Ela é um fruto do Espírito Santo, que brota do coração de Cristo ressuscitado. Somente o encontro com o Senhor pode dar esta alegria, não é uma decisão ética ou a adesão a uma ideia (SPADARO, 2016, p. 106, tradução nossa).3 3 Do original: Che cosa sono per Papa Francesco il gaudium, la laetitia? Il termine “gioia” (nelle sue varie declinazioni: alegría, gozo…) è uno dei più ricorrenti del vocabolario bergogliano. [...] Di quale gioia sta parlando qui Papa Francesco? Essa è un frutto dello Spirito Santo, che sgorga dal cuore di Cristo risorto. Solo l’incontro con il Signore può dare questa gioia, non una decisione etica o l’adesione a una idea. Jonas Emerim Velho O aggiornamento da teologia matrimonial na Exortação Amoris Laetitia 7/14 Jonas Emerim Velho O aggiornamento da teologia matrimonial na Exortação Amoris Laetitia 7/14 Trata-se, então, de um documento pontifício sobre o amor na família, e expressão da preo- cupação da Igreja em fazê-la viver na alegria desse amor, mesmo em meio às imperfeições. A partir disso, observa-se a teologia do matri- mônio presente na Exortação, caracterizada por três aspectos: seus fundamentos bíblicos, sua perspectiva histórico-salvífica e sua abordagem querigmática. caridade, escrito por São Paulo, na Primeira Carta aos Coríntios, versículos 4 a 7 do capítulo 13. O que chama a atenção é a explicação exegética dos termos paulinos, a partir do texto grego original” (PEREIRA, 2016, p. 19). 2.1 Fundamentos bíblicos de Amoris Laetitia O Papa Francisco cita a Sagrada Escritura em todos os capítulos de Amoris Laetitia. São 270 citações bíblicas em todo o documento. Dessas, 100 encontram-se no primeiro capítulo, intitulado “À luz da Palavra”. É opção do pontífice iniciar com as Escrituras, tendo nelas a inspiração para todo o documento: “começarei por uma abertura inspirada na Sagrada Escritura, que lhe dê o tom adequado” (AL 6). Nota-se que o documento pontifício se funda- menta em textos bíblicos mais práticos, como é o caso das exortações de Paulo no hino à caridade da primeira Carta aos Coríntios. “Não se trata de afirmações romântico-idealistas, ao contrário, são muito realistas e, com isso, muito verdadeiras, deixando de lado todo o palavrório superficial a respeito do amor” (KASPER, 2019, p. 47). No primeiro capítulo, Francisco lembra que a Bíblia está cheia de famílias, com histórias de amor e de crise, desde as primeiras páginas, com Adão e Eva no Gênesis, até as últimas, com as bodas do Cordeiro e sua Esposa no Apocalipse. O Salmo 128 é citado integralmente, e Mateus 19,4 é citado para falar do desígnio do princípio que Cristo evoca ao falar do matrimônio. 2 O matrimônio na Exortação Amoris Laetitia As qualidades citadas são: paciência (makrothymein), atitude de serviço (chrêstéuomai), não invejar (zêlóo), humil- dade (peuperéuomai), delicadeza (aschêmonein), desprendimento e autocontrole (paroksýnomai) e alegria (cháirei). “É uma contribuição extremamen- te rica e preciosa para a vida cristã dos esposos. É um tratado sobre a beleza da vida cotidiana do amor, inimiga do realismo. É muito necessário ler esse capítulo para entender melhor o conteúdo da Exortação” (SISTACH, 2017, p. 27). 2.2 Evangelho da família: abordagem querigmática do matrimônio A teologia de Amoris Laetitia não é de afas- tar-se da doutrina católica sobre o matrimônio e família, mas de aprofundá-la, dando-lhe novo significado. É característico do pontificado de Francisco o convite a redescobrir a beleza da fé, a “alegria do Evangelho”. “No fundo, trata-se de ‘libertar’ a ‘doutrina’ de leituras que a impedem de ser fonte de inspiração e sentido para os casais cristãos. Essa perspectiva não relativiza o dogma, mas o transforma em vida” (MORI, 2018, p. 118). Para Francisco, o matrimônio não é mais visto como simples contrato exterior, mas como um chamado divino: “O matrimônio sacramental é, ao contrário, a resposta a um chamado, [...] uma resposta à chamada específica para viver o amor conjugal como sinal imperfeito do amor entre Cristo e a Igreja” (AL 72). Para o pontífice também, “a decisão de se casar e formar uma família deve ser fruto de um discernimento vocacional” (AL 72). A ênfase agora está na livre resposta do ser humano ao chamado divino. O Papa ao iniciar o capítulo terceiro no qual aborda diretamente a teologia matrimonial, lem- bra que ela deve recuperar o caráter de “anúncio e ternura” (AL 59), e que “toda a formação cristã é, primeiramente, aprofundamento do querigma” (AL 58). É o primeiro anúncio, o querigma, que deve ressoar sempre de novo, também no que tange à doutrina sobre a família e o matrimônio. A perspectiva da aliança, assumida pelo Vatica- no II para compreensão do matrimônio, se torna evidente na Exortação. Ao falar da indissolubili- dade, Francisco não a nega como propriedade fundamental do matrimônio, mas deixa de falar com uma linguagem jurídico-canônica para falar em uma aliança de vida, que exige dos esposos um compromisso ético. “Nesse sentido, o sa- cramento do matrimônio é ‘baixado’ ao nível de uma grandeza histórica” (FONTANA, 2018, p. 92), e é compreendido como realidade histórica, que caminha entre os perigos da vida real, e vai se construindo de maneira gradual e processual. Assim também na teologia do matrimônio, Francisco propõe o retorno ao conteúdo e à linguagem do querigma, para que a doutrina sobre a família seja apresentada e compreen- dida como “Evangelho da família”, boa-nova do amor, pois “todo edifício da doutrina, formulado e transmitido, assenta-se sobre essa fonte e a partir dele pode e deve renovar-se” (PASSOS, 2015, p. 29). 2.1 O matrimônio como aliança e a centralidade do amor Amoris Laetitia em seu capítulo terceiro, apre- senta como que uma síntese do ensinamento da Igreja sobre o matrimônio. No núcleo do ca- pítulo está a seção com o título “O sacramento do matrimônio”. Francisco acentua a doutrina de que Cristo elevou a união do homem e da mulher a sinal sacramental de seu amor pela Igreja; é, então, ação da graça e não somente obra humana. “O sacramento do matrimônio não é uma convenção social, um rito vazio ou o mero sinal externo de um compromisso. O sacramento é um dom para a santificação e a salvação dos esposos” (AL 72). No capítulo terceiro, a Bíblia volta a ser citada amplamente, colocando o leitor com o olhar fixo em Jesus, e a partir dele, na vocação da família. Francisco aponta o matrimônio como realidade positiva, contra, por exemplo, os maniqueus, que proibiam o casamento, citando 1Tm 4,4 e Hb 13,4. Também afirma a indissolubilidade do matrimô- nio em Mt 19,6, entendida como um dom e não um jugo. E, recordando a carta aos Colossenses 1,16, afirma que tudo foi criado por e para Cristo; assim, o matrimônio natural só se compreende à luz do seu cumprimento sacramental, e o olhar compassivo de Cristo deve inspirar o cuidado pastoral da Igreja (VELHO, 2021, p. 30). Ainda no número 72, a Exortação apresenta o matrimônio como vocação, o que significa ressaltar o aspecto do dom e da iniciativa divina. Os esposos são chamados por Deus para que, na vida conjugal, respondam a uma iniciativa do próprio Deus. A Familiaris Consortio descreve-o como vocação, mas No capítulo quarto, sobre o amor no matrimô- nio, Francisco propõe características do amor verdadeiro, “destacando-as do conhecido hino à 8/14 Teocomunicação, Porto Alegre, v. 53, n. 1, p. 1-14, jan.-dez. 2023 \| e-44037 duas consequências principais. “De um lado liber- ta o matrimônio de uma visão demasiado jurídica e, de outro, exige um esforço hermenêutico sério no sentido de definir o amor” (FONTANA, 2018, p. 92). Em Amoris Laetitia Francisco ajuda os casais a descobrir o valor do amor e a entender o que implica amar concretamente, quando medita o hino paulino ao amor, por exemplo. Não mais fundamentando-se em Agostinho, a Exortação no capítulo 4 busca em Tomás de Aquino, em sua doutrina sobre as paixões e o amor, a fun- damentação para o amor conjugal, destacando seu valor antropológico e moral. 2.1 O matrimônio como aliança e a centralidade do amor a sua compreensão de vocação ainda parecia muito presa a uma ideia de estado de vida. A concepção de vocação de Francisco soa muito mais ‘leve’ e está muito mais baseada no encontro pessoal e alegre com o Evangelho e com a pessoa de Jesus Cristo (FONTANA, 2018, p. 89). A teologia matrimonial anterior ao Concílio Vaticano II estava amparada na concepção de Agostinho. A preocupação da teologia era em justificar o contrato realizado pelos cônjuges. Agostinho considerava totalmente impossível que o ser humano, corrompido pela queda no pecado, pudesse praticar a sexualidade total- mente sem pecado. [...] Em virtude da grande autoridade de Agostinho, sua “teoria da com- pensação” (os bens matrimoniais compensam as carências da sexualidade) e a identificação de “sacramento” e “indissolubilidade” deter- minam a teologia matrimonial dos próximos séculos (NOCKE, 2012, p. 329). 4 Gustavo Irrazábal fala sobre a debilidade argumentativa de Amoris Laetitia, quando trata da integração de casais em situações complexas na vida eclesial. Segundo o documento, pessoas em estado objetivo de pecado grave, subjetivamente não estariam nessa situação, logo, poderiam até mesmo estarem aptas a receberem frutuosamente os sacramentos. “Este opta implicitamente por uma separação – de inspiração neoescolástica – entre a dimensão objetiva e subjetiva do fazer. No plano objetivo se encontra a norma geral e sua eventual transgressão, o pecado ‘objetivo’. No plano subjetivo se situa o pecado formal, quer dizer, o aspecto da culpabilidade e os fatores atenuantes da mesma (os clássicos ‘impedimentos’)” (IRRAZÁBAL, 2016, p. 163, tradução nossa). Do original: éste implícitamente opta por una separación −de inspiración neoescolástica− entre la dimensión objetiva y subjetiva del obrar. En el plano objetivo se ubica la norma general y su eventual transgresión, el pecado “objetivo”. En el plano subjetivo se sitúa el pecado formal, es decir, el aspecto de la culpabilidad y los factores atenuantes de la misma (los clásicos “impedimentos”). O mesmo autor, ao abordar o tema do discernimento, à luz da misericórdia, pedido por Francisco diante das situações complexas, afirma se tratar de um discurso não completo. Pois, hora o discernimento não pode aplicar a lei de maneira geral, e hora o discernimento pessoal tem validez objetiva. “Porém em realidade, o Papa todavia não tem alcançado a claridade argumentativa que se requer quando o que está em questão é o conteúdo autêntico da lei, no sentido de quando é a verdadeira vontade de Deus” (IRRAZÁBAL, 2017, p. 200, tradução nossa). Do original: Pero en realidad, el Papa todavía no ha alcanzado la claridad argumentativa que se requiere cuando lo que está en cuestión es el contenido auténtico de la ley, en el sentido de cuál es la verdadera Voluntad de Dios. Outro crítico ao texto de Amoris Laetitia foi o cardeal Raymond Burke, patrono da Ordem Soberana e Militar de Malta, dos Estados Unidos. Ele e outros três cardeais (Carlo Caffarra, Joachim Meisner e Walter Brandmüller) enviaram uma carta ao Papa Francisco, intitulada “Criar clareza. Alguns nós por resolver em Amoris Laetitia – Um apelo”. Nela, solicitam que fosse esclarecido aquilo que consideram confuso no documento, sobretudo no oitavo capítulo. Apresentam cinco dúvidas a serem respondidas: a) Se a expressão “em certos casos” da nota 351 da Exortação pode ser aplicada a quem vive em novas núpcias, mesmo que se diferencie do ensinamento de Familiaris Consortio n. 84 e de Sacramentum Caritatis n. 29, admitindo essas pessoas aos sacramentos da reconciliação e à sagrada comunhão; b) o ensinamento da Encíclica Veritatis Splendor de João Paulo II, n. 79, que afirma que existem normas morais absolutas, sem qualquer exceção, que proíbem atos intrinsecamente maus, continua válido após Amoris Laetitia n. 304?; c) depois de Amoris Laetitia n. 301, pode-se ainda afirmar que uma pessoa que vive em contradição com um mandamento da lei de Deus, se encontra em situação objetiva de pecado grave?; d) após as afirmações de Amoris Laetitia n. 302, relativas às circunstâncias atenuan- tes da responsabilidade moral, ainda se deve ter como válido o ensinamento da Veritatis Splendor n. 81, segundo o qual as circunstâncias ou as intenções nunca poderão transformar um ato intrinsecamente mau pelo seu objeto, em um ato subjetivamente bom?; e) após Amoris Laetitia n. 303, ainda se deve ter como válido o ensinamento de Veritatis Splendor n. 56, que afirma que a consciência jamais está autorizada a legitimar exceções às normas morais absolutas que proíbem ações intrinsecamente más pelo próprio objeto? (Cf. BURKE et alii, 2016). As solicitações não foram respondidas pelo pontífice. 2.2 Evangelho da família: abordagem querigmática do matrimônio Acentua o Papa que “o nosso ensinamento sobre o matrimônio e a família não pode deixar de se inspirar e transfigurar à luz deste anúncio de amor e ternura, se não quiser tornar-se mera Os capítulos 4 e 5 da Exortação colocam o amor como realidade central na vida matrimonial. O amor é abordado de maneira concreta, humana, histórica e bíblica. Colocar o amor no centro tem Jonas Emerim Velho O aggiornamento da teologia matrimonial na Exortação Amoris Laetitia 9/14 Jonas Emerim Velho O aggiornamento da teologia matrimonial na Exortação Amoris Laetitia 9/14 defesa de uma doutrina fria e sem vida” (AL 59). Percebe-se, assim, que a teologia da Exortação é de retorno ao essencial, de repensar a substância da doutrina do matrimônio a partir do “coração do Evangelho”, ou do “Evangelho da família”, superando formulações teológicas abstratas e duras que não transmitam e não façam encontrar a boa-nova do amor de Deus e seu chamado para o ser humano viver no amor. Para isso, foi neces- sária uma nova maneira de formular a doutrina. enxergar os mais distantes do ideal evangélico. Esse núcleo mais fundamental permanece intacto no todo e nas partes da Exortação. A Amoris Laetitia foi recebida também com crí- ticas por parte de teólogos e ministros ordenados. A mudança de paradigma, e no entendimento de alguns, a doutrina, proposta por Francisco, trouxe desconfiança, sobretudo no âmbito da teologia moral.4 Grupos católicos progressistas e conservadores acolheram o texto de maneiras diferentes. Porém, suas orientações permanecem, lentamente, sendo estudadas e assimiladas no âmbito da pastoral familiar da Igreja católica. Francisco entende a doutrina e a tradição da Igreja como um sistema aberto que pretende colocar as verdades de fé a serviço da vida. [...] dizer que a doutrina é um sistema aberto não é romper com a verdade que ela possui e visa comunicar, mas sim entender que essa verdade deve ser situada no tempo e no espaço como um modo de compreender e expressar certos conteúdos da fé (PASSOS, 2015, p. 41). 3 Orientações de Amoris Laetitia para os casais cristãos viverem na alegria do amor Da teologia matrimonial de Amoris Laetitia, decorrem orientações ou caminhos que o casal cristão é chamado a percorrer para viver o ma- trimônio como Evangelho, como itinerário de plenitude de vida, para a alegria do amor. Para Francisco, é o amor conjugal a alma do matrimô- nio, e que deve estar no centro das preocupações da Igreja no acompanhamento dos cônjuges. Seguindo essa perspectiva, compreende-se que a teologia do matrimônio de Amoris Laetitia não muda a doutrina, muda sua formulação, sua linguagem e sua interpretação, mas não seu conteúdo substancial. O que há é um esforço de rever o que é periférico para afirmar o núcleo central. O documento recupera a substância da doutrina matrimonial. O amor permanece como regra máxima para todos os cristãos, para os unidos no matrimônio, e a partir dele se deve Assim, para o Papa, uma pastoral familiar efi- caz há que acompanhar os fiéis casados no 10/14 Teocomunicação, Porto Alegre, v. 53, n. 1, p. 1-14, jan.-dez. 2023 \| e-44037 Teocomunicação, Porto Alegre, v. 53, n. 1, p. 1-14, jan.-dez. 2023 \| e-44037 10/14 ao Evangelho: “É pedido a nós um esforço mais responsável e generoso, que consiste em apre- sentar as razões e os motivos para se optar pelo matrimônio e a família, de modo que as pessoas estejam mais bem preparadas para responder à graça que Deus lhes concede” (AL 35). sentido de ajudá-los a cultivar e desenvolver o amor, muito mais do que insistindo numa manutenção meramente jurídica do vínculo; isso porque sendo o amor conjugal a “alma” do casamento, um apostolado matrimonial que se ocupasse apenas do “corpo” – fidelidade, indissolubilidade, prole... – correria o risco de, por fim, estar garantindo meramente a susten- tação de um cadáver (ALMEIDA, 2017, p. 104). No número 49, a Exortação diz que o Evan- gelho não deve ser “doutrinado” para ser jogado como pedras nos outros, a fim de condená-los, tratando da situação das famílias fragilizadas. O Evangelho é, antes, força que cura, que dá vida ao ser humano. Nesse sentido, “a relativização da norma diante do anúncio do Evangelho e da força da graça e da misericórdia expressa a abordagem mistagógico-espiritual da Exortação” (JUNGES, 2018, p. 16). 3 Orientações de Amoris Laetitia para os casais cristãos viverem na alegria do amor A perspectiva do Evangelho da família e do matrimônio, abandona uma linguagem jurí- dica e moral, bem como a matriz hermenêutica agostiniana, buscando fundamentos também na teologia de Tomás de Aquino: “No capítulo 4, [...], o Papa busca resgatar a doutrina de Tomás sobre as paixões e sobre o amor, destacando seu valor antropológico e moral” (FONTANA, 2018, p. 92). Nesse sentido, uma pastoral familiar eficaz acompanha os casais no caminho de cresci- mento e amadurecimento do amor, enfatizan- do a vivência possível do amor conjugal, sem idealizações da doutrina matrimonial, e nem do próprio cônjuge. A partir de agora destacam-se três perspectivas para esse acompanhamento: o matrimônio como caminho histórico-salvífico, a dimensão erótica do amor conjugal e a espiri- tualidade do amor exclusivo e libertador. 3.2 A dimensão erótica do matrimônio O tema da sexualidade e do amor erótico são tratados, respectivamente, nos capítulos quatro e cinco de Amoris Laetitia, no contexto do amor no matrimônio e sua fecundidade. Insiste-se na vocação última da vida matrimonial e familiar que é chamada a ser um lugar onde se vive o amor na alegria. Há, então, uma visão positiva acerca da missão a ser vivida no âmbito familiar (ALMEIDA, 2017, p. 521). Do ponto de vista fenomenológico, o amor erótico não se reduz à posse do corpo do outro, após a admiração e o contato de intimidade. Ao se desejar alguém, nunca se deseja mantendo-se por inteiro fora do próprio desejo: “o desejo nos envolve; o indivíduo é cúmplice de seu desejo, e por ele toma consciência de seu ser-um-cor- po” (MARZANO, 2012, p. 936). No amor erótico, como compreende o Papa Francisco, “o gozo em si não é o ápice da relação, mas sim, o sentido do abandonar-se no outro, isto é, a abertura e a entrega que o “eu” encontra no “tu”, expansão do mistério da falta, da perda e do encontro” (ALMEIDA; CASTRO; SANTOS, 2020, p. 577). Este dinamismo intrinsecamente humano e divino, supõe compromisso mútuo, renovado todos os dias da vida dos cônjuges. No capítulo quarto da Amoris Laetitia a di- mensão erótica do amor é tratada por Francisco em total dependência e vínculo com o que se entende por amor apaixonado (n. 142-162). Ana- lisam-se as dimensões humanas presentes na experiência do amor, tais como: as emoções (n. 143), o sentido moral (n. 145), o ato livre (n. 146), a necessidade de um caminho pedagógico e formativo (n. 147-148) e o sentimento de alegria presente no amor (n. 149). Percebe-se, neste modo de tratar a realidade humana do amor, uma leitura antropológica a partir da condição real e concreta da pessoa. Para o Papa Francisco, o amor verdadeiro e integral se realiza em um constante empenho dos envolvidos, integrando tanto as forças mais desconhecidas das psiquê, como o olhar, a proximidade, o encanto e a ter- nura de um abraço. “O amor em sua expressão erótica é, primeiramente, uma força que brota da condição pessoal e interior do sujeito. Ela se prolonga em uma relação amorosa de entrega e comunhão recíprocas (AL, n. CASTRO; SANTOS, 2020, p. 576). Nota-se que, ao enfatizar que o casal se torna um sinal imperfeito, leva-se em conta as limitações das relações conjugais, além da realidade sacra- mental que se estende por toda a vida. Assim, em Amoris Laetitia é assumida uma perspectiva mais bíblica e existencial. São consideradas as imperfeições do casal, abandonando uma visão muito idealista do matrimônio. Diante disso, a Igreja deve ajudar os noivos a não se perderem pelas convenções sociais que estão em torno do casamento, e nem pelas idealizações próprias da paixão. Ao contrário, os cônjuges são chamados a descobrir e discernir sua vocação. Para o pontífice, a sexualidade compreendida a partir da visão cristã do amor, é um presente do Criador que necessita ser cultivado, superando um uso utilitarista do corpo e das emoções do outro para obtenção de seu próprio prazer. O Papa Francisco, ao mesmo tempo em que valoriza e promove o que é constitutivo da experiência do amor humano, também denuncia seu redu- cionismo em tempos de fragmentação humana (AL, n. 50-57). Em Amoris Laetitia, a dimensão erótica do amor deve ser entendida como um dom de Deus que embeleza e enriquece o encontro dos esposos, reconhecendo e admirando a dignidade do outro. Esta admiração, própria de quem ama e se sente atraído pelo outro, no contexto matrimonial, está na base da relação com o seu dinamismo com- preendido como amor conjugal (n. 163). “O sujeito enamora-se pela pessoa inteira do outro, a partir de sua condição humano-afetiva, em busca de sua integração e realização, numa proximidade fiel e cheia de ternura (n. 164)” (ALMEIDA; CASTRO; SANTOS, 2020, p. 577). 3.1 Abordagem histórico-salvífica do matrimônio: o casal cristão como imagem imperfeita da Santíssima Trindade O que Francisco aponta é que o mais importante não é o cum- primento da norma, mas a adesão ao querigma, Jonas Emerim Velho O aggiornamento da teologia matrimonial na Exortação Amoris Laetitia 11/14 Jonas Emerim Velho O aggiornamento da teologia matrimonial na Exortação Amoris Laetitia CASTRO; SANTOS, 2020, p. 576). 3.1 Abordagem histórico-salvífica do matrimônio: o casal cristão como imagem imperfeita da Santíssima Trindade O Papa Francisco entende o matrimônio “uma resposta à chamada específica para viver o amor conjugal como sinal imperfeito do amor entre Cristo e a Igreja” (AL 72). Colocando a ênfase na vocação pessoal, ressalta-se a livre resposta do ser humano a Deus, “distanciando-se, implici- tamente, de outras definições do matrimônio” (FONTANA, 2018, p. 91). Além disso, Amoris La- etitia apresenta uma linguagem mais espiritual para falar do matrimônio como boa-nova e como história de salvação. Outro tema em que fica evidente a mudança de linguagem de uma Exortação para a outra, é o tema do amor, que em Amoris Laetitia é tratado em tom mistagógico-espiritual. Na AL, o capítulo quarto trata do amor (núme- ros 90 a 164), considerado a joia da Exortação pelo seu canto poético ao amor, inspirado no hino à caridade de Paulo, uso que não encontra similar em outro documento do Magistério. Neste sentido é um texto mistagógico que ins- pira a vivência do amor, tendo os olhos abertos para as suas dificuldades e os remédios que a graça propõe (JUNGES, 2018, p. 17). AL tem uma linguagem mistagógico-espiritual de cunho exortativo para suscitar alegria e con- solação, centrada na boa nova do Evangelho (citado 41 vezes), explicitado no querigma (4 ve- zes), assumindo a perspectiva da misericórdia (35 vezes) e do discernimento (35 vezes) para refletir e propor soluções para as situações difíceis e irregulares quanto às exigências do matrimônio (JUNGES, 2018, p. 15). No entanto, a concepção de Francisco apre- senta uma linguagem mais leve e concreta: “O matrimônio é uma vocação, sendo uma resposta ao chamado específico para viver o amor conjugal como sinal imperfeito do amor entre Cristo e a Igreja” (AL 72). No número 123 de Amoris Laetitia Francisco acrescenta um outro tom à afirmação teológica de que o casal, pelo sacramento do matrimônio, participa do amor de Cristo pela Igreja, dizendo que o casal se torna um “sinal imperfeito do amor de Cristo e a Igreja” (AL 123). O casamento é entendido em perspectiva histórica e mistagógica, em que o querigma do matrimônio deve acontecer nas coordenadas concretas da vida do casal. 5 Do original: Anclado en una antropología que se aleja de aquellos dualismos que privilegiaban el alma en detrimento del cuerpo y sus expresiones, Francisco propone una espiritualidad capaz de integrar la interioridad y la corporalidad, el culto y el compromiso social, la unión con Dios, los vínculos familiares y la comunión eclesial. 6 Do original: La espiritualidad conyugal también se realiza en la tensión de sostener simultáneamente la mutua pertenencia y la sana autonomía. La exclusividad del amor reclama la decisión cotidiana de seguir eligiéndose para transitar juntos la aventura de amarse hasta el final. vivência diária da Igreja doméstica (AL 318) (PASSOS, 2018, p. 117). vivência diária da Igreja doméstica (AL 318) (PASSOS, 2018, p. 117). isso exige, naturalmente, que a entrega amorosa, seja sincera e real. Assim, a união dos corpos é a apoteose na qual se expressa a união dos corações (PAGOLA, 2018, p. 32). Os números 319 e 320 da Exortação tratam da espiritualidade do amor exclusivo e libertador. O Papa recorda como a fidelidade do casal reflete a fidelidade do próprio Deus. “No matrimônio, vive-se também o sentido de pertencer com- pletamente a uma única pessoa. Os esposos assumem o desafio de envelhecer e gastar-se juntos, e assim refletem a fidelidade de Deus” (AL 319). Os cônjuges são chamados a renovarem, a cada dia, diante de Deus, essa pertença do coração um ao outro. Contudo, 3.3 Espiritualidade conjugal: um amor exclusivo e libertador No capítulo nono de Amoris Laetitia, o Papa Francisco trata do tema da espiritualidade con- jugal e familiar. Segundo o pontífice, tema fun- damental para que os casais cristãos possam alegrar-se pelo matrimônio vivido e dar teste- munho de alegria a outros que encontrarem. Assim, o casal deve cultivar uma “espiritualidade específica que se desenrola no dinamismo das relações da vida familiar” (AL 313), e que aju- de os cônjuges a contemplarem um ao outro. Essa, deve cultivar-se em um itinerário que não entenda os assuntos cotidianos do casal como um obstáculo para a intimidade com Deus. Pelo contrário, Francisco entende a espiritualidade conjugal como integradora: a espiritualidade conjugal também se realiza na tensão de assegurar simultaneamente a mútua pertença e a sã autonomia. A exclusi- vidade do amor reclama a decisão cotidiana de seguir elegendo-se para transitar juntos a aventura de amar-se até o final (RUIZ, 2017, p. 89, tradução nossa).6 Segundo Francisco o amor do casal torna-se experiência de libertação, “quando um descobre que o outro não é seu, mas tem um proprietário muito mais importante, o seu único Senhor” (AL 320). É preciso cultivar um realismo espiritual, que faz com que o cônjuge “não pretenda que o outro o satisfaça completamente em suas exigências” (AL 320). É a experiência de “deixar de esperar dessa pessoa que é próprio apenas do amor de Deus” (AL 320). É um despojamento interior que o casal é chamado a realizar, para que possa amar com liberdade interior, o que será fonte de alegria para ambos. Ancorado em uma antropologia que deixa aqueles dualismos que privilegiam a alma em detrimento do corpo e suas expressões, Francisco propõe uma espiritualidade capaz de integrar a interioridade e a corporeidade, o culto e o compromisso social, a união com Deus, os vínculos familiares e a comunhão eclesial (RUIZ, 2017, p. 90, tradução nossa).5 Nesse sentido, para o pontífice “A Eucaristia é o sacramento da Nova Aliança, em que se atualiza a ação redentora de Cristo (cf. Lc 22, 20). Consta- tamos, assim, os laços íntimos que existem entre a vida conjugal e a Eucaristia” (AL 318). Os dois documentos reconhecem que a espiritualidade familiar agrega, sob a perspectiva do divino, todas as dimensões da vida conjugal, 3.2 A dimensão erótica do matrimônio 163-164)” (ALMEIDA; Na Amoris Laetitia o vínculo conjugal supõe um amor que envolve totalmente as subjetividades distintas, chegando ao ponto de se tornarem uma só carne, mediante o amor erótico, um caminho de entrega e de mútua doação. Tudo 12/14 Teocomunicação, Porto Alegre, v. 53, n. 1, p. 1-14, jan.-dez. 2023 \| e-44037 Considerações finais Com base em pesquisa na teologia matrimonial recente, percebe-se, na Exortação Amoris Laetitia, a continuidade com a perspectiva do Vaticano II em um retorno a uma antropologia integral. Assim, o aggiornamento da doutrina matrimonial cató- lica fez-se perceber. A compreensão da pessoa além de ser um caminho místico de sentido profundo do cotidiano, se faz no exercício da oração, onde a vida eucarística introduz a família em uma dimensão pascal que renova o mistério do amor que uniu e une o casal na Jonas Emerim Velho O aggiornamento da teologia matrimonial na Exortação Amoris Laetitia humana, própria do personalismo, influenciou os Padres Conciliares na elaboração da Constitui- ção Pastoral Gaudium et Spes, bem como todo o magistério no pós-concílio. Contudo, a ênfase na procriação, própria da perspectiva agostinia- na, e nos deveres do matrimônio, continuaram presentes nos documentos pontifícios sobre a família e o matrimônio. ALMEIDA, André Luiz B.; CASTRO, Robson Ribeiro de O.; SANTOS, Thales Martins. Amor e sexualidade na teologia cristã: uma interpretação ético-teológica so- bre o sentido do amor erótico à luz da Amoris Laetitia. Revista Eclesiástica Brasileira, Petrópolis, v. 80, n. 317, p. 564-582, set./dez. 2020. ALMEIDA, André Luiz B.; CASTRO, Robson Ribeiro de O.; SANTOS, Thales Martins. Amor e sexualidade na teologia cristã: uma interpretação ético-teológica so- bre o sentido do amor erótico à luz da Amoris Laetitia. Revista Eclesiástica Brasileira, Petrópolis, v. 80, n. 317, p. 564-582, set./dez. 2020. ALMEIDA, André Luiz B.; CASTRO, Robson Ribeiro de O.; SANTOS, Thales Martins. Amor e sexualidade na teologia cristã: uma interpretação ético-teológica so- bre o sentido do amor erótico à luz da Amoris Laetitia. Revista Eclesiástica Brasileira, Petrópolis, v. 80, n. 317, p. 564-582, set./dez. 2020. AMORIM, Leonardo C. da S. de O. Pessoa e comuni- dade. O individualismo religioso contemporâneo face ao personalismo de Emmanuel Mounier e ao aspecto comunitário da teologia de Karl Barth. 129 f. Disserta- ção (Mestrado em Teologia) – Faculdade de Teologia, Pontifícia Universidade Católica do Rio de Janeiro, Rio de Janeiro, 2010. O Papa Francisco leva adiante a compreensão do matrimônio mais como aliança e amor, do que como dever e contrato; mais como história de salvação e caminho mistagógico. Assim, o ma- trimônio é um chamado, uma vocação ao amor, que vai crescendo e amadurecendo no cotidiano dos cônjuges, e esses vão se tornando imagens imperfeitas do amor de Cristo pela Igreja. Considerações finais A va- lorização do amor erótico como impulso para o encontro e união do casal, enaltece a finalidade unitiva do matrimônio, e entende a vivência da sexualidade conjugal como expressão de total entrega, e não somente lugar de procriação. O casal cristão é convidado pelo pontífice, a culti- var uma espiritualidade que o ajude a chegar à experiência libertadora de saber que, em última instância, só em Deus terá a realização última de seus anseios. BÍBLIA. Português. Bíblia de Jerusalém. Nova edição ver. e ampl. São Paulo: Paulus, 2002. BURKE, Raymond et alii. Criar clareza. Alguns nós por resolver em Amoris Laetit–a - Um apelo. In: Chiesa. Roma, 14 nov. 2016. Disponível em: https://chiesa. espresso.repubblica.it/articolo/1351410.html. Acesso em: 8 fev. 2023. BURKE, Raymond et alii. Criar clareza. Alguns nós por resolver em Amoris Laetit–a - Um apelo. In: Chiesa. Roma, 14 nov. 2016. Disponível em: https://chiesa. espresso.repubblica.it/articolo/1351410.html. Acesso em: 8 fev. 2023. DENZINGER, Heinrich; HÜNERMANN, Peter. Compêndio dos símbolos, definições e declarações de fé e moral. São Paulo: Paulinas, 2007. FONTANA, Leandro Luis B. Edificar o matrimônio no amor. Amoris Laetitia em questão: aspectos bíblicos, teológicos e pastorais. In: FERNANDES, Leonardo Agostini (org.). Amoris Laetitia em questão. São Paulo: Paulinas, 2018. p. 87-96. FUMAGALLI, Aristide. Caminhar no amor. A teologia moral do Papa Francisco. Brasília: Edições CNBB, 2019. FRANCISCO. Exortação Apostólica Evangelii Gaudium: sobre o anúncio do Evangelho no mundo atual. Roma: Tipografia Vaticana, 2013. Assim, a teologia matrimonial presente na Amoris Laetitia torna-se caminho para uma com- preensão do amor conjugal como lugar de alegria. Essa, que vem da certeza de saber-se amado por um Deus misericordioso, que leva em conta as fragilidades e os limites humanos, encontra-se em viver, no matrimônio, uma vida de doação completa, também por meio da vivência da se- xualidade conjugal. A alegria, tema constante no pontificado de Francisco, deve ser característica predominante do casal cristão, em um esforço contínuo de testemunhar o amor entre Cristo e a Igreja, embora de maneira imperfeita, mas sabendo-se sustentado pela graça divina. FRANCISCO. Exortação Apostólica pós-sinodal Amo- ris Laetitia: Sobre o amor na família. Brasília: Edições CNBB, 2016. GARCIA, Jaime (org.). Diccionario de San Agustín. San Agustín a traves del tiempo. Tradução de Constantino Ruiz-Garrido. Monte Carmelo: Madrid, 2001. IRRAZÁBAL, Gustavo. Amoris Laetitia y los divorciados en nueva unión. Revista Teología, [S. l.], v. 53, n. 120, p. 151-173, ago. 2016. IRRAZÁBAL, Gustavo. La misericordia según Francisco. ALMEIDA, André Luiz Boccato de. O discernimento da consciência na Exortação Apostólica Amoris Laetitia. Revista Eclesiástica Brasileira, Petrópolis, v. 77, n. 307, p. 520-535, 2017. Considerações finais Valor y limites de um discurso. Revista Teología, [S. l.], v. 54, n. 122, p. 181-204, maio 2017. MOUNIER, Emmanuel. O personalismo. São Paulo: Centauro Editora, 2004. JUNGES, José Roque. Os documentos eclesiais pós-si- nodais Familiaris Consortio de Wojtyla e Amoris Laetitia de Bergoglio como respostas aos desafios da pastoral matrimonial. Revista do Instituto Humanitas Unisinos, São Leopoldo, n. 133, v. 15, p. 3-21, 2018. Referências ALMEIDA, André Luiz Boccato de. O discernimento da consciência na Exortação Apostólica Amoris Laetitia. Revista Eclesiástica Brasileira, Petrópolis, v. 77, n. 307, p. 520-535, 2017. KASPER, Walter. A mensagem de Amoris Laetitia: um debate amigável. Tradução de Alfred J. Keller. São Paulo: Loyola, 2019. KASPER, Walter. O Evangelho da família. Prior Velho: Paulinas Editora, 2014. Presbítero católico do clero da Diocese de Criciúma, em Criciúma, SC, Brasil. Mestre em Teologia sistemática pela Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), em Porto Alegre, RS, Brasil. Doutorando em Teologia pela mesma universidade. MARZANO, M. Sexualidade – Filosofia da sexualidade. In: MARZANO, M. (org.). Dicionário do corpo. São Paulo: Loyola; São Camilo, 2012. p. 935-939. MARZANO, M. Sexualidade – Filosofia da sexualidade. In: MARZANO, M. (org.). Dicionário do corpo. São Paulo: Loyola; São Camilo, 2012. p. 935-939. MORI, Geraldo Luiz D. Teologia e pastoral na Amoris Laetitia. Amoris Laetitia em questão: aspectos bíblicos, teológicos ‘e pastorais. In: FERNANDES, Leonardo Agostini (org.). Amoris Laetitia em questão. São Paulo: Paulinas, 2018. p. 109-129. Jonas Emerim Velho KASPER, Walter. O Evangelho da família. Prior Velho: Paulinas Editora, 2014. KASPER, Walter. O Evangelho da família. Prior Velho: Paulinas Editora, 2014. Endereço para correspondência Jonas Emerim Velho Rua Dom Paulo Evaristo Arns, 550 Michel, 88803-090 Criciúma, SC, Brasil MÜLLER, Gehrard Ludwig. Dogmática católica: teoria e prática da teologia. Tradução de Volney Berkenbrock. Petrópolis: Vozes, 2015. NOCKE, Franz-Josef. Doutrina Específica dos Sacra- mentos. In: SCHNEIDER, Theodor (org.). Manual de Dogmática. 5. ed. Petrópolis: Vozes, 2012. v. 2, p. 205-338. Os textos deste artigo foram revisados pela SK Revisões Acadêmicas e submetidos para validação do(s) autor(es) antes da publicação. LIMA, Luiz Fernando; SANCHES, Mário Antonio. A conju- galidade do amor esponsal: Um traço característico da Amoris Laetitia. Revista Eclesiástica Brasileira, Petrópolis, v. 77, n. 307, p. 536-555, jul./set. 2017. PAGOLA, José A. Originalidade do matrimônio. São Paulo: Paulinas, 2018. PASSOS, João Décio. As fontes da Amoris Laetitia. São Paulo: Paulus, 2018. PEREIRA, Ney B. A Amoris Laetitia e sua fundamentação bíblica. Encontros Teológicos, Florianópolis, v. 31, n. 1, jan./abr. 2016. PERES, Daniel Cipriano. Educar na alegria do amor: por um processo pastoral de iniciação à vida matrimonial à luz da Amoris Laetitia. 2020. 160 f. Dissertação (Mestrado em Teologia) – Faculdade de Teologia, Pontifícia Univer- sidade Católica do Rio de Janeiro, Rio de Janeiro, 2020. RUIZ, Andrea Sánchez. Espiritualidad matrimonial. Yo em ellos y tú em mí. Revista Teología, [S. l.], v. 54, n. 123, p. 81-100, ago. 2017. SESBOÜÉ, Bernard. O homem, maravilha de Deus: ensaio de antropologia cristológica. São Paulo: Paulinas, 2021. SCHILLEBEECKX, Edward. O Matrimônio. Realidade terrestre e mistério de salvação. Vozes: Petrópolis, 1969. SCOLA, Angelo. O mistério nupcial. Bauru: Edusc, 2003. SISTACH, Lluís M. Como aplicar a Amoris Laetitia. Tra- dução de Hugo C. da S. Cavalcante. São Paulo: Fons Sapientiae, 2017. SPADARO, Antonio. Amoris Laetitia: struttura e sig- nificato dell’Esortazione apostólica post-sinodale di Papa Francesco. La Civiltà Cattolica, [S. l.], v. II, n. 3980, p. 105-128, 2016. VELHO, Jonas E. As Exortações pós-sinodais Familiaris Consortio e Amoris Laetitia: continuidade e desconti- nuidade no cuidado pastoral às famílias em situação irregular. 2021. 120 f. Dissertação (Mestrado em Teolo- gia) – Faculdade de Teologia, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, 2021.
https://openalex.org/W2952964632	https://digital.csic.es/bitstream/10261/208590/1/1-s2.0-S0370269319304174-main.pdf	English	null	Inclusive cross sections for one- and multi-nucleon removal from Sn, Sb, and Te projectiles beyond the N = 82 shell closure	Physics letters. B	2,019	cc-by	7,450	Inclusive cross sections for one- and multi-nucleon removal from Sn, Sb, and Te projectiles beyond the N = 82 shell closure V. Vaquero a, A. Jungclaus a,∗, J.L. Rodríguez-Sánchez b, J.A. Tostevin c, P. Doornenbal d, K. Wimmer e,d, S. Chen d,f, E. Nácher a, E. Sahin g, Y. Shiga h, D. Steppenbeck d, R. Taniuchi d,e, Z.Y. Xu i, T. Ando e, H. Baba d, F.L. Bello Garrote g, S. Franchoo j, A. Gargano k K. Hadynska-Klek g, A. Kusoglu l,m, J. Liu i, T. Lokotko i, S. Momiyama e, T. Motobayashi d, S. Nagamine e, N. Nakatsuka n, M. Niikura e, R. Orlandi o, T. Saito e, H. Sakurai d,e, P.A. Söderström d, G.M. Tveten g, Zs. Vajta p, M. Yalcinkaya l a Instituto de Estructura de la Materia, CSIC, E-28006 Madrid, Spain b Universidad de Santiago de Compostela, E-15782 Santiago de Compostela, Spain c Department of Physics, University of Surrey, Guildford, Surrey GU2 7XH, United Kingdom d RIKEN Nishina Center, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan e Department of Physics, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan f School of Physics and State Key Laboratory of Nuclear Physics and Technology, Peking University, Bejing 10 g Department of Physics, University of Oslo, NO-0316 Oslo, Norway h Department of Physics, Rikkyo University, Tokyo, Japan i Department of Physics, The University of Hong Kong, Pokfulam, Hong Kong j Institut de Physique Nucléaire Orsay, IN2P3-CNRS, 91406 Orsay Cedex, France k Istituto Nazionale di Fisica Nucleare, Complesso Universitario di Monte S. Angelo, I-80126 Napoli, Italy l Department of Physics, Faculty of Science, Istanbul University, Vezneciler/Fatih, 34134, Istanbul, Turkey m ELI-NP, Horia Hulubei National Institute of Physics and Nuclear Engineering, 077125 Magurele, Romania n Department of Physics, Faculty of Science, Kyoto University, Kyoto 606-8502, Japan o Advanced Science Research Center, Japan Atomic Energy Agency, Tokai, Ibaraki 319-1195, Japan p MTA Atomki, P.O. Box 51, Debrecen H-4001, Hungary a r t i c l e i n f o Article history: Received 25 February 2019 Received in revised form 23 April 2019 Accepted 17 June 2019 Available online 20 June 2019 Editor: D.F. Geesaman Keywords: Inclusive knockout cross sections Intranuclear cascade model Eikonal reaction theory Article history: Received 25 February 2019 Received in revised form 23 April 2019 Accepted 17 June 2019 Available online 20 June 2019 Editor: D.F. Geesaman Inclusive one- and multi-nucleon removal cross sections have been measured for several Sn, Sb and Te isotopes just beyond the N = 82 neutron shell closure. The beams were produced in the projectile ﬁssion of a 238U beam at the Radioactive Isotope Beam Factory at RIKEN. The experimental cross sections are compared to predictions from the most recent version of the Liege intranuclear cascade model. Although the overall agreement is good, severe discrepancies are observed for the cases of one- and two-neutron removal from 134Sn and 135Sb projectiles and one-proton knockout from all measured N = 84 isotones. These discrepancies, as well as the relevance of quasi-elastic reaction channels to the one-neutron removal cross sections, are discussed. In addition, the measured inclusive one-proton knockout cross section for the semi-magic 134Sn projectile is compared to eikonal direct reaction theory calculations to assess if the suppression factors to these calculated cross sections, deduced from data on reactions of lighter projectile nuclei, are also applicable to heavy nuclei. Keywords: Inclusive knockout cross sections Intranuclear cascade model Eikonal reaction theory © 2019 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. Contents lists available at ScienceDirect Contents lists available at ScienceDirect * Corresponding author. E-mail address: andrea.jungclaus@csic.es (A. Jungclaus). Physics Letters B 795 (2019) 356–361 Physics Letters B 795 (2019) 356–361 * Corresponding author. E-mail address: andrea.jungclaus@csic.es (A. Jungclaus). obtained from a comparison of the experimental inclusive and ex clusive one-nucleon knockout cross sections to theoretical direct reaction calculations. To calculate these cross sections, information https://doi.org/10.1016/j.physletb.2019.06.035 0370-2693/© 2019 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. d 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. Experiment and results The experiment was carried out at the Radioactive Isotope Beam Factory (RIBF), operated by the RIKEN Nishina Center and the Center for Nuclear Study of the University of Tokyo. A pri- mary beam of 238U at 345 MeV/u bombarded a 4-mm-thick beryl- lium target located at the entrance of the BigRIPS fragment sep- arator [18] which is sketched in Fig. 1. Fission products around 136Te were selected and puriﬁed by employing the Bρ-E-Bρ method through combination of magnetic rigidity (Bρ) selection and two wedge-shaped aluminium degraders. The particle iden- tiﬁcation was performed on an event-by-event basis using the E-Bρ-TOF method, where the energy loss E was measured by an ionization chamber located at the focal plane F7, Bρ was deter- mined from position measurements using parallel plate avalanche counters (PPACs) and the time of ﬂight (TOF) was measured with two plastic scintillators located at the focal points F3 and F7. The atomic number (Z) and the mass-over-charge (A/Q) ratio of each ion were determined with this method [19]. The resulting particle identiﬁcation plot is shown in Fig. 2a). While a discrepancy with the model calculations is expected, e.g. due to many-body correlation effects beyond those of trun- cated-basis shell-model calculations, the magnitude of the ob- served Rs from the model calculations is not yet understood quan- titatively. The observed inclusive cross section systematics have nevertheless been used, see e.g. [5–7], to deduce spectroscopic factors by comparison of the calculations with measured ﬁnal- state exclusive removal cross sections, taking into account an Rs value consistent with the systematics. In recent years the ﬁrst one- nucleon knockout experiments have been performed in heavier regions of the nuclear chart [8,9], in particular for nuclei around doubly-magic 132Sn [10–13]. However, before structure informa- tion can be deduced from such experiments, it must be clariﬁed whether data and calculations for heavy nuclei conform to the sup- pression factor, Rs, behavior observed in the lighter mass regions, as collected in Refs. [1,2]. After the selection and identiﬁcation, the secondary beams were transported to the focal point F8 where they impinged on a 534-mg/cm2 C target. The energies of the reaction products of interest were in the range 162-170 MeV/u, 138-145 MeV/u and 112-117 MeV before, at the center and behind the target, re- spectively. 1. Introduction The reaction dynamics has generally been modeled assuming the sudden (fast collisions) and eikonal (for- ward scattering) approximations [3,4]. A systematic comparison between the experimental and calculated inclusive (to all bound ﬁ- nal states) one-nucleon knockout cross sections, for a large number of light and medium-mass projectile nuclei, evidenced a signiﬁcant overestimation of the cross section by the calculations [1,2]. The overestimation is more pronounced the larger the binding of the removed nucleon – driven by the neutron-proton asymmetry of the system, S = Sn −Sp for neutron removal and S = Sp −Sn for proton removal, with Sp (Sn) the proton (neutron) separation energy. These inclusive cross section systematics have been pre- sented as a suppression factor Rs = σexp/σth as a function of the separation energy asymmetry S [1,2]. on the structure of the projectile initial and residual nucleus ﬁnal states is combined with an approximate description of the reaction dynamics. In most cases this structure information is taken from shell-model calculations employing appropriate model spaces and effective interactions. The reaction dynamics has generally been modeled assuming the sudden (fast collisions) and eikonal (for- ward scattering) approximations [3,4]. A systematic comparison between the experimental and calculated inclusive (to all bound ﬁ- nal states) one-nucleon knockout cross sections, for a large number of light and medium-mass projectile nuclei, evidenced a signiﬁcant overestimation of the cross section by the calculations [1,2]. The overestimation is more pronounced the larger the binding of the removed nucleon – driven by the neutron-proton asymmetry of the system, S = Sn −Sp for neutron removal and S = Sp −Sn for proton removal, with Sp (Sn) the proton (neutron) separation energy. These inclusive cross section systematics have been pre- sented as a suppression factor Rs = σexp/σth as a function of the separation energy asymmetry S [1,2]. 1. Introduction such as Be or C, have proved to be a useful tool to study the shell structure of nuclei far from the valley of stability [1,2]. In- formation concerning active shells at and near the neutron and proton Fermi surfaces in exotic nuclei, and their occupancies, is obtained from a comparison of the experimental inclusive and ex- clusive one-nucleon knockout cross sections to theoretical direct reaction calculations. To calculate these cross sections, information In the last twenty years, one-nucleon knockout reactions from intermediate energy radioactive ion beams on light target nuclei, 0370-2693/© 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativec SCOAP3 V. Vaquero et al. / Physics Letters B 795 (2019) 356–361 357 Fig. 1. Schematic view of the BigRIPS and ZeroDegree spectrometers (adopted from Ref. [18]). The material along the beam line between the focal planes F7 and F8, which has been considered in the determination of the correction factor χ in Eq. (2), is shown enlarged in the upper part of the ﬁgure. Fig. 1. Schematic view of the BigRIPS and ZeroDegree spectrometers (adopted from Ref. [18]). The material along the beam lin has been considered in the determination of the correction factor χ in Eq. (2), is shown enlarged in the upper part of the ﬁgu Fig. 1. Schematic view of the BigRIPS and ZeroDegree spectrometers (adopted from Ref. [18]). The material along the beam line between the focal planes F7 and F8, which has been considered in the determination of the correction factor χ in Eq. (2), is shown enlarged in the upper part of the ﬁgure. information – limited to the one-body projectile density. For one- proton knockout from the semi-magic 134Sn projectile, the experi- mental cross section is also compared to the eikonal model, direct reaction calculations, as discussed above [3,4], in which the pro- jectile structure is taken into account through the single-nucleon overlap functions with the ﬁnal states of interest and the reaction dynamics are described based on the complex nucleon- and resid- ual nucleus-target optical potentials. on the structure of the projectile initial and residual nucleus ﬁnal states is combined with an approximate description of the reaction dynamics. In most cases this structure information is taken from shell-model calculations employing appropriate model spaces and effective interactions. 2. Experiment and results From this data, an experimen- tal loss factor, ϵline = 0.946(14), has been deduced for 136Te which includes both losses due to reactions on beam line detectors and the particle identiﬁcation eﬃciency of the ZeroDegree spectrom- eter. In a second step, the losses due to reactions in the C target, ϵtarget, have been determined with the target inserted based on the number of projectile ions detected in BigRIPS and ZeroDegree (sep- arately for each charge state and taking into account the respective ZeroDegree transmission) and ϵline. The value of ϵtarget = 0.931(21) determined in this way is in perfect agreement with the result of a LISE++ calculation, ϵLI SE target = 0.9309. Further details about the data analysis procedure are provided in Ref. [21]. detected in the ZeroDegree spectrometer in three different charge states, namely fully-stripped, hydrogen-like and helium-like. This results in rather complex A/q distributions as illustrated in Fig. 2c) which shows the A/q distribution for ions with a reconstructed Z in the range Z = 51.5-52.5. This ﬁgure demonstrates that the A/q resolution of the ZeroDegree spectrometer is suﬃcient to enable a reliable determination of the number of ions for the reaction prod- ucts populated following the removal of up to six neutrons. The cross section for the knockout of a number of neutrons, xn, and protons, yp, from the projectile can be determined from the number of projectile ions impinging on the target, Npro, the number of reaction products in the knockout channel of interest, Nrp, and the number of C atoms per cm2 in the target, n: σrp = Nrp n · Npro (1) σrp = Nrp n · Npro (1) with n = d · N A/Mmol, calculated from the thickness d = 534(27) mg/cm2 and the molar mass Mmol of the target and the Avogadro constant N A. Assuming that the losses due to reactions on beam line detectors (plastic detectors, PPACs and MUSICs, see Fig. 1) as well as the eﬃciency of these detectors is the same for both ion species, the ratio Nrp n·Npro in Eq. 2. Experiment and results (1) can be substituted by the ratio between the respective numbers of ions detected in the ZeroDe- gree spectrometer, N Z D rp n·N Z D pro , and two correction factors, Trel and χ: Following the approach sketched above 0pxn and 1pxn removal cross sections have been determined for all projectiles which have been produced and identiﬁed in BigRIPS with suﬃcient statistics. The results are summarized in Fig. 4 and will be discussed in the following. σrp = N Z D rp n · N Z D pro · Trel · χ (2) (2) Trel is the ratio between the ZeroDegree transmissions for the reaction product and the projectile, Trel = Trp/T pro, and the fac- tor χ accounts for the production of the nucleus of interest in reactions on other than the target material. Note that the cross section has to be calculated separately for each charge state (com- pare Fig. 2) according to Eq. (2) since Trel varies. To determine Trel the spatial distribution of the projectile ions in horizontal di- rection at the F5 momentum-dispersive focal plane is used. Fig. 3 shows the distributions of 136Te ions separately for those events in which fully stripped 130−135Te ions, populated via the removal of one to six neutrons from the 136Te projectile, were detected in the ZeroDegree spectrometer. They are compared to the correspond- ing distribution when fully stripped 136Te ions were detected in the ZeroDegree spectrometer, in each case normalized to the right 2. Experiment and results In each case, the latter has been downscaled so that both curves coincide on the right wing of the distributions. The resulting values of Trel are quoted for each case. Fig. 3. Distributions of 136Te ions in x direction at the dispersive F5 focal plane for those events in which the isotopes 130−135Te, populated following the removal of one to six neutrons, were detected in the ZeroDegree spectrometer (red lines) com- pared to the distribution obtained when unreacted 136Te ions were detected (black lines). In each case, the latter has been downscaled so that both curves coincide on the right wing of the distributions. The resulting values of Trel are quoted for each case. Fig. 2. a) BigRIPS and b) ZeroDegree particle identiﬁcation plots, the latter for inci- dent 136Te ions detected and identiﬁed in BigRIPS. c) Projection of the matrix shown in b) in the range Z = 51.5-52.5. The peaks corresponding to the removal of one to six neutrons from the 136Te projectile ions detected in the three different charge states are labelled by numbers. Fig. 3. Distributions of 136Te ions in x direction at the dispersive F5 focal plane for those events in which the isotopes 130−135Te, populated following the removal of one to six neutrons, were detected in the ZeroDegree spectrometer (red lines) com- pared to the distribution obtained when unreacted 136Te ions were detected (black lines). In each case, the latter has been downscaled so that both curves coincide on the right wing of the distributions. The resulting values of Trel are quoted for each case. Fig. 2. a) BigRIPS and b) ZeroDegree particle identiﬁcation plots, the latter for inci- dent 136Te ions detected and identiﬁed in BigRIPS. c) Projection of the matrix shown in b) in the range Z = 51.5-52.5. The peaks corresponding to the removal of one to six neutrons from the 136Te projectile ions detected in the three different charge states are labelled by numbers. wing of the distributions which is not cropped by the ZeroDegree acceptance. After this normalization, Trel is then simply obtained as the ratio between the integrals of the two curves. The second correction factor in Eq. (2), χ, takes into account that the nuclei of interest are not only produced in reactions taking place in the C target but also in the detector material along the beam line. 2. Experiment and results Finally, the reaction products as well as the elastically scattered beam ions were identiﬁed by the ZeroDegree spectrome- ter [18] using again the previously described E-Bρ-TOF method. Three slightly different ZeroDegree settings have been used during the experiment. As an example Fig. 2b) shows the ZeroDegree par- ticle identiﬁcation following the interaction of 136Te ions with the C target from a run in which this nucleus moved on the central tra- jectories in both BigRIPS and ZeroDegree. As clearly visible in this ﬁgure, the 136Te ions, as well as all other reaction products, are In this Letter, we report on the measurement of one- and multi- nucleon removal cross sections from a number of neutron-rich nuclei beyond the N = 82 shell closure, which have been pro- duced with energies around 165 MeV/u at the Radioactive Isotope Beam Facility (RIBF) at RIKEN. The experimental cross sections are compared to the results of calculations performed with the Liege intranuclear cascade model [14–17]. This approach, that describes the nuclear collisions based on a cascade of in-medium, two-body nucleon-nucleon collisions, involves only minimal nuclear structure V. Vaquero et al. / Physics Letters B 795 (2019) 356–361 358 Fig. 2. a) BigRIPS and b) ZeroDegree particle identiﬁcation plots, the latter for inci- dent 136Te ions detected and identiﬁed in BigRIPS. c) Projection of the matrix shown in b) in the range Z = 51.5-52.5. The peaks corresponding to the removal of one to six neutrons from the 136Te projectile ions detected in the three different charge states are labelled by numbers. Fig. 3. Distributions of 136Te ions in x direction at the dispersive F5 focal plane for those events in which the isotopes 130−135Te, populated following the removal of one to six neutrons, were detected in the ZeroDegree spectrometer (red lines) com- pared to the distribution obtained when unreacted 136Te ions were detected (black lines). In each case, the latter has been downscaled so that both curves coincide on the right wing of the distributions. The resulting values of Trel are quoted for each case. Fig. 3. Distributions of 136Te ions in x direction at the dispersive F5 focal plane for those events in which the isotopes 130−135Te, populated following the removal of one to six neutrons, were detected in the ZeroDegree spectrometer (red lines) com- pared to the distribution obtained when unreacted 136Te ions were detected (black lines). 2. Experiment and results More precisely, χ is the ratio between the reactions on the target and the reactions on all material between the ion identiﬁcation in Bi- gRIPS, i.e. the MUSIC ionization chamber at F7, up to and including the target. As shown in Fig. 1 there are several plastic and PPAC de- tectors on the beam line in which the reaction of interest can take place. For the present experimental conditions, a value χ = 0.83(7) has been obtained with the help of LISE++ calculations [20]. The reliability of this approach has been investigated using the data taken with an empty target frame. From this data, an experimen- tal loss factor, ϵline = 0.946(14), has been deduced for 136Te which includes both losses due to reactions on beam line detectors and the particle identiﬁcation eﬃciency of the ZeroDegree spectrom- eter. In a second step, the losses due to reactions in the C target, ϵtarget, have been determined with the target inserted based on the number of projectile ions detected in BigRIPS and ZeroDegree (sep- arately for each charge state and taking into account the respective ZeroDegree transmission) and ϵline. The value of ϵtarget = 0.931(21) determined in this way is in perfect agreement with the result of a LISE++ calculation, ϵLI SE target = 0.9309. Further details about the data analysis procedure are provided in Ref. [21]. wing of the distributions which is not cropped by the ZeroDegree acceptance. After this normalization, Trel is then simply obtained as the ratio between the integrals of the two curves. The second correction factor in Eq. (2), χ, takes into account that the nuclei of interest are not only produced in reactions taking place in the C target but also in the detector material along the beam line. More precisely, χ is the ratio between the reactions on the target and the reactions on all material between the ion identiﬁcation in Bi- gRIPS, i.e. the MUSIC ionization chamber at F7, up to and including the target. As shown in Fig. 1 there are several plastic and PPAC de- tectors on the beam line in which the reaction of interest can take place. For the present experimental conditions, a value χ = 0.83(7) has been obtained with the help of LISE++ calculations [20]. The reliability of this approach has been investigated using the data taken with an empty target frame. 3. Discussion To describe the experimental 0pxn and 1pxn removal cross sections shown in Fig. 4 calculations were performed using two different versions of the Liege intranuclear cascade model (INCL) [14]. This model, which originally had been developed for the de- scription of spallation reactions induced by nucleons, has been extended a few years ago to reactions induced by light ions [15] and in this latter version it can be applied to the experiment discussed here. In this standard version of the model, identical Woods-Saxon type density distributions are used for protons and neutrons. To describe the de-excitation process following the ini- tial cascade stage, the ABLA07 statistical de-excitation model is V. Vaquero et al. / Physics Letters B 795 (2019) 356–361 359 V. Vaquero et al. / Physics Letters B 795 (2019) 356–361 V. Vaquero et al. / Physics Letters B 795 (2019) 356–361 359 Fig. 4. Comparison between experimental inclusive removal cross sections and the results of calculations performed with the INCL code for a) the 0pxn and b) the 1pxn removal channels. The results obtained following the standard INCL approach [15] are shown as dashed blue lines while the calculations considering realistic proton and neutron densities from HFB calculations and fuzziness parameters f = 0.3 for neutrons and f = 0.5 for protons [17] are shown as solid black lines. In each case the neutron separation energy Sn in MeV [22] of the nucleus populated following a) one-neutron or b) one-proton knockout is quoted. The experimental cross sections for 0pxn removal from 112Sn shown in a) are taken from Ref. [8]. Fig. 4. Comparison between experimental inclusive removal cross sections and the results of calculations performed with the INCL code for a) the 0pxn and b) the 1pxn removal channels. The results obtained following the standard INCL approach [15] are shown as dashed blue lines while the calculations considering realistic proton and neutron densities from HFB calculations and fuzziness parameters f = 0.3 for neutrons and f = 0.5 for protons [17] are shown as solid black lines. In each case the neutron separation energy Sn in MeV [22] of the nucleus populated following a) one-neutron or b) one-proton knockout is quoted. The experimental cross sections for 0pxn removal from 112Sn shown in a) are taken from Ref. [8]. clei, both the magnitude and the gentle odd-even staggering of the cross sections is nicely reproduced by both calculations. 3. Discussion In contrast, none of them correctly describes the measured cross sec- tions for one- and two-neutron removal from the N = 84 isotones 134Sn and 135Sb, while for the heavier isotones 136Te and 137I the modiﬁcations of the INCL model discussed above clearly improve the agreement with experiment. Taking into account the peculiar structure of nuclei such as 134Sn, with only two valence neutrons above the N = 82 shell gap, in combination with the low neu- tron separation energy, Sn, of the 1n daughter, the failure of the calculations is easily understood [13]. In these cases only the re- moval of one of the two valence neutrons leads to the population of bound states in the daughter nuclei and thus contributes to the one-neutron removal cross section, while due to the large shell gap the knockout of a neutron from the closed N = 82 core pop- ulates core-excited states with energies well above the neutron separation energy. These highly-excited states then mainly decay via neutron emission and thus contribute to the measured two- neutron removal cross section. The INCL model, which ignores the shell structure of the nucleus and assumes a continuous energy distribution of the nucleons, is not able to correctly distinguish between knockout from the valence space on the one hand side and removal from the closed core on the other, but reproduces well the sum of the one- and two-neutron removal cross sections as well as the ones for the removal of more than two neutrons. So the conclusion from Fig. 4a) is that for the knockout of neu- trons, i.e. the less bound nucleon species in the neutron-rich nuclei employed [23]. Very recently several reﬁnements have been intro- duced in the description of the cascade stage of the model aiming for an improvement of the agreement with experiment, in partic- ular for the one-proton knockout channel [16,17]. Two important modiﬁcations have been applied: ﬁrst, more realistic proton and neutron radial density distributions are employed which are ob- tained either from Hartree-Fock-Bogoliubov (HFB) calculations with a Skyrme interaction [17] or shell model calculations [16]. This re- ﬁnement may become relevant in the case of heavy neutron-rich nuclei such as the ones studied in the present work. The sec- ond modiﬁcation intents to partially compensate for the neglect of quantum-mechanical effects in the naive INCL picture of the nu- cleus. Table 1 Table 1 Calculation of the inclusive one-proton removal cross section from 134Sn, σth, based on the excitation energies, Ex, and spectroscopic factors, C2S, predicted by shell model calculations (see text for details). Iπ Ex (MeV) C2S σsp (mb) σth(α) (mb) 9/2+ 1 0.00 9.3 3.26 31.2 9/2+ 2 0.81 0.3 3.18 1.0 1/2− 1 0.57 1.9 3.53 6.9 3/2− 1 1.18 1.2 3.46 4.2 3/2− 2 1.62 2.5 3.40 8.7 sum 15.2 52.0 Turning now to the one-proton knockout cross sections, Fig. 4b) clearly shows that both calculations fail to reproduce the experi- mental values for all three studied N = 84 projectiles, i.e. 134Sn, 135Sb and 136Te. Note, however, that in this case the reﬁnements, which have been introduced in the modiﬁed version of the INCL code, have a much stronger effect as compared to the case of one-neutron knockout, reducing the calculated one-proton knock- out cross sections by roughly a factor of two. Already in the past, a similar overestimation of the cross sections for one-proton re- moval from heavy nuclei by the INCL model has been reported, see for example Refs. [8,16]. In Ref. [24], Glauber model calculations coupled to the ABLA07 code have been performed to describe the one-proton knockout from various Sn projectiles on a C target at higher energies as compared to the present work and also here the calculations yielded far too high cross sections. In that work, this deﬁciency was cured by an arbitrary increase of the excitation en- ergy of the knockout residue after the cascade stage by 7 MeV. In this way it was possible to adjust the calculated cross sections to experiment. A similar approach, namely an ad hoc increase of the excitation energy before the deexcitation stage, was also followed in the INCL calculations presented in Ref. [25] in order to improve the agreement for a large set of experimental one-proton and one- neutron knockout cross sections. In this case, however, not a con- stant value as in Ref. [24] but in each case the difference between projectile and daughter separation energies was added to the INCL excitation energy at the end of the cascade stage (for details see Ref. [25]). Table 1 As discussed in the introduction, this model approach has the property that it connects measured knockout cross sections with theoretically-predicted spectroscopic information, namely the spectroscopic factors, C2S(α, jπ). How- ever, to apply the model in the region around 132Sn, it should be clariﬁed if the experimental to theoretical inclusive one-nucleon removal cross section ratio (Rs) systematics of Refs. [1,2] are ap- propriate also for these heavy nuclear systems. The theoretical inclusive one-nucleon removal cross section, σth, is calculated as the sum of the partial cross sections, Eq. (3), to each bound ﬁnal state of the reaction product. It is assumed that excited ﬁnal states above the neutron separation energy de- cay exclusively by particle emission. So, this calculation requires knowledge of the energies and spectroscopic strengths of the ﬁ- nal states of the daughter nucleus and has to rely on nuclear structure calculations, performed for example in the frame of the nuclear shell model. For most of the one-nucleon knockout reac- tions studied in the present work, i.e. one-neutron knockout from N > 82 and one-proton knockout from Z > 50 nuclei, the calcu- lation of the inclusive cross sections involves large uncertainties due to the unknown excitation energies of the many core-excited states populated in the daughter nuclei. Therefore, unfortunately, in these cases no meaningful conclusion can be drawn from the measured cross sections. The situation is different in the case of one-proton knockout from proton-magic 134Sn projectiles, in which the experimental cross section can be compared to the theoretical model value calculated using Eq. (3). Based on spherical Hartree- Fock and shell model calculations it is assumed that bound states in 133In are populated after knockout from the 1p3/2, 1p1/2, and 0g9/2 orbitals. Shell-model calculations were carried out employ- ing the realistic effective interaction for the N ≥82, Z ≤50 valence space, as were discussed recently in Ref. [26]. For the 1p3/2 and 1p1/2 proton-hole single particle energies, relative to the 0g9/2 or- bital, the experimental energies of the (3/2−) and (1/2−) states in 131In, namely 1353 and 365 keV [27,28], were employed. Since no 133In excited states information is available from experiment, the removal-reaction calculations use both the shell model excitation energies and spectroscopic factors, listed in Table 1. Table 1 It is important to notice, however, that any ad hoc in- crease of the excitation energy of the knockout residue not only leads to the desired decrease of the one-proton knockout cross sec- tion, but necessarily implies at the same time an increase of the probability for neutron emission and thus higher cross sections for other reaction channels, in particular 1p1n and 1p2n (one-proton knockout followed by the emission of one or two neutrons), for which unfortunately no experimental results have been reported in Refs. [24,25]. The overall good agreement between calculation and experiment observed in Fig. 4b) for the 1pxn channels with x > 0, and in particular the values measured for the 1p1n removal from 135Sb and 136Te, suggest that although seemingly allowing to cure the discrepancy for the one-proton knockout channel, an ad hoc increase of the excitation energy in the INCL calculations is not the right approach to follow in order to uncover the origin of the widely recognized problem the INCL model has in reproducing cross sections for the removal of the more bound nucleon species. one-nucleon removal reaction residue in a particular ﬁnal state re- ﬂects the parentage of this conﬁguration in the ground-state wave function of the projectile. The partial cross section for the removal of a nucleon from a single-particle orbital jπ , leading to a given ﬁ- nal state α with excitation energy E⋆α in the mass A −1 residue is given by σth(α) = (A/(A −1))N · C2S(α, jπ) · σsp( j, S⋆ α) (3) (3) where C2S(α, jπ) is the spectroscopic factor and σsp( j, S⋆α) the single-particle cross section which depends on the effective sepa- ration energy S⋆α = Sn,p + E⋆α [2]. As discussed in the introduction, this model approach has the property that it connects measured knockout cross sections with theoretically-predicted spectroscopic information, namely the spectroscopic factors, C2S(α, jπ). How- ever, to apply the model in the region around 132Sn, it should be clariﬁed if the experimental to theoretical inclusive one-nucleon removal cross section ratio (Rs) systematics of Refs. [1,2] are ap- propriate also for these heavy nuclear systems. where C2S(α, jπ) is the spectroscopic factor and σsp( j, S⋆α) the single-particle cross section which depends on the effective sepa- ration energy S⋆α = Sn,p + E⋆α [2]. 3. Discussion A fuzziness parameter is introduced to mimic the fact that in the quantum-mechanical square-well problem, the density outside the well does not vanish, in contrast to the classical INCL picture. The full details and the reasoning behind the applied changes are given in Refs. [16,17]. In the present work, calculations were per- formed using the HFB densities and standard fuzziness parameters of f = 0.3 for neutrons and f = 0.5 for protons [17]. The re- sults of the calculations using the standard and reﬁned versions of the model are shown as dashed blue and solid black lines, re- spectively, in Fig. 4. This ﬁgure shows an overall good agreement between the calculations and the experimental results. In particu- lar for the 0pxn removal from the N = 83 projectiles 133Sn, 134Sb, and 135Te as well as the stable 112Sn [8] (left column in Fig. 4a), i.e. the cases in which nuclear structure effects are washed-out due to the high neutron-separation energy of the 1n daughter nu- V. Vaquero et al. / Physics Letters B 795 (2019) 356–361 360 under study, the INCL model describes the experimental results re- markably well as long as nuclear structure effects are negligible. This is even more notable considering that a signiﬁcant fraction of the total calculated cross section corresponds to the quasi-elastic channel, i.e. events in which the projectile is ﬁrst excited to high excitation energies followed by the evaporation of one or several nucleons. For example, in the case of the one-neutron knockout reactions studied in the present work, roughly one third of the to- tal cross section corresponds to such two-step processes. Note that these are not considered in the eikonal direct reaction model and therefore, if they indeed turn out to be signiﬁcant in the region of the nuclear chart discussed in the present work, will need to be taken into account when extracting nuclear structure information from measured one-nucleon knockout cross sections. Table 1 Calculation of the inclusive one-proton removal cross section from 134Sn, σth, based on the excitation energies, Ex, and spectroscopic factors, C2S, predicted by shell model calculations (see text for details). Table 1 For all three orbitals listed above, at least 92% of the full strength is carried by As outlined in the introduction, besides the classical reaction models such as INCL, eikonal direct reaction theory has been used extensively for the calculation of one-nucleon knockout cross sec- tions [3,4]. A basic assumption is that, in the fast, single-nucleon removal from near the surface of a fast-moving projectile with mass A impinging on a light target, the remaining A −1 nucle- ons act as spectators. As a consequence, the probability to ﬁnd the V. Vaquero et al. / Physics Letters B 795 (2019) 356–361 361 the ﬁrst two states of each spin, lying below the neutron separa- tion energy of 133In (Sn = 3.13(21) MeV [22]). approach which has been suggested to cure the incapacity of the model to correctly describe the removal of deeply bound nucleons, does not address the origin of this problem. Finally, the experimen- tal inclusive cross section for one-proton removal from semi-magic 134Sn was compared with calculations based on eikonal direct re- action theory with structure information from the nuclear shell model. The limit this places on the derived suppression factor, Rs, is lower than from the systematics derived from similar analyses of inclusive cross section data for lighter nuclei, and alerts that more experimental information is needed before such one-nucleon removal reaction systematics should be used to deduce spectro- scopic information in the region of heavy nuclei around 132Sn. A theoretical cross section of σth = 52.0 mb is obtained. Re- garding the fourth orbital of the Z = 28–50 shell, namely 0 f5/2, the single-hole energy of this orbital is experimentally unknown and thus no reliable prediction can be made as to whether knock- out will lead to bound states in 133In. We therefore exclude it from the calculation, likewise the small missing strengths from the or- bitals considered above, and thus take the calculated theoretical cross section as a lower limit, σth > 52.0 mb. From this and the ex- perimental value, σexp = 13(2) mb, we determine an approximate upper limit on the suppression factor, Rs = σexp/σth < 0.25(4). The associated separation energy asymmetry, S, calculated from the cross-section weighted average of the S⋆α values to the bound ﬁnal states is S = 13.1 MeV. Comparison with the light nu- cleus Rs systematics [1,2], using the approximate parametrization of Ref. Instrum. Methods B 317 (2013) 323. [20] D. Bazin, et al., Nucl. Instrum. Methods A 482 (2002) 307. [21] Victor Vaquero Soto, PhD thesis, Universidad Autónoma de Madrid, 2018. [21] Victor Vaquero Soto, PhD thesis, Universidad Autónoma de Madrid, 2018. [22] M. Wang, et al., Chin. Phys. C 36 (2012) 1603. [23] A. Kelic, M.V. Ricciardi, K.-H. Schmidt, Joint ICTP-IAEA Advanced Workshop on Model Codes for Spallation Reactions, Report INDC(NDC)-0530, IAEA, Trieste, Italy, 2008, p. 181. [23] A. Kelic, M.V. Ricciardi, K.-H. Schmidt, Joint ICTP-IAEA Advanced Workshop on Model Codes for Spallation Reactions, Report INDC(NDC)-0530, IAEA, Trieste, Italy, 2008, p. 181. Table 1 [6], one would expect an Rsys s ≈0.4 for such a value of S. In the range S = 12-14 MeV, four Rs values have been de- rived, the two most accurate for one-proton knockout being for 10Be (Rs = 0.42(2) [29]) and 36Si (Rs = 0.39(2) [5]). These values signiﬁcantly exceed the limit suggested from the present analysis. Acknowledgements We thank the staff of the RIKEN Nishina Center accelerator complex for providing high-intensity beams to the experiment. This work was supported by the Spanish Ministerio de Economía y Competitividad under contracts FPA2014-57196-C5-4-P and FPA2017-84756-C4-2-P. J.A.T. acknowledges the support of the Sci- ence and Technology Facilities Council (UK) grant ST/L005314/1 and R.O. that of JSPS KAKENHI Grant No. 26887048. G.M.T. grate- fully acknowledges funding of this research from the Research Council of Norway, Project Grant No. 222287. g y gg p y We note that, if one employs a single-hole energy for the 0 f5/2 orbital (relative to 0g9/2) of 2.6 MeV, as has been used in the literature [27,30–33], then the shell model calculation used here attributes a spectroscopic factor of 4.7 to the sixth 5/2−state at an excitation energy of 2.68 MeV, below Sn, so that Rs would be fur- ther reduced. Spherical Skyrme Hartree-Fock calculations, on the other hand, using the SkX and Sly4 interactions, place this 0 f5/2 hole energy at 4-5 MeV [34,35]. An energy of 3.8 MeV is ex- pected on the basis of the nuclear monopole Hamiltonian which was adjusted to a large number of experimental energies of parti- cle and hole states outside double magic cores all over the chart of nuclides by Duﬂo and Zuker [36]. Furthermore, the Rs analysis presented above relies on high purities of the proton-hole states in 131In (similar to the ones measured for neutron-hole and neutron- particle states in 131Sn and 133Sn, respectively [37,38]) and that the shell-model calculations provide a realistic treatment of the effects of nucleon-nucleon correlations upon the valence orbitals. Clearly, more exclusive experimental information is required to validate these assumptions in order that one-nucleon removal reactions might be used to extract spectroscopic information in the region around 132Sn. As such data become available for nuclei south-east of 132Sn, then alternative shell-model approaches using extended valence spaces, see e.g. Refs. [39–41], can be used to assess the systematic uncertainties inherent in the nuclear structure model description presented here. 4. Summary 24] J.L. Rodríguez-Sánchez, et al., Phys. Rev. C 96 (2017) 034303. 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https://openalex.org/W3195694152	https://pedagogia.umsida.ac.id/index.php/pedagogia/article/download/280/743	Indonesian	null	Inclusive Practices: Strengthening Character Through Social Participation of Deaf Students	Pedagogia	2,020	cc-by	7,671	RESEARCH ARTICLE published: 21 April 2020 doi: 10.21070/pedagogia.v9i2.280 RESEARCH ARTICLE published: 21 April 2020 doi: 10.21070/pedagogia.v9i2.280 Keywords: Inclusive Education, Character Education, Social Participation, Deaf Deaf Keywords: Inclusive Education, Character Education, Social Participation, Deaf Deaf Keywords: Inclusive Education, Character Education, Social Participation, Deaf Deaf Penelitian ini bertujuan untuk mengamati dan menganalisis praktek partisipasi sosial mahasiswa tunarunggu dalam mewujudkan penguatan karakter di kampus inklusif Uni- versitas Lambung Mangkurat Banjarmasin. Responden penelitian sebanyak 31 orang dalam satu kelas, terdiri dari 5 orang mahasiswa tunarungu dan 26 orang mahasiswa sebaya lainnya. Partisipasi sosial diteliti melalui tiga elemen kunci yaitu: hubungansosial pertemanan, penerimaan teman sekelas, dan persepsi mahasiswa. Penelitian menggu- nakan pendekatan kuantitatif dengan metode survey. Teknik pengumpulan data meng- gunakan wawancara, angket, dan dokumentasi. Teknik analisis data menggunakan statistik deskriptif. Hasil penelitian menunjukkan keberhasilan penguatan pendidikan karakter melalui partisipasi sosial dalam setting inklusif bagi mahasiswa tunarunggu den- gan teman sebayanya atau sebaliknya. Edited by: Rifki Afandi Reviewed by: Emy Pratiwi Correspondence: Amka Amka amka.plb@ulm.ac.id Received: 7 April 2020 Accepted: 14 April 2020 Published: 21 April 2020 Inclusive Practices: Strengthening Character Through Social Participation of Deaf Students Praktek Inklusif : Penguatan Karakter Melalui Partipasi Sosial Mahasiswa Tunarungu Amka Amka, Mirnawati Mirnawati Program Studi Pendidikan Khusus, Universitas Lambung Mangkurat,, Banjarmasin, Indonesia Program Studi Pendidikan Khusus, Universitas Lambung Mangkurat,, Banjarmasin, Indonesia This study aims to observe and analyze the practice of social participation of students with hearing impairment in realizing character strengthening in the inclusive campus of Lambung Mangkurat University, Banjarmasin. The research respondents were 31 peo- ple in one class, consisting of 5 deaf students and 26 other peer students. Social par- ticipation is researched through three key elements, namely: friendship social relations, acceptance of classmates, and student perceptions. This research uses a quantitative approach with a survey method. Data collection techniques using interviews, question- naires, and documentation. The data analysis technique used descriptive statistics. The results showed the success of strengthening character education through social partici- pation in an inclusive setting for students with hearing impairment with their peers or vice versa. ISSN 2548 2254 (online) ISSN 2089 3833 (print) ISSN 2548 2254 (online) ISSN 2089 3833 (print) Edited by: Rifki Afandi Reviewed by: Emy Pratiwi *Correspondence: Amka Amka amka.plb@ulm.ac.id Received: 7 April 2020 Accepted: 14 April 2020 Published: 21 April 2020 Pendidikan Karakter Tema pendidikan karakter seakan tidak pernah selesai untuk didiskusikan. Dari waktu ke waktu terus saja menjadi sorotan bila dikaitkan dengan proses dan hasil pendidikan berupa peri- laku siswa sehari-hari. Berbagai rumusan terus saja ditawarkan oleh para pakar pendidikan, psikologi, dan ilmu sosial lainnya. Rumusan yang ditawarkan sangat beragam tekanannya, ter- gantung dari sudut pandang dan latar belakang keilmuan. Namun satu hal yang menjadi kesep- akatan bahwa karakter selalu dikaitkan dengan persoalan manusia yang baik dalam berbicara dan bertindak. Pendidikan karakter bertujuan menghasilkan kualitas manusia bernilai baik dan benar, berdimensi universal, tidak sektoral, dapat diterima di setiap tempat dan waktu, yang diwujudkan dalam tindakan bernilai manfaat bagi orang lain dan lingkungan. Jones and Stoodley (1999) dan Williams (2000), berpendapat bahwa pendidikan karakter ditujukan pada pengembangan ”kualitas pribadi,” atau pengembangan komunitas dalam konteks kehidupan. Dari perspektif kualitas pribadi, pendidikan karakter adalah pengajaran yang bertujuan men- gajarkan ”mengetahui yang baik, mencintai yang baik, dan melakukan yang baik” Bebeau et al. (1999) . Schaps and Lewis (1999) mengonseptualisasikan pendidikan karakter sebagai instruksi yang mencari pengembangan ”peduli, berprinsip, dan bertanggung jawab”. Enam karakteristik dasar tujuan pendidikan yang sering digambarkan adalah: kebiasaan moral, alasan moral, keba- jikan, nilai-nilai, pengambilan keputusan individu, dan tanggung jawab dalam konteks kehidu- pan. Martinson (2003); Brogan and Brogan (1999); Jones and Stoodley (1999); Lickona (1999) Pendidikan karakter bukanlah sekedar untuk pengembangan pikiran dan perasaan, tetapi lebih dari itu adalah pendidikan karakter berpusat pada hati, untuk melahirkan kekuatan piki- ran positif, kepekaan perasaan, dan kekuatan perilaku baik Azis (2012). Kekuatan karakter adalah dasar dari pengembangan dan pertumbuhan hidup yang optimal. Karakter yang baik bukan hal yang tunggal tetapi jamak, bersifat positif yang ditunjukkan dalam pikiran, perasaan, dan perilaku seseorang. Jika masyarakat benar-benar peduli dengan karakter yang baik di kalan- gan anak muda, kita harus menilai kekuatan dan memperhatikan bagaimana mereka berkem- bang. Pendidik dan orang tua sibuk mengukur kemampuan akademik anak dan memantau kemajuan belajar. Kami berharap bahwa suatu hari nanti sekolah juga akan menilai kekuatan karakter siswa dan juga mencatat kemajuan perkembangan mereka, pendidik dan pembuat kebijakan harus memperhatikan kekuatan karakter tertentu Park and Peterson (2009) . Peneli- tian secara konsisten menunjukkan bahwa kekuatan ”hati” seperti cinta dan syukur, keber- samaan, lebih kuat daripada kesejahteraan dan kekuatan pikiran yang bersifat individu, seperti kreativitas, berpikir kritis , dan penghargaan keunggulan Park and Peterson (2008); Park et al. (2004). Kami telah menemukan bahwa prestasi akademik siswa dipengaruhi oleh serangkaian kekuatan karakter. Citation: Amka A and Mirnawati M (2020) Inclusive Practices: Strengthening Character Through Social Participation of Deaf Students. PEDAGOGIA:Jurnal Pendidikan. 9:2. Agustus 2020 \| Volume 9 \| Issue 2 PEDAGOGIA:Jurnal Pendidikan \| ojs.umsida.ac.id/index.php/ 243 Inclusive Practices: Strengthening Character Through Social Participation of Deaf Students Amka et al. Pendidikan Inklusif Pelayanan pendidikan terhadap anak berkebutuhan khusus telah berubah derastis dalam beber- apa dekade terakhir. Mendidik anak-anak penyandang kebutuhan khusus telah menjadi bagian penting dari sekolah regular di banyak negara. Perkembangan pelayanan pendidikan anak berkebutuhan khusus digambarkan dengan istilah ’inklusi’. Menurut Rafferty et al. (2001) , inklusi mengacu pada ’proses mendidik anak-anak penyandang cacat di ruang kelas reguler di sekolah lingkungan mereka. Di Indonesia setiap warga negara berhak mendapatkan pen- didikan Dasar (1945). Layanan pendidikan yang setara dan tanpa diskriminasi kepada semua peserta didik adalah praktik pendidikan yang dijadikan kebijakan dalam sistem pendidikan Indonesia (UU No. 20 Tahun 2003). Pemberian hak pendidikan kepada setiap warga ner- aga adalah upaya mendukung inisiatif menyelenggarakan “Pendidikan Untuk Semua” yang dicanangkan oleh Perserikatan Bangsa-Bangsa dan deklarasi Salamanca (UNESCO, 1994) yang menegaskan bahwa cara terbaik untuk mencapai “Pendidkkan Untuk Semua” adalah melalui praktek inklusif di sekolah regular. Pendidikan inklusif berkaitan dengan upaya mengatasi hambatan yang mencegah partisipasi dan pembelajaran semua anak, terlepas dari ras, jenis kelamin, latar belakang sosial, gender, kecacatan, dan prestasi hasil belajar di sekolah Booth and Ainscow (2002). Lembaga pendidikan penyelenggara pendidikan inklusi mempunyai daya tarik tersendiri bagi orang tua anak berkebutuhan khusus, data terbaru menunjukkan bahwa semakin banyak orang tua memilih menyekolahkan anaknya yang berkebutuhan khusus di sekolah penyeleng- gara pendidikan inklusif, sehingga jumlah anak berkebutuhan khusus yang terdaftar di seko- lah penyelenggara pendidikan inklusif telah mengalami peningkatan secara substansial dalam beberapa dekade terakhir Ferguson (2008). Komitmen terhadap pendidikan inklusif di selu- ruh dunia terus meningkat Sharma et al. (2017) . Beberapa hasil penelitian menegaskan bahwa pendidikan inklusif dipandang sebagai upaya kolektif untuk memberi setiap siswa di komunitas sekolahnya berupa hak untuk memiliki Falvey and Givner (2005) . Pendidikan inklusif bertolak belakang dengan paradigma pendidikan terpisah yang membagi siswa dan memberikan pener- imaan bersyarat berdasarkan pengukuran kemampuan, pendidikan inklusif bertujuan untuk merangkul semua orang dan menjadikan sekolah sebagai tempat bagi semua siswa Tarman- syah (2007). Komitmen ini berarti bahwa sekolah harus memasukkan sistem yang melibatkan sikap positif terhadap inklusi dan serangkaian strategi agar berhasil untuk mengikutsertakan semua siswa. Selain itu, pendidikan inklusif membutuhkan upaya bersama oleh guru, kelu- arga, siswa dan kepala sekolah untuk merancang kebijakan inklusif, membangun budaya positif dan menerapkan praktik yang meningkatkan tingkat pengajaran dan pembelajaran yang efek- tif Ainscow (1999) . Dengan demikian pendidikan inklusif merupakan filosofi hidup, belajar bersama, bermanfaat bagi semua orang, termasuk mahasiswa penyandang tunarunggu. Pendidikan Karakter Di antara siswa sekolah menengah, kekuatan karakter dari ketekunan, cinta, syukur, harapan, dan perspektif memprediksi prestasi akademik. Hasil serupa juga ditemukan di kalangan mahasiswa. Belajar terjadi tidak hanya kepada seseorang tetapi di antara mereka, dan kekuatan karakter dapat memfasilitasi proses pembelajaran Park and Peterson (2009). j Kekuatan karakter adalah aspek kepribadian yang dihargai secara moral. Baumrind and Competence and character through life (1998) mencatat, “Dibutuhkan karakter yang baik untuk menghendaki yang baik, dan kompetensi untuk melakukan yang baik”. Banyak pro- gram pendidikan tinggi dan sosial saat ini fokus pada membantu kaum muda memperoleh keterampilan dan kemampuan akademik seperti berpikir kritis. Ini membantu kaum muda mencapai tujuan hidup mereka, dan tentu saja penting, namun tanpa karakter yang baik, indi- vidu mungkin kurang memiliki keinginan untuk melakukan hal yang benar. Kekuatan karak- ter, ketika dilakukan, tidak hanya mencegah hasil kehidupan yang tidak diinginkan Botvin et al. (1995) tetapi penting untuk mewujudkan kehidupan peribadi yang berkembang sehat dalam hidupnya Colby and Damon (1992); Weissberg and Greenberg (1998). Semakin banyak bukti menunjukkan bahwa kekuatan karakter yang spesifik, misalnya harapan, kebaikan, kecer- dasan sosial, kontrol diri, dan pengendali efek negatif dari stres dan trauma, mencegah atau membatasi masalah. Selain itu, kekuatan karakter membantu kaum muda untuk berkembang mencapai keberhasilan pendidikan, kepemimpinan, toleransi dan penilaian keanekaragaman, kemampuan untuk menunda kepuasan, dan kebaikan Park et al. (2004) . Membangun keku- Agustus 2020 \| Volume 9 \| Issue 2 PEDAGOGIA:Jurnal Pendidikan \| ojs.umsida.ac.id/index.php/ PEDAGOGIA:Jurnal Pendidikan \| ojs.umsida.ac.id/index.php/ 244 244 Inclusive Practices: Strengthening Character Through Social Participation of Deaf Students Amka et al. atan karakter dipengaruhi oleh pola pengasuhan dan alam. Berbagai pengaruh berkontribusi pada pengembangan karakter seperti keluarga, sekolah, teman sebaya, dan komunitas Steger et al. (2007). Oleh karena itu dalam konteks pendidikan inklusif, upaya membangun partisipasi sosial anak berkebutuhan khusus dan anak sebaya lainya perlu diperhatikan oleh para pendidik dalam menguatkan pendidikan karakter bagi komunitas siswa secara inklusif. Partisipasi Sosial Banyak negara meninggalkan sistem sekolah khusus dan orang tua memilih sekolah regu- lar untuk anak mereka bersekolah Meijer et al. (2006). Walaupun orang tua ini memiliki motif berbeda untuk memilih pengaturan pendidikan reguler untuk anak mereka, mereka memilih sekolah biasa karena kemungkinan anak mereka untuk berpartisipasi secara sosial dalam kelompok sebaya. Orang tua berharap bahwa anaknya dapat partisipasi sosial Scheep- stra et al. (1999). Menurut Koster et al. (2010), istilah ’partisipasi sosial’ dapat dijelaskan bahwa Partisipasi sosial siswa berkebutuhan khusus dalam pendidikan reguler adalah adanya kon- tak/interaksi positif antara anak-anak ini dan teman-teman sekelasnya; penerimaan mereka oleh teman sekelas mereka; hubungan sosial/pertemanan antara mereka dan teman sekelas mereka dan persepsi murid bahwa mereka diterima oleh teman sekelas mereka. Meskipun par- tisipasi sosial menjadi salah satu motif dari orang tua, penelitian telah menunjukkan bahwa di sekolah reguler tidak secara otomatis mengarah pada peningkatan jumlah kontak dan pertem- anan dengan teman sebaya de Monchy et al. (2004). Siswa dengan berbagai jenis kecacatan mengalami kesulitan dalam memperoleh posisi sosial yang baik dalam pendidikan reguler. Beberapa penelitian menunjukkan bahwa anak-anak dengan kebutuhan khusus di sekolah biasa kurang diterima oleh teman-teman sebayanya, memiliki lebih sedikit persahabatan dan kurang sering menjadi bagian dari jaringan di kelas Bramston et al. (2002); Kuhne and Wiener (2000); Frostad and Pijl (2007); Yu et al. (2005). Karena hasil ini, tampak jelas bahwa partisipasi sosial layak mendapat perhatian lebih ketika menerapkan pendidikan inklusif. Karena motif utama orang tua dalam memilih sekolah reguler adalah kemungkinan bagi anak mereka untuk berin- teraksi secara sosial, dimensi sosial dipandang sebagai aspek penting dalam melaksanakan pen- didikan inklusif dengan sukses. Untuk menyelidiki apakah pendidikan inklusif berhasil, kami berpendapat untuk mengukurnya dalam hal hasil sosial siswa. Sebagian besar orang tua anak berkebutuhan khusus beranggapan bahwa mereka akan mendapatkan keuntungan positif dalam hal akademik jika menyekolahkan anak mereka di sekolah reguler penyelenggara pendidikan inklusif FREDERICKSON et al. (2004). Disisi lain, motif utama orang tua anak berkebutuhan khusus lebih memilih menyekolahkan anaknya di sekolah reguler penyelenggara pendidikan inklusif daripada di sekolah khusus atau di sekolah luar biasa karena orang tua beranggapan bahwa peluang sosial anak berkebutuhan khusus akan meningkat di sekolah inklusi Scheepstra et al. (1999). Orang tua sangat berharap anak mereka yang berkebutuhan khusus dapat membangun hubungan positif dengan teman sebaya di lingkungan sekolah, karena segala hambatan yang dialami anak berkebutuhan khusus berdampak pada kemampuannya dalam membina hubungan sosial yang baik dengan lingkun- gan sekitarnya. Mahasiswa Tunarungu Kebijakan pendidikan inklusif di Indonesia telah diberlakukan di perguruan tinggi (Peratu- ran Menteri Riset dan Teknologi no. 46 tahun 2017) Menteri (2017) . Berdasarkan regulasi tersebut Universitas Lambung Mangkurat Banjarmasin menerima berbagai jenis mahasiswa berkebutuhan khusus, termasuk mahasiswa tunarunggu. Tunarungu adalah seseorang yang mengalami kekurangan atau kehilangan kemampuan mendengar baik sebagian atau seluruh- nya, karena diakibatkan tidak berfungsinya alat pendengaran sebagian atau seluruh, dan mem- bawa dampak kehidupan secara kompleks Haenuddin (2013). Hambatan pendengaran menye- babkan anak sangat minim mendapatkan input bahasa, sehingga tunarungu mengalami kesuli- tan dalam berkomunikasi secara verbal. Pada umumnya, komunikasi tunarungu menggunakan bahasa isyarat, hal demikian pada akhirnya membatasi pergaulan tunarungu dengan sesama tunarungu saja Nur’Aeni (2017). Pendidikan Inklusif Pendidikan inklusif tidak hanya fokus pada hambatan yang dihadapi siswa tetapi juga, seperti Booth and Ainscow (2002) menyasarankan, berfokus pada pengembangan budaya, kebi- jakan dan praktik dalam sistem dan lembaga pendidikan, agar mereka dapat menanggapi ker- agaman siswa mereka dan memperlakukan mereka secara setara. Di sekolah inklusif keragaman disambut dan dianggap sebagai sumber pembelajaran dan bukan masalah. Fakta yang paling penting adalah bahwa pendidikan inklusif adalah proses yang berkelanjutan dan bukan tahap yang dapat kita capai pada saat tertentu.Secara umum, pendidikan inklusif adalah masalah yang kompleks dan kontroversial, sebuah topik yang sering menimbulkan perdebatan sengit di antara para pemangku kepentingan yang berbeda. Ainscow et al. (2000) menyatakan bahwa ada ketidakpastian dan kebingungan seputar makna ’pendidikan inklusif’ Petrou (2005) .Kehadi- ran siswa penyandang cacat memberikan kesempatan bagi semua siswa untuk belajar perbe- daan dan menunjukkan empati bagi seorang individu terhadap penyandang cacat yang berbeda Agustus 2020 \| Volume 9 \| Issue 2 PEDAGOGIA:Jurnal Pendidikan \| ojs.umsida.ac.id/index.php/ PEDAGOGIA:Jurnal Pendidikan \| ojs.umsida.ac.id/index.php/ 245 245 Inclusive Practices: Strengthening Character Through Social Participation of Deaf Students Amka et al. dengan rasa kasihan atau simpati. Siswa dapat belajar tentang perbedaan dan bermanfaat men- dukung individu penyandang cacat Shapiro (1994). Partisipasi Sosial Sebagaimana hasil penelitian menunjukkan bahwa siswa dengan kebutuhan khusus tidak secara otomatis mengarah pada peningkatan persahabatan di antara siswa den- gan kebutuhan khusus dan siswa tanpa kebutuhan khusus Buysse et al. (2002); Guralnick et al. (2007) menunjukkan bahwa hampir seperempat dari siswa dengan kebutuhan khusus Agustus 2020 \| Volume 9 \| Issue 2 PEDAGOGIA:Jurnal Pendidikan \| ojs.umsida.ac.id/index.php/ 246 Inclusive Practices: Strengthening Character Through Social Participation of Deaf Students Amka et al. mengalami kesulitan serius membentuk hubungan dengan rekan-rekan mereka. Beberapa penelitian telah menemukan bahwa siswa dengan gangguan spektrum autis dan siswa den- gan kelainan perilaku serius sangat sulit untuk membangun hubungan dengan teman sebaya dan berisiko terisolasi di kelas Chamberlain et al. (2007); de Monchy et al. (2004); Harrell et al. (1997). Beberapa penelitian di atas menunjukkan bahwa anak berkebutuhan khusus di sekolah penyelenggara pendidikan inklusif masih mengalami kesulitan dalam membina hubungan sosial dengan teman sebaya, kecenderungan sosial mereka masih terbatas pada teman sebaya yang juga mengalami kebutuhan khusus. Mereka kekurangan kontak dengan teman sebaya, keterampilan sosial mereka tidak berkembang serta membentuk konsep diri yang negatif Ben- der and Wall (1994). Konsep diri negatif mengarah ke eksternalisasi misalnya agresi dan inter- nalisasi misalnya kecemasan Durrant et al. (1990). Anak-anak dengan kebutuhan khusus cen- derung memegang posisi sosial yang terpisah di ruang kelas dari waktu ke waktu, yang menyi- ratkan bahwa isolasi adalah fenomena yang cukup stabil. Berdasarkan fenomena-fenomena yang diuraikan di atas, maka penting untuk memantau dan mengevaluasi sosial anak berke- butuhan khusus di lembaga pendidikan penyelenggara pendidikan inklusif mulai dari jenjang sekolah dasar sampai jenjang perguruan tinggi. Mengingat keberhasilan penyelenggaran pen- didikan inklusif bagi anak berkebutuhan khusus tidak terbatas pada ketercapaian pemberian layanan akademik semata, namun juga penting untuk mengembangkan hubungan sosial selu- ruh masyarakat sekolah. Dimensi sosial dipandang sebagai aspek penting dalam melaksanakan pendidikan inklusif dengan sukses Koster et al. (2009). Kriteria sebenarnya untuk berhasil menerapkan sistem yang lebih inklusif pada akhirnya tergantung pada apa yang terjadi di sekolah dan ruang kelas Ainscow (2007). Keberhasilan penerapan pendidikan inklusif bergantung pada keberadaan sistem pendukung, yang meliputi pelatihan guru, sumber daya untuk sekolah, dukungan sosial, dan partisipasi masyarakat, mis- alnya mengembangkan hubungan kolaboratif di antara staf dan dengan orang tua, serta hubun- gan kolaboratif dengan organisasi yang terlibat dalam masyarakat Kantavong (2018) . Per- masalahan yang dihadapi dalam mengevaluasi aspek sosial inklusi adalah ambiguitas konsep yang digunakan oleh peneliti. Seperti yang dijelaskan oleh Koster et al. Partisipasi Sosial (2010) , berbagai kon- sep diadopsi untuk menggambarkan dimensi sosial inklusi. Tiga konsep payung - yaitu, par- tisipasi sosial, integrasi sosial dan inklusi sosial - sering digunakan oleh para peneliti. Namun, ada ketidakjelasan tentang maknanya. Oleh karena itu, Koster et al. (2009) merekomendasikan menggunakan konsep ”partisipasi sosial”. Beberapa peneliti menggambarkan partisipasi sosial sebagai jumlah pertemanan antar siswa Harper et al. (1999); Hunt et al. (1996) , sedangkan yang lain menekankan pentingnya interaksi antara siswa atau konsep diri sosial mereka Kamps et al. (1999), atau cenderung untuk membatasi deskripsi partisipasi sosial mereka pada peneri- maan teman sebaya terhadap siswa dengan kebutuhan khusus Odom (2000). Analisis literatur mengungkapkan bahwa terdapat empat tema utama untuk mengukur partisipasi sosial yaitu: persepsi diri sosial siswa, penerimaan oleh teman sekelas, pertemanan / hubungan dan kontak / interaksi Koster et al. (2009), selanjutnya diadopsi dalam penelitian ini, namun peneliti hanya memungkinkan untuk fokus pada 3 tema partisipasi sosial yaitu hubungan pertemanan sosial, persepsi diri sosial, dan penerimaan oleh teman sebaya dalam satu kelas belajar. Penelitian ini mengkaji dan mendeskripsikan partisipasi sosial mahasiswa berkebutuhan khusus tunarunggu di program studi pendidikan khusus (PKh) Fakultas Keguruan dan Ilmu Pendidikan (FKIP) Universitas Lambung Mangkurat (ULM) Banjarmasin Indonesia sebagai perguruan tinggi penyelenggara pendidikan inklusif. METODE Penelitian ini dilakukan dengan menggunakan pendekatan kuantitatif yaitu mengungkap par- tisipasi sosial mahasiswa berkebutuhan khusus tunarungu Program Studi Pendidikan Khusus Fakultas Keguruan dan Ilmu Pendidikan Universitas Lambung Mangkurat. Metode pengumpu- lan data menggunakan metode survey. Menurut Sugiyono (2016) bahwa metode survey digu- nakan untuk mendapatkan data dari tempat tertentu yang alamiah (bukan buatan). Penggunaan Agustus 2020 \| Volume 9 \| Issue 2 PEDAGOGIA:Jurnal Pendidikan \| ojs.umsida.ac.id/index.php/ 247 247 Inclusive Practices: Strengthening Character Through Social Participation of Deaf Students Amka et al. metode survey akan memudahkan peneliti untuk memperoleh data untuk diolah dengan tujuan memecahkan masalah yang menjadi tujuan akhir suatu penelitian. Tema payung tiga dari empat tema partisipasi sosial yang diajukan Koster et al. (2009) yaitu hubungan pertemanan sosial, persepsi diri sosial, dan penerimaan oleh teman sebaya dalam satu kelas belajar, digunakan dan dikembangkan sebagai instrumen penelitian, dengan 5 opsi: sangat setuju, setuju, ragu-ragu, kurang setuju, dan tidak setuju. Sumber data pada penelitian ini adalah segala bentuk infor- masi baik berupa ucapan, ataupun tindakan dari responden terkait partispasi sosial mahasiswa berkebutuhan khusus tunarungu sasaran. Responden dalam penelitian ini adalah mahasiswa berkebutuhan khusus tunarungu sebanyak 5 orang, dan teman sekelas 26 orang. Pengumpulan data dilakukan bekerja sama dengan para volunteer, adapun teknik pengumpulan data yang digunakan ini adalah wawancara dan angket (kuesioner) serta dokumentasi. Teknik analisis data dalam penelitian ini menggunakan statistik deskriptif dengan rumus: Nilai = skor perolehan skor maksimal × 100% Teknik analisis data dalam penelitian ini menggunakan statistik deskriptif dengan rumus: Nilai = skor perolehan skor maksimal × 100% Nilai yang diperoleh kemudian dimaknai sesuai dengan panduan dibawah iniTabel 1 : Nilai = p skor maksimal × 100% Nilai yang diperoleh kemudian dimaknai sesuai dengan panduan dibawah iniTabel 1 : skor maksimal Nilai yang diperoleh kemudian dimaknai sesuai dengan panduan dibawah iniTabel 1 : [Table 1 about here.] [Table 1 about here.] Penguatan Karakter Dalam Hubungan sosial/pertemanan antara mahasiswa berkebutuhan khusus tunarungu dengan teman sekelas mereka. Penguatan pertemanan antara mahasiswa berkebutuhan khusus tunarungu dengan teman seke- las, menunjukkan hasil pada Tabel 2 . PEDAGOGIA:Jurnal Pendidikan \| ojs.umsida.ac.id/index.php/ [Table 2 about here.] (2009) Seperti yang dialami oleh Disca, salah seorang tunarungu yang mengalami masalah sosial, salah satunya karena bersekolah di sekolah reguler/ umum ia dapat mengerti bahasa temannya tetapi temannya tidak dapat mengerti bahasanya. komunikasi adalah proses di mana suatu ide dialihkan dari sumber kepada satu penerima atau lebih dengan maksud untuk mengubah tingkah laku yang pada gilirannya akan tiba pada saling pengertian yang mendalam. Pengalaman-pengalaman interaksi sosial yang seringkali sulit dan mengecewakan bagi tunarungu seperti kesulitan dalam memahami pembicaraan lawan bicara, keliru dalam mempersepsikan ekspresi dan bahasa tubuh orang lain, begitu pun sebaliknya lawan bicara tidak mampu memahami maksud dari tunarungu. Yull (2004) dalam W.R. (2009) Seperti yang dialami oleh Disca, salah seorang tunarungu yang mengalami masalah sosial, salah satunya karena bersekolah di sekolah reguler/ umum ia dapat mengerti bahasa temannya tetapi temannya tidak dapat mengerti bahasanya. p y p g y Kondisi demikian membentuk anak tunarungu menjadi pribadi yang kurang bahkan tidak memiliki rasa percaya diri. Rasa percaya diri adalah satu di antara aspek-aspek kepribadian yang penting dalam kehidupan manusia. Rasa percaya diri sangat membantu manusia dalam perkembangan kepribadiannya. Karena itulah rasa kepercayaan diri sangat dibutuhkan manu- sia dalam menjalani hidupnya termasuk pada anak tunarungu. Dalam hubungan dengan par- tisipasi sosia, mahasiswa tunarungu sebagian besar bersikap ragu-ragu atau tidak setuju memi ta bantuan kepada teman sekelasnya atas masalah yang dihadapi. Hal tersebut terjadi kemungk- inan disebabkan karena mahasiswa tunarungu terkendala dalam menyampikan masalahnya ke teman sekelas, mahasiswa tunarungu tidak realistis akan kondisi dirinya yang mengalami keter- batasan sehingga mutlak membutuhkan orang lain sebagai makhluk sosial. Bahwa ada hubun- gan yang signifikan antara percaya diri dengan kemampuan komunikasi anak tunarungu di SDLB-B Karya Mulia II Surabaya. Kepercayaan diri adalah keyakinan untuk melakukan sesuatu pada diri sebagai karakteristik pribadi yang mencakup keyakinan akan kemampuan diri, opti- mis, objektif, bertanggung jawab, rasional, dan realistis (Gufron & Rini, 2014) dalam Suryani (2010). Pada perkembangan sosialnya, umumnya individu tuli memiliki masalah pada penyesuaian sosial. Penyesuaian sosial merupakan kapasitas untuk bereaksi secara efektif atau adekuat ter- hadap kenyataan yang ada di lingkungannya sehingga individu akan mampu untuk memenuhi tuntutan sosial dengan cara yang dapat diterima dan memuaskan bagi dirinya maupun lingkun- gannya Nurihsan and Agustin (2000) . [Table 2 about here.] Wujud dari keberhasilan penyesuaian sosial ini antara lain, kemampuan individu dalam menjalin komunikasi dengan orang lain, menyelaraskan antara tuntutan dirinya dengan tuntutan lingkungan, memenuhi aturan kelompok masyarakat dan mampu menciptakan suatu relasi yang sehat dengan orang lain, mengembangkan persaha- batan, berperan aktif dalam kegiatan sosial. Selain itu, menghargai nilai-nilai, hukum-hukum sosial dan tradisi atau dapat dikatakan mampu bertindak sesuai dengan norma yang berlaku dan lain sebagainya. Penguatan Karakter Melalui Persepsi Mahasiswa Berkebutuhan Khusus Tunarungu Terhadap Penerimaan Teman Sekelas Persepsi mahasiswa berkebutuhan khusus tunarungu terhadap penerimaan teman sekelas ditunjukkan pada Tabel 3 . Penguatan Karakter Melalui Persepsi Mahasiswa Berkebutuhan Khusus Tunarungu Terhadap Penerimaan Teman Sekelas Persepsi mahasiswa berkebutuhan khusus tunarungu terhadap penerimaan teman sekela ditunjukkan pada Tabel 3 . [Table 2 about here.] Tabel 2 menunjukkan bahwa mahasiwa tunarungu menyatakan hubungan sosial atau pertem- anan antara mahasiswa berkebutuhan khusus tunarungu dengan teman sekelas adalah sangat setuju 10%, setuju 60%, ragu-ragu, 8.5%, tidak setuju 21.5%, dan sangat tidak setuju 0%. Peneli- tian ini menemukan penguatan karakter melalui partisipasi sosial cukup baik yaitu sebesar 70% (10%+60%) yang dibuktikan dalam praktek hubungan sosial/pertemanan antara mahasiswa berkebutuhan khusus tunarungu dengan teman sekelas mereka. Penguatan karakter melalui hubungan sosial pertemanan antara mahasiswa berkebutuhan khusus tunarungu dengan teman sekelas mereka dikatakan cukup baik atau kuat, sehingga dapat mengatasi masalah maha- siswa tunarungu yang mengalami kesulitan dalam menjalin pertemanan dan tidak percaya diri dalam berinteraksi. Kondisi hubungan sosial pertemanan menunjukkan kekuatan karakter dalam pertemanan untuk mengantisipasi terjadinya masalah hambatan pendengaran. Ham- batan pendengaran pada mahasiswa tunarungu berdampak pada bahasa dan komunikasinya. Mahasiswa tunarungu kesulitan dalam berinteraksi karena kemampuan interaksi sosial mem- butuhkan kemampuan berbahasa yang baik sementara mahasiswa tunarungu kesulitan dalam berbahasa lisan (verbal), kesulitan komunikasi tersebut berakibat terhambatnya keterampi- lan sosial dan hubungan sosial yang sesuai Antia et al. (2011) . Mahasiswa tunarungu berko- munikasi dengan menggunakan bahasa isyarat sementara teman sekelas mereka tidak semua mengerti dan memahami maksud dari setiap isyarat yang dilakukan tunarungu. Para peneliti dan praktisi secara khusus juga menunjukkan keprihatinan terhadap interaksi sosial anak den- gan gangguan pendengaran yang belajar di sekolah inklusi, karena keterbatasan komunikasi dan interaksi teman sebaya Kluwin (2002). y Kondisi demikian pada akhirnya menjadikan interaksi tidak berkesan, tidak menye- nangkan, karena tidak adanya koneksi dalam pembicaraan tersebut, sementara syarat ter- jadinya interaksi sosial yang baik adalah ketika didalamnya lawan bicara bisa saling memahami maksud dari pembicaraan. Everett M. Rogers seorang pakar Sosiologi Pedesaan Amerika yang telah memberi banyak perhatian pada studi riset komunikasi, khususnya dalam hal penyebaran inovasi seperti dikutip oleh Cangara (2006: 19) dalam Bintoro (2011) membuat definisi bahwa Agustus 2020 \| Volume 9 \| Issue 2 PEDAGOGIA:Jurnal Pendidikan \| ojs.umsida.ac.id/index.php/ 248 Inclusive Practices: Strengthening Character Through Social Participation of Deaf Students Amka et al. komunikasi adalah proses di mana suatu ide dialihkan dari sumber kepada satu penerima atau lebih dengan maksud untuk mengubah tingkah laku yang pada gilirannya akan tiba pada saling pengertian yang mendalam. Pengalaman-pengalaman interaksi sosial yang seringkali sulit dan mengecewakan bagi tunarungu seperti kesulitan dalam memahami pembicaraan lawan bicara, keliru dalam mempersepsikan ekspresi dan bahasa tubuh orang lain, begitu pun sebaliknya lawan bicara tidak mampu memahami maksud dari tunarungu. Yull (2004) dalam W.R. Penguatan Karakter Melalui Penerimaan Teman Sekelas Terhadap Mahasiswa Berkebutuhan Khusus Tunarungu Penguatan karakter melalui penerimaan teman sekelas terhadap mahasiswa berkebutuhan tunarungu ditunjukkan padaTabel 4 . g Penguatan karakter melalui penerimaan teman sekelas terhadap mahasiswa berkebutuhan tunarungu ditunjukkan padaTabel 4 . [Table 3 about here.] Penguatan Karakter Melalui Penerimaan Teman Sekelas Terhadap Mahasiswa Berkebutuhan Khusus Tunarungu Penguatan karakter melalui penerimaan teman sekelas terhadap mahasiswa berkebutuhan tunarungu ditunjukkan padaTabel 4 . [Table 3 about here.] Tabel 3 menunjukkan bahwa mahasiswa tunarungu menyatakan persepsinya terhadap peneri- maan teman sekelas mereka adalah sangat setuju 16%, setuju 66%, ragu-ragu, 4%, tidak setuju 14%, dan sangat tidak setuju 0%. Penelitian ini menemukan penguatan karakter melalui par- tisipasi sosial yang baik atau kuat sebanyak 82% (16%+66%), yang dibuktikan dalam pratek dimensi persepsi mahasiswa berkebutuhan khusus tunarungu terhadap penerimaana teman sekelas.Tanggapan mahasiswa berkebutuhan khusus tunarungu terhadap penerimaan teman sekelasnya termasuk dalam kategori karakter baik atau kuat. Mahasiwa tunarungu merasakan adanya sikap yang baik teman sekelas terhadap mereka. Pada umumnya teman sekelas mema- hami kondisi dan hambatan mahasiswa tunarungu. Mahasiswa tunarungu beranggapan bahwa teman sekelas memperlakukan mereka dengan baik, namun disisi lain beberapa dari mahasiswa tunarungu beranggapan bahwa terdapat teman sekelas yang tidak mau menerima tanggapan dari mahasiswa tunarungu, dan ada teman sekelas yang tidak ingin mengajaknya ke kantin. Agustus 2020 \| Volume 9 \| Issue 2 PEDAGOGIA:Jurnal Pendidikan \| ojs.umsida.ac.id/index.php/ 249 Inclusive Practices: Strengthening Character Through Social Participation of Deaf Students Amka et al. Kondisi demikian tidak lepas dari kondisi beberapa teman sekelas yang kesulitan dalam berko- munikasi dengan mahasiswa tunarungu, bukan tidak ingin mengajak namun mereka bingung bagaimana cara mengajak mahasiswa tunarungu berhubung komunikasi lisan juga sulit dipa- hami oleh mahasiswa tunarungu sementara mereka tidak bisa berbahasa isyarat. Empati mempengaruhi apakah individu tersebut akan memberikan maaf atas kesalahan yang dilakukan oleh individu lain ataukah tidak. Menurut Gagan (1983) dalam Silfiasari (2017) empati berarti kemampuan untuk merasakan apa yang dirasakan oleh orang lain. Ketika siswa regular mengerti perasaan dari temannya yang merupakan siswa berkebutuhan khusus, maka akan muncul rasa menghargai. Siswa regular akan memahami bagaimana keadaan dari siswa berkebutuhan khusus yang mempunyai keterbatasan-keterbatasan. Dari sini rasa penghargaan dan rasa empati akan muncul, jadi ketika siswa berkebutuhan khusus melakukan kesalahan maka siswa regular akan memaafkan siswa berkebutuhan khusus. Tidak mengherankan, jika mahasiswa tunarungu beranggapan mengalami kesulitan dalam menjalin berteman dengan teman sebaya. Banyak siswa tunarungu melaporkan bahwa meskipun mereka berpartisipasi dalam kegiatan sosial dengan teman sebaya yang mendengar, namun hubungan mereka bersi- fat jangka pendek dan santai, mereka merasa aman secara emosional hanya dengan teman- teman yang juga tunarungu, meskipun juga terdapat beberapa siswa tunarungu secara emo- sional aman dengan teman sekelas yang mendengar Stinson (1999). Faktor-faktor yang tam- paknya mempengaruhi hubungan sosial yang baik adalah kesempatan untuk berinteraksi den- gan teman-teman dengar, dukungan guru untuk interaksi, dan peluang untuk terlibat dengan teman sebaya dalam kegiatan ekstrakurikuler Stinson (1999). [Table 4 about here.] Tabel 4 menunjukkan bahwa sebanyak 23 orlangahasiwa menyatakan penerimaan mereka seba- gai teman sekelas terhadap mahasiswa tunarungu sangat setuju 67%, setuju 30%, ragu-ragu, 3%, tidak setuju 0%, dan sangat tidak setuju 0%. Penelitian ini menemukan penguatan karakter melalui partisipasi sosial sebesar 97% (67%+30%) yang dibuktikan paktek dalam hal peneri- maan teman sekelas terhadap mahasiswa tunarungu. Penerimaan teman kelas terhadap mahasiswa berkebutuhan khusus tunarungu termasuk dalam kategori penguatan karakter sangat baik. Hal ini dibuktikan melalui teman sekelas den- gan menunjukkan pemahaman yang baik terkait kondisi mahasiswa tunarungu. Pemahaman tersebut menjembatani terbentuknya penerimaan yang positif pula. Sebagain besar mahasiswa reguler, teman sekelas tidak pernah menunjukkan rasa iri terhadapa mahasiswa tunarungu yang mendapatkan perhatian dari dosen, ikut membantu mahasiswa tunarungu yang men- galami kesulitan, Dengan senang hati menerima keberadaan mahasiswa tunarungu dalam kelompok belajar, bersedia mendengarkan mahasiswa tunarungu bercerita dengan menggu- nakan bahasa isyarat maupun oral, tidak memebalas saat mahasiswa tunarungu marah, men- ganggap mahasiswa tunarungu sebagai teman yang sama dengan teman yang lainnya, serta tidak menghindari mahasiswa tunarungu. Adanya penerimaan yang baik teman kelas terhadap mahasiswa tunarungu menunjukkan tercapainya salah satu indikator keberhasilan penyelenggaraan pendidikan inklusif di Program Studi Pendidikan Khusus Universitas Lambung Mangkurat. Menurut penelitian Mastropieri and Scruggs (1994) faktor keberhasilan dari sekolah inklusif adalah penerimaan atmosfer yang baik, yang dalam hal ini adalah keberadaan teman sebaya. Penelitian dari Miller & Miller dalam Hasan and Handayani (2014) menyebutkan bahwa teman dapat memberikan dukun- gan agar siswa berkebutuhan khusus dapat belajar dengan baik. Penelitian Bond & Castagnera dalam Hasan and Handayani (2014) menjelaskan bahwa teman sebaya dapat membantu siswa berkebutuhan khusus dalam komunikasi dan penyesuaian diri di sekolah. Ketika siswa berkebu- tuhan khusus mengalami kesulitan dalam belajar, maka siswa regular akan bersedia membantu Agustus 2020 \| Volume 9 \| Issue 2 PEDAGOGIA:Jurnal Pendidikan \| ojs.umsida.ac.id/index.php/ 250 250 Inclusive Practices: Strengthening Character Through Social Participation of Deaf Students Amka et al. dengan senang hati. Siswa regular membantu siswa berkebutuhan khusus untuk menyesuaikan diri di sekolah dan berinteraksi dengan semua komponen-komponen yang ada di sekolah. Salah satu contoh adalah ketika siswa berkebutuhan khusus ketinggalan penjelasan dari guru di kelas, maka siswa regular membantu menjelaskan dengan bahasa sehari-hari yang mudah dimengerti oleh siswa berkebutuhan khusus. Siswa regular membantu siswa berkebutuhan khusus belajar sebagai teman kepada teman, memberikan rasa nyaman sehingga siswa berkebutuhan khusus lebih cepat mengerti tentang pembelajaran di kelas ketika dijelaskan oleh teman sebayanya. [Table 4 about here.] Tetapi, ketika interaksi yang positif tidak bisa tercapai, maka interaksi negatiflah yang akan muncul dalam suatu pertemanan. Magee and Smith (2013) menyatakan ketika siswa regular mengerti ketidakmampuan dari siswa berkebutuhan khusus dan mengerti pentingnya untuk membantu mereka, maka lingkungan komunikasi yang aman dan lancar akan tercipta antara siswa regular dan siswa berkebutuhan khusus, sehingga akan meminimalisir terjadinya konflik dalam hubungan pertemanan mereka. KESIMPULAN Pendidikan inklusif dengan memasukkan semua anak berkebutuhan khusus di sekolah umum, termasuk kampus Universitas Lambung Mangkurat Banjarmasin, telah memberikan keuntun- gan positif bagi mahasiswa penyandang tunarungu dan mahasiswa sebaya lainnya. Mereka secara bersama-sama dapat berpartisipasi sosial melalui hubungan pertemanan antara maha- siswa berkebutuhan khusus tunarungu dengan teman sekelas mereka, persepsi mahasiswa berkebutuhan khusus tunarungu terhadap Penerimaan Teman Sekelas, dan penerimaan teman sekelas terhadap mahasiswa berkebutuhan khusus tunarungu. 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Mainstream- special school inclusion partnerships: pupil, parent and teacher perspectives. Inter- national Journal of Inclusive Education 8, 37–57. doi: 10.1080/1360311032000159456. Mastropieri, M. A. and Scruggs, T. E. (1994). Text versus hands-on science curriculum: Implications for students with disabilities. Remedial and Special Education 15, 72– 85. doi: https://doi.org/10.1177/074193259401500203. Frostad, P. and Pijl, S. J. (2007). Does being friendly help in making friends? The relation between the social position and social skills of pupils with special needs in mainstream education. European Journal of Special Needs Education 22, 15–30. doi: 10.1080/08856250601082224. Meijer, C. J., Soriano, V., and Watkins, A. (2006). 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Scheepstra, A. J. M., Nakken, H., and Pijl, S. J. (1999). Con- tacts with classmates: the social position of pupils with Down’s syndrome in Dutch mainstream education. Euro- pean Journal of Special Needs Education 14, 212–220. doi: 10.1080/0885625990140303. Kantavong, P. (2018). Understanding inclusive education practices in schools under local government jurisdiction: a study of Khon Kaen Municipality in Thailand. Inter- national Journal of Inclusive Education 22, 767–786. doi: 10.1080/13603116.2017.1412509. Shapiro, J. P. (1994). No pity: People with disabilities forging a new civil rights movement (New York: Random House), 1–400. Kluwin, T. N. (2002). Social Processes and Outcomes of In- School Contact Between Deaf and Hearing Peers. 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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. Stinson, M. (1999). Considerations in educating deaf and hard-of-hearing students in inclusive settings. Journal of Deaf Studies and Deaf Education 4, 163–175. doi: 10.1093/deafed/4.3.163. Sugiyono (2016). Metode Penelitian Pendidikan Pendekatan Kuantitatif, Kualitatif, dan R&D. (Bandung: Alfabeta). Copyright © 2020 Amka and Mirnawati. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or repro- duction 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 reproduc- tion is permitted which does not comply with these terms. Suryani, S. F. (2010). Inferioritas dan kepercayaan Diri Pada Penyandang Tuna Rungu. Falkutas Pskologi, 1–5. Tarmansyah (2007). Inklusi Pendidikan Untuk Semua (Jakarta: Depdiknas). Weissberg, R. P. and Greenberg, M. T. (1998). School and community competence-enhancement and preven- tion programs. Journal of Mental Health 7, 479–492. Agustus 2020 \| Volume 9 \| Issue 2 253 Inclusive Practices: Strengthening Character Through Social Participation of Deaf Students Amka et al. LIST OF TABLES 1 Kriteria penilaian penguatan karakter melalui partisipasi sosial mahasiswa tunarungu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 2 Penguatan Karakter Dalam PartisipasiSosial MahasiswaTunarungu di Univer- sitas Lambung Mangkurat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256 3 Penguatan Karakter Melalui Persepsi mahasiswa tunarungudi Universitas Lam- bung Mangkurat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 4 Penerimaan Teman Sekelas Terhadap MahasiswaBerkebutuhan Khusus Tunarungu di UniversitasLambung Mangkurat . . . . . . . . . . . Agustus 2020 \| Volume 9 \| Issue 2 PEDAGOGIA:Jurnal Pendidikan \| ojs.umsida.ac.id/index.php/ 254 PEDAGOGIA:Jurnal Pendidikan \| ojs.umsida.ac.id/index.php/ 254 Agustus 2020 \| Volume 9 \| Issue 2 PEDAGOGIA:Jurnal Pendidikan \| ojs.umsida.ac.id/index.php/ 255 PEDAGOGIA:Jurnal Pendidikan \| ojs.umsida.ac.id/index.php/ Agustus 2020 \| Volume 9 \| Issue 2 PEDAGOGIA:Jurnal Pendidikan \| ojs.umsida.ac.id/index.php/ 256 PEDAGOGIA:Jurnal Pendidikan \| ojs.umsida.ac.id/index.php/ Agustus 2020 \| Volume 9 \| Issue 2 PEDAGOGIA:Jurnal Pendidikan \| ojs.umsida.ac.id/index.php/ 257 PEDAGOGIA:Jurnal Pendidikan \| ojs.umsida.ac.id/index.php/ 257 Agustus 2020 \| Volume 9 \| Issue 2 PEDAGOGIA:Jurnal Pendidikan \| ojs.umsida.ac.id/index.php/ 258 PEDAGOGIA:Jurnal Pendidikan \| ojs.umsida.ac.id/index.php/ 258 CONTEMPORARY LIVES OF MORAL COMMITMENT (New York: Free Press). . . . . . . . 258 Agustus 2020 \| Volume 9 \| Issue 2 Amka et al. Inclusive Practices: Strengthening Character Through Social Participation of Deaf Students Inclusive Practices: Strengthening Character Through Social Participation of Deaf Students TABLE 1 \| Kriteria penilaian penguatan karakter melalui partisipasi sosial mahasiswa tunarungu Persentase Kriteria 91% -100% Sangat baik 76% - 90% Baik 61% - 75% Cukup 51% - 60% Sedang ≤50% Kurang TABLE 1 \| Kriteria penilaian penguatan karakter melalui partisipasi sosial mahasiswa tunarungu Agustus 2020 \| Volume 9 \| Issue 2 Inclusive Practices: Strengthening Character Through Social Participation of Deaf Students Amka et al. TABLE 2 \| Penguatan Karakter Dalam PartisipasiSosial MahasiswaTunarungu di Universitas Lambung Mangkurat No Pastisipasi sosial Persentase Sangat setuju Setuju Ragu Tidak setuju Sangat tidak setuju 1 Hubungan sosial/pertemanan antara mahasiswa berkebutuhan khusus tunarungu dengan teman seke- las mereka. 10 60 8,5 21,5 0 Kriteria Penguatan kurang sedang kurang kurang kurang TABLE 2 \| Penguatan Karakter Dalam PartisipasiSosial MahasiswaTunarungu di Universitas Lambung Mangkurat Agustus 2020 \| Volume 9 \| Issue 2 Inclusive Practices: Strengthening Character Through Social Participation of Deaf Students Amka et al. TABLE 3 \| Penguatan Karakter Melalui Persepsi mahasiswa tunarungudi Universitas Lambung Mangkurat No Pastisipasi sosial Persentase Sangat setuju Setuju Ragu Tidak setuju Sangat tidak setuju 1 Persepsi mahasiswa berkebutuhan khusus tunarungu terhadap penerimaan teman sekelas mereka 16 66 4 14 0 Kriteria Penguatan kurang cukup kurang kurang kurang TABLE 3 \| Penguatan Karakter Melalui Persepsi mahasiswa tunarungudi Universitas Lambung Mangkurat Agustus 2020 \| Volume 9 \| Issue 2 Inclusive Practices: Strengthening Character Through Social Participation of Deaf Students Amka et al. TABLE 4 \| Penerimaan Teman Sekelas Terhadap MahasiswaBerkebutuhan Khusus Tunarungu di UniversitasLambung Mangkurat No Pastisipasi sosial Persentase Sangat setuju Setuju Ragu Tidak setuju Sangat tidak setuju 1 Penerimaan teman kelas terhadap mahasiswa berke- butuhan khusus tunarungu. 67 30 3 0 0 Kriteria Penguatan cukup kurang kurang kurang kurang TABLE 4 \| Penerimaan Teman Sekelas Terhadap MahasiswaBerkebutuhan Khusus Tunarungu di UniversitasLambung M k t Agustus 2020 \| Volume 9 \| Issue 2 258
https://openalex.org/W2062390352	https://research.vu.nl/files/147098590/100723_Molecular_Sciences_11_12_5212_5233.pdf	English	null	Deleted in Malignant Brain Tumors-1 Protein (DMBT1): A Pattern Recognition Receptor with Multiple Binding Sites	International journal of molecular sciences	2,010	cc-by	10,846	General rights C i ht d s moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright dition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private st Y t f th di t ib t th t i l it f fit ki ti it i l i • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal y p py y p p p p • You may not further distribute the material or use it for any profit-making activity or commer • You may freely distribute the URL identifying the publication in the public portal • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ou ay o u e d s bu e e a e a o use o a y p o a g ac y o co e c a ga • You may freely distribute the URL identifying the publication in the public portal VU Research Portal Deleted in Malignant Brain Tumors-1 protein (DMBT1): A pattern recognition receptor with multiple binding sites Ligtenberg, A.J.M.; Karlsson, N.G.; Veerman, E.C.I. Deleted in Malignant Brain Tumors-1 protein (DMBT1): A pattern recognition receptor with multiple binding sites Ligtenberg, A.J.M.; Karlsson, N.G.; Veerman, E.C.I. Link to publication in VU Research Portal Link to publication in VU Research Portal citation for published version (APA) Ligtenberg, A. J. M., Karlsson, N. G., & Veerman, E. C. I. (2010). Deleted in Malignant Brain Tumors-1 protein (DMBT1): A pattern recognition receptor with multiple binding sites. International Journal of Molecular Sciences, 11(12), 5212-5233. https://doi.org/10.3390/ijms1112521 citation for published version (APA) Ligtenberg, A. J. M., Karlsson, N. G., & Veerman, E. C. I. (2010). Deleted in Malignant Brain Tumors-1 protein (DMBT1): A pattern recognition receptor with multiple binding sites. International Journal of Molecular Sciences, 11(12), 5212-5233. https://doi.org/10.3390/ijms1112521 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Download date: 24. Oct. 2024 Deleted in Malignant Brain Tumors-1 protein (DMBT1): A pattern recognition receptor with multiple binding sites Ligtenberg, A.J.M.; Karlsson, N.G.; Veerman, DOI 10.3390/ijms1112521 Publication date 2010 Document Version Final published version Published in International Journal of Molecular Sciences Take down policy Take down policy If b li th t th Take down policy f you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Take down policy f you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim down policy believe that this document breaches copyright please contact us providing details, and we will remove access to the wo vestigate your claim. E-mail address: vuresearchportal.ub@vu.nl E-mail address: vuresearchportal.ub@vu.nl E-mail address: vuresearchportal.ub@vu.nl Download date: 24. Oct. 2024 UvA-DARE (Digital Academic Repository) Link to publication Citation for published version (APA): Ligtenberg, A. J. M., Karlsson, N. G., & Veerman, E. C. I. (2010). Deleted in Malignant Brain Tumors-1 protein (DMBT1): A pattern recognition receptor with multiple binding sites. International Journal of Molecular Sciences, 11(12), 5212-5233. https://doi.org/10.3390/ijms1112521 Citation for published version (APA): Ligtenberg, A. J. M., Karlsson, N. G., & Veerman, E. C. I. (2010). Deleted in Malignant Brain Tumors-1 protein (DMBT1): A pattern recognition receptor with multiple binding sites. International Journal of Molecular Sciences, 11(12), 5212-5233. https://doi.org/10.3390/ijms1112521 General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. Disclaimer/Complaints regulations Int. J. Mol. Sci. 2010, 11, 5212-5233; doi:10.3390/ijms1112521 International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Review Deleted in Malignant Brain Tumors-1 Protein (DMBT1): A Pattern Recognition Receptor with Multiple Binding Sites Antoon J. M. Ligtenberg 1,, Niclas G. Karlsson 2 and Enno C. I. Veerman 1 1 Periodontology and Oral Biochemistry, Academic Centre for Dentistry Amsterdam, G. Mahlerlaan 3004, 1081LA, Amsterdam, The Netherlands; E-Mail: e.veerman@acta.nl 2 Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden Box 440, 40530 Gothenburg, Sweden; E-Mail: niclas.karlsson@medkem.gu.se Author to whom correspondence should be addressed; E-Mail: a.ligtenberg@acta.nl; Tel.: +31-(0)-20-5980-896. Received: 11 November 2010; in revised form: 9 December 2010 / Accepted: 9 December 2010 / Published: 17 December 2010 Abstract: Deleted in Malignant Brain Tumors-1 protein (DMBT1), salivary agglutinin (DMBT1SAG), and lung glycoprotein-340 (DMBT1GP340) are three names for glycoproteins encoded by the same DMBT1 gene. All these proteins belong to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins: a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. In addition to SRCR domains, all DMBT1s contain two CUB domains and one zona pellucida domain. The SRCR domains play a role in the function of DMBT1s, which is the binding of a broad range of pathogens including cariogenic streptococci, Helicobacter pylori and HIV. Mucosal defense proteins like IgA, surfactant proteins and lactoferrin also bind to DMBT1s through their SRCR domains The binding OPEN ACCESS Int. J. Mol. Sci. 2010, 11, 5212-5233; doi:10.3390/ijms1112521 International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms OPEN ACCESS Disclaimer/Complaints regulations UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:16 Dec 2021 1. Introduction: Saliva as a Protective Fluid Mucosal surfaces are the largest and most important interface between the human body and its environment, comprising a total area of approximately 300 m2 [1]. Interaction with the environment is of vital importance for the uptake of nutrients and oxygen, but the external interaction of mucosal surfaces also poses a threat the body. These threats include antigenic, mitogenic and toxic stimuli in food and in the air as well as pathogenic bacteria and viruses. To overcome these constant challenges, the immune system has developed extensive innate and adaptive responses. The oral tissues, a part of the mucosal immune system, are constantly covered by saliva, which harbors a similar set of antimicrobial proteins as other mucosal fluids [2]. A major role of saliva is to maintain the natural oral microbial ecosystem. The function and secretion of saliva can be disturbed after radiation therapy for head and neck cancer, by auto-immune diseases affecting glandular tissues such as Sjögren’s syndrome [3,4] or as a side-effect of numerous drugs. A reduced saliva secretion leads to a significant increase in oral bacteria as well as to a shift in composition of the oral microflora. At the same time, the oral complications increase in number and severity [5]. Reduced saliva secretion may also affect general health. For instance, the majority of sedated patients in intensive-care units show a shift in the oral microflora from Gram-positive to Gram-negative species, which subsequently may spread into the respiratory tract causing pulmonary afflictions [6]. Antoon J. M. Ligtenberg 1,, Niclas G. Karlsson 2 and Enno C. I. Veerman 1 2 Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden Box 440, 40530 Gothenburg, Sweden; E-Mail: niclas.karlsson@medkem.gu.se 2 Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden Box 440, 40530 Gothenburg, Sweden; E-Mail: niclas.karlsson@medkem.gu.se Author to whom correspondence should be addressed; E-Mail: a.ligtenberg@acta.nl; Tel.: +31-(0)-20-5980-896. * Author to whom correspondence should be addressed; E-Mail: a.ligtenberg@acta.nl; Tel.: +31-(0)-20-5980-896. Received: 11 November 2010; in revised form: 9 December 2010 / Accepted: 9 December 2010 / Published: 17 December 2010 Received: 11 November 2010; in revised form: 9 December 2010 / Accepted: 9 December 2010 / Published: 17 December 2010 Received: 11 November 2010; in revised form: 9 December 2010 / Accepted: 9 December 2010 / Published: 17 December 2010 Abstract: Deleted in Malignant Brain Tumors-1 protein (DMBT1), salivary agglutinin (DMBT1SAG), and lung glycoprotein-340 (DMBT1GP340) are three names for glycoproteins encoded by the same DMBT1 gene. All these proteins belong to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins: a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. In addition to SRCR domains, all DMBT1s contain two CUB domains and one zona pellucida domain. The SRCR domains play a role in the function of DMBT1s, which is the binding of a broad range of pathogens including cariogenic streptococci, Helicobacter pylori and HIV. Mucosal defense proteins like IgA, surfactant proteins and lactoferrin also bind to DMBT1s through their SRCR domains. The binding motif on the SRCR domains comprises an 11-mer peptide in which a few amino acids are essential for binding (GRVEVLYRGSW). Adjacent to each individual SRCR domain are glycosylation domains, where the attached carbohydrate chains play a role in the binding of influenza A virus and Helicobacter pylori. The composition of the carbohydrate chains is not only donor specific, but also varies between different organs. These data demonstrate a role for DMBT1s as pattern recognition molecules containing various peptide and carbohydrate binding motifs. Keywords: dental caries; innate immunity; mucosal protection; SRCR domains Int. J. Mol. Sci. 2010, 11 Int. J. Mol. Sci. 2010, 11 5213 2. Adherence and Oral Infections As a result of the continuous flow of saliva in our mouth, planktonic bacteria are swallowed, making microbial adherence one of the key selective criteria in the oral cavity [7,8]. Salivary proteins in solution may inhibit adherence by competition with bacterial binding sites on proteins coated at dental surfaces. Binding of salivary proteins to bacteria may result in bacterial agglutination which is, therefore, considered as a mechanism for clearance of bacteria and protection against dental caries [9–11]. One of the important bacteria agglutinating proteins is Deleted in Malignant Brain Tumors-1 protein (DMBT1) also known as Salivary Agglutinin (DMBT1SAG) [12,13]. 3. Deleted in Malignant Brain Tumors-1 (DMBT1), Salivary Agglutinin (DMBT1SAG) and Glycoprotein 340 (DMBT1GP340) 3. Deleted in Malignant Brain Tumors-1 (DMBT1), Salivary Agglutinin (DMBT1SAG) and Glycoprotein 340 (DMBT1GP340) Streptococcus mutans is considered the major cause for dental caries. In search for S. mutans agglutinating substances, Ericson and Rundegren isolated a 300–400 kDa glycoprotein from parotid saliva [12] that showed calcium-dependent binding to S. mutans. This protein was named salivary agglutinin (DMBT1SAG). It also binds a number of both Gram-positive and Gram-negative bacteria, as well as to host proteins including IgA, complement factor C1q, bovine and human lactoferrin, albumin and lysozyme [14–19]. Several studies have shown that the protein core of DMBT1SAG is identical with lung glycoprotein 340 (DMBT1GP340) and Deleted in Malignant Brain Tumors-1 (DMBT1) [20–22]. The amino acid sequences characterized so far are identical to the one deduced from the DMBT1 gene [23,24]. DMBT1SAG and DMBT1gp-340 cross react with monoclonal antibodies raised against the DMBT1s isolated from the different sources. Both proteins show calcium-dependent binding to S. mutans and SP-D [21]. DMBT1GP340 was purified from bronchoalveolar lavage fluid of Int. J. Mol. Sci. 2010, 11 5214 patients with alveolar proteinosis [20]. It binds to surfactant proteins A (SP-A) and D (SP-D) [25], members of the collectin family that play an important role in innate immunity by binding to specific carbohydrate structures on the surface of pathogenic micro-organisms [26]. DMBT1SAG and DMBT1GP340 thus represent DMBT1 isoforms that are encoded by the DMBT1 gene on chromosome 10q26.13. The DMBT1 gene shows frequent genomic rearrangements and/or loss of expression in two common types of malignant brain tumors, but also in diverse epithelial cancer types, including lung, gastric, esophageal, colon, breast, and skin cancer [27–36]. A search through the human genome revealed only one copy of the gene. Genetic polymorphism results in variants with different numbers of SRCR exons. In addition, different isoforms may arise through alternative splicing or differential post-translational modifications such as glycosylation. 4. The Domain Organization of DMBT1 The most typical feature of DMBT1 is that it consists of protein domains (Figure 1). DMBT1 is a member of the scavenger receptor cysteine-rich (SRCR) superfamily, which is characterized by the presence of one or more SRCR domains [23,24]. In between each of the multiple SRCR domains, all located to the N-terminal end of the DMBT1s, are short serine-threonine-rich amino acid motifs of 20–24 amino acids in length, called SRCR interspersed domains (SIDs). The largest naturally occurring allele known to date contains 13 of these SRCR-SID units, while the shortest known displays only eight. This genetic variability in normal individuals raises the question if size variations of DMBT1GP340, DMBT1SAG and variants from other tissues are due to alternative splicing and post-translational processing or are alternatively determined by genetic differences. The stretch of tandem repeated SRCR domains with their interspersed SIDs are followed by two CUB domains separated by a 14th SRCR domain, and at the C-terminal end of the gene is a Zona Pellucida domain. Except for the SIDs, these domains are all known to be involved in ligand binding [37–39]. DMBT1 orthologs have been identified in various mammalian organisms such as mouse (dmbt1, CRP-ductin, vomeroglandin, muclin, apactin), rat (dmbt1, ebnerin, pancrin), rabbit (hensin), cow (bovine gall bladder mucin), pig (dmbt1) and rhesus monkey (H3) (Table 1). These proteins all have in common that they consist of SRCR, CUB and ZP domains although the number and order of these domains may be different (Figure 1). Table 1. DMBT1 synonyms and orthologs in different organisms. Organism Name Function Human DMBT1 gp-340 SAG Tumor suppression Epithelial cell differentiation Innate immunity Mouse CRP-ductin Vomeroglandin Muclin apactin Mucosal defense Epithelial differentiation Pheromone perception Sorting receptor Rabbit Hensin Terminal differentiation of kidney epithelial intercalated cells and embryonic stem cells Rat Ebnerin Pancrin Liver regeneration Taste perception Table 1. DMBT1 synonyms and orthologs in different organisms. Int. J. Mol. Sci. 2010, 11 5215 Table 1. Cont. Organism Name Function Pig Porcine dmbt1 Cow Bovine gallbladder mucin Cholesterol-binding Gallstone formation Rhesus monkey H3 Hormone-responsive Endometrial regeneration in ovulation cycle Table 1. Cont. Table 1. Cont. Figure 1. Domain organization of DMBT1 and DMBT1 orthologs. This picture was adapted from J.Mollenhauer [35]. DMBT1/8 kb.2 and DMBT1/6 kb.1 represent the largest and the smallest human variant, respectively, that have been recovered so far. The DMBT1 prototype features 13 scavenger receptor cysteine rich (SRCR) domains, separated by SIDs. 5. SRCR Domains The most prevalent domains in DMBT1 are the SRCR domains [38]. The SRCR superfamily consists of both membrane bound and secreted proteins which play a role in ligand binding [40]. SRCR proteins are present in multicellular animals along the entire animal kingdom, with their earliest appearance in sponges [38,41,42]. In addition, they also appear in protozoan parasites such as Cryptosporidium, Toxoplasma and Plasmodium and algae of the genus Chlamidomonas [43,44]. The organism with the highest number of SRCR domains is the purple sea urchin, Strongylocentrotus purpuratus, which contains 218 genes comprising altogether 1,095 SRCR domains [45]. For these SRCR proteins, a role as innate immune receptors was suggested since this organism also has a high number of innate immune receptors such as Toll-like receptors. Despite their presence in a large variety of animals [42], SRCR domains have retained a high degree of conservation. SRCR domains are ~100–110 amino acids long and can be subdivided into two groups: members of group A contain SRCR domains with six cysteine residues, which are encoded by two exons; members of group B, to which DMBT1 belongs, usually contain SRCR domains with eight cysteine residues and are encoded by a single exon [38]. The position of the cysteines and their disulfide bond pattern are well conserved within each SRCR domain. The disulfide bond pattern of a group B SRCR domain is C1–C4, C2–C7, C3–C8 and C5–C6 [42]. 4. The Domain Organization of DMBT1 The SRCR domains are followed by a short Thr-rich region, a CUB domain, a 14th SRCR domain, a Ser-Thr-Pro-rich region, a second CUB domain, and a Zona Pellucida domain. The DMBT1 orthologs from the rabbit (Hensin), Mouse (CRP-ductin-alfa), rat (Ebnerin) and pig have varying numbers of SRCR domains, followed by one or more CUB domains, and share a C-terminal SRCR domain, CUB domains and Zona Pellucida domain. Int. J. Mol. Sci. 2010, 11 5216 6. CUB Domains The CUB domain is a 100–110 residue-spanning extracellular domain named after the three proteins, in which it was first recognized: Complement subcomponents (C1s/C1r), embryonic sea urchin protein (Uegf; sea urchin epidermal growth factor), and bone morphogenetic protein 1 (Bmp1). CUB domains are found in numerous proteins that are mainly involved in developmental processes [39]. Almost all CUB domains contain four conserved cysteines, which probably form two disulfide bridges (C1–C2, C3–C4). The structure of the CUB domain has been predicted to be a beta-barrel similar to that of immunoglobulins [46]. 8. Glycosylation Patterns of DMBT1SAG The carbohydrate part of DMBT1SAG accounts for 25–40% of the molecular weight [23,52]. DMBT1s contains up to 14 potential N-linked glycosylation sites [23], and numerous potential O-linked sites situated primarily in the SIDs. The SRCR domains, however, contain only few potential O-glycosylation sites (Ser and Thr residues). Presumably, the high density of glycans forces the SIDs in an extended conformation, as has been demonstrated for mucins [53], thereby creating a molecule with alternating stretched SIDs and globular SRCR domains. The dominating O-linked structures in DMBT1SAG are extended core 1 and core 2 oligosaccharides, either neutral or monosialylated (Table 2). These structures are extended by fucosylated oligo-N-acetyllactosamine units. Sialylation and fucosylation are found terminating the N-acetyllactosamine chains as Sialyl-Lex or Leb/Ley, respectively. Sialylated structures tend to be shorter than fucosylated structures, suggesting that sialylation regulates the N-acetyllactosamine extension. Glycosylation of proteins is controlled by genetically encoded glycosyltransferases [54]. The Se gene, which determines the presence of blood group antigens in secretory fluids, encodes a α1,2-fucosyltransferase. This enzyme couples a fucose to galactose (Fucα1-2Gal), which is the first step in the generation of blood group antigens. Consequently, non-secretors have a different carbohydrate composition compared to secretors. Non-secretors have Lea and Lex structures on DMBTSAG; secretors also have Leb and Ley and ABH structures, in addition to Lea and Lex, on DMBT1SAG [13,55]. DMBT1SAG from secretors has a higher molecular mass than DMBT1SAG from non-secretors [55]. Although evidence has been obtained that blood group antigens such as ABH and the Lea antigens function as ligands for bacterial receptors, and thus might be involved in bacterial binding [56–58], only a few papers describe a correlation between blood group status and the susceptibility to caries [59]. Glycosylation of lung DMBT1GP340 is different from that of salivary DMBT1SAG. Lung DMBT1GP340 completely lacks ABH and Le antigens [55] and its content of 2,3 linked sialic acid residues seems to be lower than that of salivary DMBT1SAG [60]. It is not clear, however, to what extent this difference is related to individual variation in glycosylation, as the preparations were from different donors. Differences are also found for tear DMBT1 when compared to saliva DMBT1SAG. In contrast to saliva, DMBT1 in tears had no sialyl-Lex. [61]. Also, time-dependent variations in glycosylation patterns have been reported. The reactivity of DMBT1SAG with antibody MECA-79, which recognizes the L-selectin ligand SO3-6GlcNAc [62,63], fluctuates during the ovulation cycle. 7. Zona Pellucida Domains The zona pellucida (ZP) domain was first recognized in the sperm receptor proteins ZP2 and ZP3 [39,47]. These proteins are, along with ZP1, responsible for sperm adhesion to the zona pellucida, the glycoprotein membrane surrounding the plasma membrane of the oocyte. The ZP domain is a ~260 amino acid module which contains eight conserved cysteines, forming four disulfide bridges. The disulfide bonding pattern suggests that the ZP domain consists of two subdomains. In addition to the conserved cysteines, a few aromatic or hydrophobic amino acids are absolutely invariant [48]. ZP domains are usually present in glycosylated proteins containing other domains and usually located at the C-terminal of the polypeptide, which is also the case for DMBT1s. The functions of proteins containing a ZP domain vary tremendously. They are found in glycoproteins involved in development, acoustic perception, immunity, and cancer [49]. ZP domains have been suggested to play a role in protein oligomerization [50]. This is consistent with the model that DMBT1SAG forms aggregates [51,52]. Int. J. Mol. Sci. 2010, 11 5217 8. Glycosylation Patterns of DMBT1SAG It is low shortly after menstruation and reaches a maximum a few days after ovulation. Also, during pregnancy and lactation, reactivity is high. These results suggest that sulfation of DMBT1SAG is hormonally regulated [63]. Int. J. Mol. Sci. 2010, 11 5218 Table 2. Overview of glycan chains found on DMBT1. Oligosaccharide Structure Method Source Reference: core 1 sialylated-core 1 Gal1-3GalNAcα1-Ser/Thr NeuAcα2-3 Gal1-3GalNAcα1-Ser/Thr LC-MS2 Tears/Saliva [61,64] disialylated-core 1 NeuAc2-3Gal1-3(NeuAc2-6)GalNAcα1-Ser/Thr core 2 Gal1-3(GlcNAcβ1-6)GalNAcα1-Ser/Thr LC-MS2 Tears/Saliva [61,64] sialylated-core 2 NeuAcα2-3 Gal1-3(GlcNAcβ1-6)GalNAcα1-Ser/Thr (Le-a) Gal1-4(Fuc1-3)GlcNAc immunoblotting Saliva [13] (Le-x) Gal1-3(Fuc1-4)GlcNAc immunoblotting Saliva [13,22] (Le-b) Fuc1-2 Gal1-4(Fuc1-3)GlcNAc immunoblotting/LC-MS2 Saliva [13,64] (Le-y) Fuc1-2Gal1-3(Fuc1-4)GlcNAc immunoblotting/LC-MS2 Saliva [13,64] H antigen A antigen  B antigen Fuc1-2 Gal1-4GlcNAc GalNAc1-3(Fuc1-2 )Gal1-4GlcNAc Gal1-3(Fuc1-2 )Gal1-4GlcNAc immunoblotting/LC-MS2 immunoblotting immunoblotting Saliva Saliva Saliva [13,64] [13] [13] (Sialyl-Lex) NeuA-α2-3Galβ1-3(Fucα1-4)GlcNAc immunoblotting saliva, not lung [22] Sialyl-Lea NeuAc2-3 Gal1-4(Fuc1-3)GlcNAc Immunoblotting/LC-MS2 Tears [61]] NeuAc2-3 lectin blotting Saliva, not lung [60] NeuAc2-6 lectin blotting lung, saliva [60] SO3-6GlcNAc Immunoblotting Saliva [63] Table 2. Overview of glycan chains found on DMBT1. Table 2. Overview of glycan chains found on DMBT1. 5219 Int. J. Mol. Sci. 2010, 11 Int. J. Mol. Sci. 2010, 11 9.1. Binding Sites on the SRCR Domains For DMBT1 SAG, the SRCR domains are identified as bacteria-binding sites. Proteolytic cleavage of DMBT1SAG with Lys-C results in a protein fragment of 1722 amino acids, only containing SRCR domains and SIDs, which still binds to S. mutans [65]. The SRCR domains in this protein fragment show a high degree of homology, which justifies the design of a consensus sequence. Based on this consensus sequence, synthetic peptides were designed consisting of the fragments between the cysteine residues covering the full SRCR domain and SID fragments. Bacterial binding was restricted to a 16-mer peptide (QGRVEVLYRGSWGTVC) representing the second fragment in the SRCR domain and therefore named SRCRP2 [65]. This peptide binds S. mutans and a number of other bacterial species including Streptococcus gordonii, Staphylococcus aureus, Escherichia coli and H. pylori [66]. Bacterial adhesion to the peptide correlates with adhesion to the full molecule (Figure 2) [67]. Figure 2. Correlation between bacteria binding to DMBT1SAG and SRCRP2 [67]. N- and C-terminal truncation resulted in an 11-mer peptide still able to bind bacteria, therefore named DMBT1 pathogen binding site 1 (DMBT1pbs1) [66]. Although bacterial binding within the SRCR domains is only restricted to the 11-mer peptide, within the peptide different residues are essential for binding of different bacteria. Alanine scanning analysis demonstrated that eight residues out of 11 were involved in binding of different bacterial species (GRVEVLYRGSW). Of these eight residues, four were always present in the binding motif (GRVEVLYRGSW). Of the 14 SRCR domains in the full length molecule, 12 contain the eight amino acids involved in binding giving Figure 2. Correlation between bacteria binding to DMBT1SAG and SRCRP2 [67]. Figure 2. Correlation between bacteria binding to DMBT1SAG and SRCRP2 [67]. N- and C-terminal truncation resulted in an 11-mer peptide still able to bind bacteria, therefore named DMBT1 pathogen binding site 1 (DMBT1pbs1) [66]. Although bacterial binding within the SRCR domains is only restricted to the 11-mer peptide, within the peptide different residues are essential for binding of different bacteria. Alanine scanning analysis demonstrated that eight residues out of 11 were involved in binding of different bacterial species (GRVEVLYRGSW). Of these eight residues, four were always present in the binding motif (GRVEVLYRGSW). Of the 14 SRCR domains in the full length molecule, 12 contain the eight amino acids involved in binding giving 5220 Int. J. Mol. Sci. 2010, 11 bacterial binding of DMBT1 a multivalent character. 9.2. Binding Sites on the Glycan Chains Studies of bacterial binding sites on SAG were first mainly focused on carbohydrate ligands, following the concept that bacteria primarily recognize carbohydrate ligands on mucosal surfaces [76,77]. These studies revealed that DMBT1SAG-mediated agglutination of S.mutans is inhibited by high concentrations of fucose and lactose. In line with these results, it was shown that the Lea-antigen (Galβ1, 3(Fucα1, 4)GlcNAc) was involved in S.mutans binding, harboring an essential role for the terminal fucose [13]. Other studies demonstrated that sialic acid plays a role in binding of DMBT1SAG to S. sanguis and S. mutans [52,78]. H. pylori can bind Leb structures and sialic acid on DMBT1SAG with its BabA and SabA adhesins, respectively [64]. 9.1. Binding Sites on the SRCR Domains One of the two deviating SRCR domains is the 14th SRCR domain (GRVEIYHGGTW), located between two CUB domains and as such probably not involved in bacterial binding. SRCR domains in other proteins have previously been implicated in bacteria binding [68–70]. The bacteria binding site locates in a similar region of the group A SRCR domains in MARCO, which is a macrophage receptor recognizing bacterial surface components such as LPS and LTA. In this case, an RXR-motif, adjacent to the SRCRP2 peptide motif, is responsible for bacterial interactions (GSSNRGRAEVYYSG) [71,72]. Similarly, the bacteria binding sites in human Spand CD6, both group B members of the SRCR superfamily that are expressed by lymphoid macrophages and lymphocytes, are also located in the SRCR domain. [73,74]. CD 163 (M130), a macrophage receptor only consisting of nine SRCR domains, also binds bacteria. Peptides homologous to the DMBT1SAG bacteria binding site were synthesized for all nine SRCR domains and tested for bacteria binding [75]. One of these nine peptides, of SRCR domain 3, showed good binding to bacteria (GRIEIKFQGRW). 10.1. Antigen I/II Polypeptides DMBT1SAG was first described as an agglutinating agent for S.mutans and other streptococci [12]. Several studies have underlined the importance of bacterial agglutination in the protection against dental caries [9,79–81]. High concentrations of DMBT1SAG on the dental surface promoted S. mutans colonization, but high concentrations in saliva inhibit its colonization [80]. DMBT1SAG binds in a calcium-dependent manner to antigen I/II polypeptides, a group of surface receptors on S. mutans and related streptococci. Antigen I/II polypeptides have been characterized under different names in a variety of streptococci, S. mutans (antigen B, P1, Pac, SpaP, MSL-1), S. sobrinus (PAg, SpaA), S. gordonii (SspA, SspB), S. intermedius (Pas), S. pyogenes (aspA) and S. agalactiae (bspD) [82,83]. Antigen I/II polypeptides on oral streptococci have been studied extensively as candidates for vaccine development [84]. These polypeptides, between 826 and 1653 amino acids long, share a common primary sequence (Figure 3). All proteins start with a 38 amino acid (aa) leader sequence that is cleaved off during secretion. The A-region, containing one to four copies of an 82 aa residues alanine-rich sequence, is found 165 aa residues downstream of this leader. This is followed by a variable region (V-region) and then by a proline-rich P-region containing 5221 Int. J. Mol. Sci. 2010, 11 one to three copies of a 39 residue sequence block. The C-terminal region of about 615 amino acids shows 64% or more sequence homology between the I/II polypeptides. Figure 3. Schematic overview of the structural organization of antigen I/II polypeptides. All proteins contain a 38 residues leader peptide. 165 amino acid residues downstream of the leader peptide is the alanine-rich region containing 1–4 repeats of an 82 residues alanine-rich repeat. This is followed by a variable (V) region, followed by proline-rich P region containing 1–3 copies of a 39 residues repeat. The C-terminus shows ≥64% homology along the antigen I/II protein family. C-terminally is the cell wall anchoring sequence (CWA). sequence (CWA). Table 3. Binding sites for DMBT1SAG on various proteins. Protein Sequence SRCRP2 binding Ref. S.mutans antigen I/II PQLKTADLPAGRDETTSFVLV [85] S.gordonii antigen I/II S.mutans antigen I/II ELKTEALTAGRPKTTSFVLV QLKTADLPAGRDETTSFVLV [86] S.gordonii SspB 390-402 S.gordonii SspB 390- T400K-402 DYQAKLAAYQTEL DYQAKLAAYQKEL + + [87] SsP(A4K-A11K) Consensus SspA and B DYQKKLAAYQKEL DYQAKLAAYQAEL + + [88] Bovine lactoferrin 480-492 SCAFDEFFSQSCA + [89,90] Human lactoferrin DCKFDEYFSQSCA [91] Human casein LLNQELLNPTHQIYPVTQPLAPVHNPISV [91] HIV V3 region CTRPNYNKRKR [92] Table 3. Binding sites for DMBT1SAG on various proteins. Table 3. 10.1. Antigen I/II Polypeptides Binding sites for DMBT1SAG on various proteins. Protein Sequence SRCRP2 binding Ref. S.mutans antigen I/II PQLKTADLPAGRDETTSFVLV [85] S.gordonii antigen I/II S.mutans antigen I/II ELKTEALTAGRPKTTSFVLV QLKTADLPAGRDETTSFVLV [86] S.gordonii SspB 390-402 S.gordonii SspB 390- T400K-402 DYQAKLAAYQTEL DYQAKLAAYQKEL + + [87] SsP(A4K-A11K) Consensus SspA and B DYQKKLAAYQKEL DYQAKLAAYQAEL + + [88] Bovine lactoferrin 480-492 SCAFDEFFSQSCA + [89,90] Human lactoferrin DCKFDEYFSQSCA [91] Human casein LLNQELLNPTHQIYPVTQPLAPVHNPISV [91] HIV V3 region CTRPNYNKRKR [92] A number of putative binding sites for DMBT1SAG were identified on antigen I/II polypeptides and other proteins (Table 3). Insight has come from the elucidation of the tertiary structure of antigen I/II [83,93]. The V-region of Ag I/II polypeptides may adopt a lectin-like conformation, supporting the suggestion that antigen I/II polypeptides bind to DMBT1SAG by recognition of carbohydrate moieties [94]. This V-region is projected from the cell surface by a stalk formed by an association between an N-terminal α-helix of the alanine-rich repeats and a C-terminal polyproline helix. The C-terminus of antigen I/II, which is in close vicinity to the cell surface, also binds DMBT1SAG [85,93]. A synthetic 20-mer peptide, corresponding to residues 1025-1044 of antigen I/II (PQLKTADLPAGRDETTSFVLV), inhibited binding to DMBT1SAG and selectively inhibited colonization of S.mutans to the tooth surface [85]. The presence of two binding sites for DMBT1SAG may also explain the intriguing observation that streptococcal binding to surface immobilized DMBT1SAG is different from binding to DMBT1SAG in solution [95,96]. Overlapping, but not identical, subsets of monoclonal antibodies against antigen P1 inhibited SAG mediated adherence and 5222 Int. J. Mol. Sci. 2010, 11 aggregation, indicating that in the adsorbed state, other domains were involved in the interaction with bacteria than in the soluble phase. Furthermore, DMBT1SAG in solution and adsorbed DMBT1SAG were differentially recognized by streptococci, which formed three phenotypic groupings according to their modes of interaction: one group binding to both surface-bound and soluble DMBT1SAG, one group binding only to surface-bound SAG, and one group interacting preferentially with soluble DMBT1SAG [95]. Deletion of antigen I/II polypeptides in S. gordonii reduced adhesion to immobilized DMBT1SAG by 40%, while deletion of Hsa—another streptococcal surface antigen—reduced adhesion by 80%. In contrast, aggregation by DMBT1SAG disappeared after deletion of antigen I/II polypeptides but was not affected by deletion of Hsa [97]. 10.1. Antigen I/II Polypeptides The differences in binding between absorbed and soluble state is not unique to DMBT1SAG, and has been reported for the protein-bacterial interaction between Actinomyces viscosus and acidic proline rich proteins as well as statherin. A. viscosus only bound to surfaces coated with these proteins, but not to the same proteins in solution. [98,99]. This is attributed to a conformational change which takes place when the proteins are adsorbed onto a surface, resulting in the exposure of previously hidden bacterial receptors, cryptitopes. Statherin is a peptide which in solution has no defined tertiary structure. Upon adsorption onto a hydroxyapatite surface, the C-terminal region folds into a α-helical conformation, which is recognized by bacterial receptors [100]. Conclusive evidence for differences in soluble and surface bound DMBT1SAG remains to be demonstrated by identifying the epitopes that are exposed only on immobilized DMBT1SAG compared to soluble DMBT1SAG, and vice versa. 10.2. Pathogen Associated Molecular Patterns Bacteria that do not possess antigen I/II polypeptides, like H. pylori and Staphylococcus aureus, also bind DMBT1SAG [22,65]. CRP-ductin, the mouse homolog of DMBT1, bind to several Gram+ and Gram- bacteria, including Haemophilus influenzae, Klebsiella oxytoca, S. aureus and Streptococcus pneumoniae [101]. As many SRCR proteins are pattern recognition receptors, DMBT1SAG might also recognize common motifs on pathogens, so–called PAMPs (pathogen associated molecular patterns). DMBT1SAG binds to lipoteichoic acid, a component on the membrane of Gram-positive bacteria, and to lipopolysaccharide (LPS), a common component on the cell membrane of Gram-negative bacteria. LPS has been found to bind to SRCRP2, suggesting DMBT1 recognizes LPS with its SRCR domains [102]. Binding studies to truncated variants of LPS from Salmonella showed that binding depended on the availability and accessibility of phosphorylated structures on LPS. In addition, sulfated substances such as dextran sodium sulfate (DSS) and the food stabilizer carrageenan also bound to DMBT1. DSS is used to induce colitis in mice in a disease model for ulcerative colitis. In a DSS-induced colitis model, Dmbt1-/- mice were more susceptible to low doses of DSS than Dmbt1+/+ mice. However, since this is a commensal bacteria induced colitis model, this effect is likely to be attributed to Dmbt1’s ability to control the natural intestinal microflora. 11. Interaction with Viruses Besides bacteria, DMBT1SAG also binds viruses, including HIV-1 [92,103] and influenza A virus [60,104]. HIV-1 infects host cells by binding to the CD4 receptor through the viral envelope glycoprotein gp120, which leads to conformational changes in gp120 allowing high affinity interaction with chemokine receptors [105,106]. DMBT1SAG shows a calcium-dependent interaction with the Int. J. Mol. Sci. 2010, 11 5223 virus gp120. The DMBT1SAG binding region on gp120 is localized to a linear, highly conserved sequence (CTRPNYNKRKR) near the stem of the V3 loop that is critical for chemokine receptor interaction during viral binding and infection [92]. Thus, DMBT1SAG most probably blocks the access of gp120 to the chemokine receptor. The N-terminal sequence of DMBT1, including the first SRCR domain and one-half of the first SID, binds in a Ca2+-dependent manner to the same HIV-1 V3 sequences previously identified to interact with full-length SAG [107]. DMBT1SAG and DMBT1GP340 also inhibit the hemagglutination activity and infectivity of Influenza A virus (IAV) [104], which is responsible for outbreaks of influenza. IAV infects cells of the respiratory tract by binding to sialic acid residues present at the cell surface. Different influenza strains recognize specifically (2,3)-linked or (2,6)-linked sialic acid. DMBT1 has broad antiviral activity against human, equine and porcine IAV strains by sialic acid mediated binding to IAV, which results in virus neutralization [60]. A variant of DMBT1SAG had significantly greater inhibitory activity against avian-like IAV strains which preferentially bound sialic acids in (2,3) linkage, than DMBT1SAG from another donor or three lung preparations of DMBT1GP340 [60]. In line with this, a greater density of (2,3)-linked sialic acids was present on the DMBT1SAG from this donor as compared with the other samples. Hence, the specificity of sialic acid linkages on DMBT1 is an important determinant of anti-IAV activity. In addition to these direct virus-neutralizing properties, DMBT1GP340 displays cooperative interactions with SP-D in viral neutralization and aggregation assays. On the other hand, in some cases DMBT1SAG strongly antagonizes the antiviral activities of SP-D by binding to its carbohydrate recognition domain, which is involved in virus binding [60]. Int. J. Mol. Sci. 2010, 11 DMBT1SAG that is responsible for S. mutans binding also mediates binding of lactoferrin [17,89]. Bovine lactoferrin residues 480–492 (SCAFDEFFSQSCA) are important for DMBT1SAG binding. Although the homologous sequence in human lactoferrin is slightly different (SCKFDEYFSQSCA), human lactoferrin also binds to DMBT1 [19]. DMBT1GP340, which originally was isolated as a soluble receptor for SP-D, also binds SP-A [20,25]. SP-A and SP-D are collagen-containing (C-type) calcium-dependent lectins called collectins [113]. SP-D forms oligomers of 4–8 subunits. Each subunit is composed of three identical polypeptides of 43 kDa held together by disulfide bonds and non-covalent interactions at the N-terminal ends of the chains. Each polypeptide consists of a short N-terminal region, followed by a collagen-like sequence, a short -helical sequence and the carbohydrate recognition domain. DMBT1GP340 binds to the carbohydrate recognition domain of SP-D through a calcium-dependent protein-protein interaction [20]. SP-A and SP-D are involved in a range of immune functions including viral neutralization, aggregation and killing of bacteria and fungi, and clearance of apoptotic and necrotic cells. 13. Role of DMBT1 in Innate Immunity Due to their broad pathogen binding spectrum, DMBT1 and its mouse homolog CRP-ductin are considered pattern recognition receptors (PRRs) [101], an ancient germline encoded defense system against pathogen invasion in both plants and animals [114,115]. PRRs recognize conserved structures of the microbial cell that are vital for survival, so–called pathogen-associated molecular patterns (PAMPs). PAMPs include mannans and zymosan of the yeast cell wall, various bacterial cell-wall components, such as lipoteichoic acid (LTA) of Gram-positive bacteria or lipopolysaccharide (LPS) of Gram-negative bacteria, and bacterial DNA [116,117]. In the human body, PRRs are found on host–pathogen interacting surfaces and expressed by epithelial cells, macrophages, granulocytes and dendritic cells or as secretory products present in mucosal fluids [115,118]. These characteristics are also applicable to DMBT1. PRRs form a diverse group of proteins including mannose-binding lectin, collectins, CD14, Toll-like receptors and Nucleotide-binding oligomerization domain (Nod) proteins [70,115,119,120]. The SRCR proteins SR-AI and SR-AII, MARCO and Sp, alsobelong to this group. 12. Interaction with Endogenous Protein Ligands DMBT1 binds to a number of endogenous proteins such as secretory IgA, MUC5B, surfactant proteins A and D, complement factor C1q, lactoferrin and albumin [16–18,21,25,89,108,109]. DMBT1SAG binds to secretory IgA in a calcium-dependent manner, resulting in a cooperative effect on bacterial aggregation [15,110]. Binding of DMBT1SAG to IgA is mediated by the same 11-mer peptide that is responsible for the broad-spectrum bacteria binding of DMBT1SAG [18]. Binding of a DMBT1SAG-IgA complex to the Pac molecule of S. mutans was demonstrated [52]. After chemical reduction, this complex was dissociated into sIgA and DMBT1SAG and concomitantly binding to Pac disappeared. DMBT1SAG also binds to C1q of the complement system, a system of plasma proteins that may react with one another to opsonize or permeabilize pathogens and induce a series of inflammatory responses [109]. By binding of SAG to C1q, the complement system is activated through the classical pathway [16]. Although DMBT1SAG is secreted in saliva and C1q is a serum component, these two fluids may mix in the oral cavity under conditions of oral inflammation, e.g., periodontal disease, or mucosal damage, thus enabling a local complement activation. DMBT1 also binds to bovine and human lactoferrin, an 80 kDa iron binding protein belonging to the transferrin family [17,19]. Lactoferrin exhibits various functions in the innate immune system. It sequesters iron from the local environment thus inhibiting microbial growth and it prevents the formation of Pseudomonas biofilms [111]. In addition, antimicrobial peptides are released upon proteolytic degradation of lactoferrin [112]. Bovine lactoferrin inhibits DMBT1SAG binding to S. mutans protein antigen Pac, which belongs to the antigen I/II family [17]. The SRCRP2 peptide of 5224 Int. J. Mol. Sci. 2010, 11 14. Conclusions Conclusively, DMBT1 plays a role in innate immunity by binding to a large panel of exogenous and endogenous ligands. SRCRP2 on the SRCR domains and sialic acids on the glycan chains have been identified as ligand binding sites, but DMBT1 may harbor many other potential ligand binding sites. These many ligand binding sites and its structural heterogeneity—both in the different tissues and between different individuals—are only two of the many surprises hidden in this molecule. Future research should focus on the precise role of DMBT1 in the innate immune system and the physiological consequences of its heterogeneity. 5225 Int. J. Mol. Sci. 2010, 11 References 1. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
W4246733089.txt	https://zenodo.org/records/2162588/files/article.pdf	de		Allgemeine analytische Methoden und Apparate	European food research & technology	1,904	public-domain	3,889	~t. N o8. v eB~.d. m b e r 1904.j~ Referate. -- Allgem. analyt. Methoden u. ApI)arate. 555 Referate. Allgemeine analytische )][ethoden und Apparate. M. l ) e n n s t e d t : Z u r v e r e i n f a e h t e n Elementaranalyse. (Chem.-Ztg. 1904, 28, 3 5 - 3 6 . ) Die Angaben yon C h a r i t s c h k o f f , nach welchen selbst bei scheinbar normalem Verlauf der Verbrennung zu vie1 Kohlenstoff und Wasserstoff geflmden werde, veranlaStel~ Verf., zahlreiche Analysen yon SchmierSlen, Petroleum u. s. w. auszufiihren. Was den Vorwurf anbelangt, daf~ unverbrannte Substanz deu Pla*inquarz fiberschreite, so kann dies nur bei mangelhafter Leitung der Verbrennung geschehen; bezfiglich des Umstandes jedoch, da$ selbst bei anscheinend normal verlaufener Verbrennung mehr als 100 °/0 Kohlenstoff und Wasserstoff gefunden Werden, was auch bereits S c h fit z e n b e r g e r 1882 bei der Analyse des kaukasischen Petroleums beobachtete, glaubt Verf. eine sehr ungezwungene ErklKrung dieser chemischen Anomalie gefunden zu haben und zwar dfirfte die Ursache derselben in der Behandlung der Absorptionsapparate zu suchen sein. Es stellte sich heraus, da$ auch in blinden Versuchen eine Zunahme des Gewichtes des Kohlensaureabsorptionsapparates stattfand, welche auf Kohlenstoff berechnet 1 - - 2 °/o betragen kann, w/ihrend die Gewichtszunahme des Chlorcalciumrohres eine normale war. Eine genaue Betrachtung der Apparate mit der Lupe liet~ erkennen, dal~ sich in dem, dem Verbrennungsrohr abgewendeten kleinen AnsatzrShrchen des Chlorealciumrohres, welches auch bei geschlossenen Hi~hnen mit der /iugeren Luft in Bertihrung bleibt, kleine, staubartige Teilehen yon Chlorcalcium befanden, nach deren Entfernung die Gewiehtszunahme des :Natronkalkrohres aufhSrte. Die mit freiem Auge nicht sichtbare Spur yon Chlorcalcimn zieht beJm Aufbewahren Feuehtigkeit an, die mit dem Rohr mitgewogen wird, und die durch den troekenen Luftstrom wfihrend der Verbrennung abet in das 57atronkalkrohr i'~bergeffihrt wird u. s. w. und dort die Gewichtsvermehrung veranlaf~t. Aber auch bei tadellos reinen AbsorptionsrShrchen kSnnen gleiche Anomalien beobaehtet werden, die auf die Feuchtigkeit tier zur Verbindung benutzten Kautschukschl/iuche zuriickzuftihren sin& Bei Anwendung getrockneter KautschukrShrchen hSrte auch bei dem blinden Versueh jede Gewiehtszunahme auf. Zweifellos siad die beobachteten Anomalien dutch diese oder ~ihnliche Ursachen hervorgerufen. Beztiglich der Kritik L i p p m a n n ' s (Chem.-Ztg. 1903, 27, 81.0) bemerkt Verf., alas das Platin lediglich als Sauerstofffibertr/iger wirkt, an Stelie des Ptatinquarzes kann mit gleichem Erfolg ein etwa 7 cm langes, 1,5 cm breites, lockenfSrmig aufgerolltes Platinblech angewendet werden, sodas die umst/hldliche Herstellung des PIatinquarzes erspart werden kann. J. ~[ayrhofer. Eugbne Sellier: 1 ] b e r d i e B e s t i m m u n g d e s A m m o n i a k s in p f l a n z l i c h e n P r o d u k t e n u n d b e s o n d e r s in d e r Z u e k e r r i i b e u n d d e n E r z e u g nissen der Zucker- und Spiritusfabrikation. (Bull. Assoc. Chim. Sucr. et Distill. 1904, 21, I063--1071 u. 11.15--1126.) --Anschliet~end an eine frfihere Abhandlung (Z. 1903, 6, 754) teilt Verf. mit, dal~ ihm bei Abfassung derselben die Arbeit yon B a u m a n n und B S m e r (Z. 1898, 1, 106) nieht bekannt gewesen sei, deren Ergebnisse rficksieht]ich des Verfahrens der Phosphorwolframsauref/~llung mit seinen Beobachtungen fibereinstimmen. Verf. hat bei seinen weiteren Versuchen aueh das Trennungsverfahren mittels Alkohols geprfift. Die F/illung der S/iureamide im Rfibensalt war auch bei Anwendung sehr growler Mengen Alkohol unvollst~ndig. Ferner versuehte er naeh dem Vorschlag yon B r e s l e r (Z. 1903, 6, 758) das Ammoniak als Phosphormolybd/~nsaueres Ammon zu bestimmen. Die F~illung einer Chlorammonium enthaltenden ZuckerlSsung mit dem B r e s l e r ' s e h e n Reagens blieb auch bei 48-stfindigem Stehen]assen unvollstfindig. Erneute Versuche mit dem auf versehiedene Arten hergestellten Reagens,. tells in salzsauerer, tells in salpetersauerer LSsung, hatten 556 R e fe r a t e. ~ Allgem. analyt. ~ethoden u. Apparate. [Zei~sehr. [d. Nahr.-Lu.Untersuehung GenulimitteL dasselbe Ergebnis. Bei der Bestimmung des Am.moniaks in dem Niedersehlag zeigte es sieh, d a g e s von Magn~esia beim Koehen vollstandig ausgetrieben wird. Die Reaktion der Salpetrigen Siiure mit den Amlden kann nieht zu einem Bestimmungsverfahren ffihren, weil aueh die im Riibensaft enthaltenen S~mren (Leuein, Tyrosin) dureh Salpetrige Saure zersetzt werden und die ~Virkung derselben auf Glutamine eine andere ist. Das Verfahren, Asparagin und Olutamin mittels Queeksilbernitrats yore Ammoniak zu trennen, gibt keine genauen Resultate, eine kleine Menge Ammoniak geht - - wahrseheinlieh unter Bildung eines wenig 15sliehen Doppelsalzes - - in den Queeksilberniedersehlag. Verf. land sehlieglieh, dag die besten Resultate dureh Destillation mit Magnesia bei 35--450 unter Yermindertem Drnek erzielt werden. Versuehe mit einer grSgeren Anzahl von Ammonsalzen zeigten, dag die Werte fiir das tiberdestillierte Ammoniak bei Destillation unter gewShnliehem nnd unter vermindertem Druek gut tibereinstimmten. Eine Ausnahme maehte das Ammoniumlaktat, bei welehem weder dureh 3fagnesia noeh dureh Soda oder Kalk das Ammoniak unter vermindertem Druek vollsfiindig ansgetrieben wurde. Die Si~ureamide werden bei 35--450 nieht zersetzt. Kalk greift jedoeh bei 30--40 o Glutamin sehr merkbar an. Baryumkarbonat und Bleihydrat bewirken unvollstfindlge Zerlegung aueh bei gewShnliehem Druek. Baryumkarbonat greift Glutamin stgrker an, als es Magnesia mr, dagegen wirkt es weniger stark auf Asparagln. Soda zersetzt Glutamin vollst~tndig unter Ammoniakbildung unterhalb 35 °. Ffir die Bestimmung des Ammoniaks in pflanzliehen Produkten ergibt sieh demnaeh folgendes Verfahren: Die Substanz wird mig wenig Wasser verdfinnt, fails viel Sfiure vorhanden ist, diese mit Soda abgestumpft und ein Tell des Extraktes verwendet, sodag der Gehalt an organiseher Substanz 10--15 g, der an Ammoniakstiekstoff 0,02 g nieht fibersteigt. Man verdfinnt mit Wasser auf 200--250 ecru, ffigt einige Dezigramme Vaseline und einen ~bersehug an Magnesia hinzu und desfillierg unter vermindertem Druek und Benutzung eines L iebig'sehen Kiihlers bei einer 500 nieht fibersteigenden Temperatur 45 Minuten lang. G. Son, tag. I . (lawa l o w s k i : g e d u k t i o n alkaliseher Kupferl5sung dutch Glyk o s e in d e r K i i l t e . (Zeitsehr. 0sterr. Apoth.-Ver. 1903, 41, 1 1 4 7 - - 1 1 4 8 . ) - Das Reagens fiir Glykosenaehweis (im Ham) in der Kglte wird in folgender Weise bereitet: 40 g kryst. Kupfersulfat und 3 g Chlorammonimn werden in 160 eem Wasser kalt gel5st, andererseits werden in einer kMten L5sm~g yon 130 g Natriumhydroxyd ir~ g00 ecru Wasser 80 g Weinstein gelSst; dieser Gauge wird die KupferlSsung allm~hlieh zugemiseht und das Ganze auf 1 1 gefiillt. Die naeh dem Absitzen erhaltene klare Flfissigkeit wird nun mit reiehlieher Menge 90- bis 92 °/o-igen Alkohols durehsehfit~elt. Es bilden sieh zwei Sehiehten, eine lelehtere blS~uliehgriine und eine sehwere tiefblaue Flfissigkeit. Die letztere, yore spez. Gew. 1,409 bis 1,412 stellt das Reagens din'. Es h~lt sieh l~tngere Zeit in dunklen Flasehen unter 18 bis 20°; auf 20 '~ erwiirmt seheidet es binnen einer halben Smnde reiehlieh Kupferoxydul aus. Glykose wirkt bei einer Temperatur unter 20 o selmell reduzierend, w/ihrend Saeeharose selbsg naeh 16-sgfindiger Einwirkung in der KMte keine Ausseheidung hervorbringt. Beliebige Verdiinnung der KupferlSsung dureh die Zuekerfliisslgkeit seh~digt die Empfindiiehkeit der Reaktion nieht. Das Reagens ist his 0,05 °/0 Glykosegehalt noeh einwandfrei empfindlieh. G. Sonntag. Emile Remy: A n a l y s e e i n e s G e m i s e h e s vonSaeeharose, Glykose und Fruktose. (Bull. Assoc. Chim. Suer. eg Distill. 1904, 21, 1002--1006.) Verf. maeht dm'auf aufmerksmn, dab bei der Benutzung der C l e r g e t ' s e h e n FormeI ffir die Analyse eines Gemisehes der drei Zuekerarten das DrehungsvermSgen der Giivulose in sauerer :L6sung eingesetzt werden mfisse, well die Liivulose in sauerer L6sung nieht dasselbe DrehungsvermSgen besitzt wie in wiisseriger GSsung und die C ] e r g e t'sehe Formel auf die Unver~nderliehkeit der Drehung von Glykose und Gi~vu- s. Band.1904.] 1. N o v e m b e r Refei ate. -- Allgem. analyt. Methoden u. Apparate. 557 lose vor und naeh der Inversion gegriindet ist. Der Fehler, der in die Reehnung geri~t, wenn dies nieht berfieksiehtigt wird, wird erst bei grogen Mengen Lgvulose betl~ehtlieh: Bei einem Gemiseh yon 50 °/o Saeeharose, 25 °/0 Gtykose und 25 °/0 Liivulose wiirde man 51,65°/o Saeeharose linden. Verf. entwlekelt eine Formel fiir die Bereehnung unter Beriieksiehtigung der ver~nderliehen Drehung der I,iivulose, bet~)nt die Notwendigkeit, bei den Ablesungen vor und nach der Inversion genau die gleiehen Temperaturgrade inneznhalten und stellt in einer Tabelle die in die Reehnung einzusetzenden Faktoren ffir versehiedene Temperatur zusammen. G. Sonntag. Bernhard Ylerk: N a c h w e i s y o n C i t r o n e n s ~ u r e . (Pharm. Ztg. 1904, 48, 894.) - - Die dureh Einwirkung yon konzentrierter Schwefelsaure auf Citronen~aure entstehende Aeetondikarbons~ure gibt naeh dem Verdiinnen mit Wasser und Neutralisation mit Natronlauge auf Zusatz friseh bereiteter LSsung yon Nitroprussidnatrium die bekannte Aeetonfiirbnng (rubinrot, naeh dem Ansftuern purpurrot in violett iib'ergehend). Weinsiture stSrt diese Reaktion dureh die Verkohlung, man kann jedoeh diesem Migstand abhelfen, wenn man statt reiner Sehwefelsg.ure eine Misehung yon 3--4 Teilen Essigsfmreanhydrid und 6--7 Teilen konzentrierter Sehwefels/iure anwendet und diese Misehung 5--10 Minuten bei 90--950 einwirken t~igt. Hierbei wird die Weinsiiure aeetyliert~ und vor Verkohlung gesehfitzt. Es k6nnen naeh diesem Verfahren noeh 0,001 g Citronensiiure naehgewiesen werden. 0,1 g einer 1 °/0-igen V,reinsgure-Citronens~.ure-Misehung wird mit 4 Tropfen Essigs~ureauhydrid und 6 Tropfen konzentrierter Sehwefelsiiure etwa 5 Minuten Iang erw~rmt, mit Wasser verdtinnt, vorsiehtig alkalis& gemache und hierauf mit Nitroprussidnatrium und Essigsiiure versetzt. J. Mayrlw] er. O. w Spindler: N e u e M o d i f i k a t i o n d e r R e a k t i o n y o n D e n i g g s . N a e h w e i s d e r W e i n s / i u r e in C i t r o n e n s i ~ u r e . (Chem.-Ztg. 1904, 28, 15--16.) Verf. versuchte die bekannte Reaktion yon D e n ig ~ s zur quantitativen Bestimmung tier Citronensfiure auszubilden, da die Bestimmung als Triealeiumsalz unbrauehbare Resultate liefert. Die Versuehe haben jedoch ergeben, dag dem gebildeten Niederschlag, den D e n i g ~ s nach der Formel [(CO. O. CH 2 . CO. CI42 . COO),~Hg]~. H g . O~. Hg~. SO~ zusammengesetzt betrachtet, je nach Konzentration der LSsungen weehselnde Mengen yon Manganhyperoxyd, gebildet dureh Einwirkung des iiberschiissigen Permanganats aaf den weil~en K6rper, beigemengt sind, sodaB die Reaktion in angegebener Form wenigstens nieht zur Mengenbestimmung angewendet werden kann. Versuehe, das Permanganat dureh andere Oxydationsmittel zu ersetzen, lieferten nur fiir Kaliumehromat ein bemerkenswertes Ergebnis insofem als bei Gegenwart der Citronensiiure ein gelb gefgrbter Niedersehlag' entsteht, wS.hrend alle anderen organisehen S~uren ohne Bildung eines Niederseblages oxydiert werden, wobei gleiehzeitig die LSsung dutch entstehendes Chromsulfat griin gef~rbt wird. AIIerdings finder aueh wie beim Permanganat eine Einwirkung des fibersehfissigen Chromates auf den Niedersehlag statt, doeh kann diese dureh Einhal~en bestimmter Versuehsbedingungen wesentlich eingesehrSmkt werden. Weinsii,ure wird unter Kohlens£ureentwiekelung oxydiert, die Flfissigkeit ist je naeh der 3/Ienge des iibersehfissigen Biehromats griin his gelbgrfin gefi~rbt, in Misehungen beider Si~uren verlaufen diese Reaktionen nebeneinander, sodal3 bei genauer Einhaltung naehstehender Vorsehrift Weins~iure neben Citronensiiure naehgewiesen werden kann: 0,5 g der fragliehen Siiure, in 10 ecru Wasser gelSst, werden mit 2 eem Queeksilbemitrat naeh D e n i g g s versetzt, aufgekocht und 2 eem der BiehromatI5sung (5:1000) zugefiigt und ohne weiteres Erhitzen stehen gelassen. Bei reiner Citronensgure tritt alsbald ein hellgelber NiedersehIag auf, wghrend die LSsung tagelang hellgelb bleibt, bei Gegenwart yon Weinsgure dagegen fi~rbt sieh die Fliissigkeit nach kurzer Zeit schmutzig braun, bei grSgeren Mengen yon ~Vein- - 558 R e f e r a t e. - - ntersuchung Allgem. analyt. ]~Iethodenu. Apparate. [Zeitsehr. [d.Nahr,- f.u. UGenul~mi~t;e]. s/iure wird Kohlens/~ureentwiekelung bemerkbar und nach dem Absetzen ist die Fliissigkeit je nach der Menge der vorhandenen Weins/ture mehr oder weniger griin gef~trbt~ Bei 5 °/0 Weins/~ure ist die Reaktion noch scharf, die Farbenver/inderung der LSsung tritt nach 5 Minuten ein; nach 2 Stunden ist die LSsung dunkelgrfin. ~Terden die Mengenverh/~Imisse ge~ndert, so tritt auch bei der Citronens/ture nachtr~glieh Oxydation und Griinf~rbung ein. Es ist desha]b bei Gegenwart kleinerer Mengen yon Weins~iure ein Kontrollversuch mit reiner Citronens~ure auszuffihren. Der ge]be KSrper enthg~lt neben der organischen S/Sure Quecksilber, Schwefels/iure, Chroms~ure und (!hrom in anderer Bindung, er besitzt wahrscheinlich eine ~hnliche Konstitution wie der KSrper yon D e n i g ~ s , seine Zusammensetzung ist abet eine wechselnde, was Verf. dureh Bestimmung des Queeksi]bers und der Chroms/iure naehweist. Gleiche Mengen Citronens/~ure f/~llen Yerschiedene Mengen Queeksilber und Chroms/iure aus, auch diese Reaktion kann daher einstweilen nicht zu einer Mengenbestimmung der Citronens/~ure verwendet werden. J. 1FIayrhofer. Domenico Ganassini: E i n e c h a r a k t e r i s t i s c h e Reaktion der freien Weins/~ure. (Boll. chim. Farm. 19(~3, 4~2, 513--516.) - - Man erhitzt die betreffende yon Minerals~uren freie Flfissigkeit mit etwas fiberschfissiger ~M:ennige kurze Zeit zum Kochen, dekantiert oder filtriert, ffigt zum Filtrat ein gteiches Volumen einer w~tsserigen 20 °/0-igen RhodankaliumlSsung, erhitzt zum Sieden und ]~ist stehen. Bei Gegenwart von freier Weins/iure (auch nur 1 °/o) schw/irzt sich die :Flfissigkeit allm/~hlich, wobei sieh ]angsam Sehwefelblei absetzt. Diese Reaktion kann aueh zum Nachweis yon Weins~ure in Weinen dienen, wobei ein braunvioletter ~Tiederschlag entsteht, w~hrend in eitronen- oder apfelsaueren LSsungen durch die Weins~ure ein rotbrauner iNiederschlag hervorgerufen wird. W. RoSa. A l o i s W i i n s e h : Z u r P r i i f u n g a u f f r e i e 1V[inerals/~ure i n M i l c h ~ s/iure. (Deutsche Gerberztg. 1903, 46, No. 11.) - - Gelegentlich elniger Untersuehungen des Vegetallns (vergl. Deutsche Gerberztg. 1902, 45, No. 117 u. 118) wurde vom Verf. gefunden, daiS Vegetalin mit Methylorangepapier trotz Anwesenheit reichlicher Mengen von freier Milehs~iure keine Rotf/~rbung zeigte, w/~hrend eine solche nach Zusatz sehr geringer ~iengen Minerals/~ure sofort eintrat. Verf. schloiS hieraus,. daiS Milchs/iure Methylorange iiberhaupt nieht vergndere und sprach die Behauptung aus, daiS man in jeder Milchs~ure einen eventuellen Gehalt an Minerals/iure mit Hilfe yon Methylorange nachweisen kSnne. Verf. ist inzwischen yon Herrn Dr. E b e r h a r d in Ludwigslust darauf aufmerksmn gemacht worden, daiS sowohl chemisch reine als aueh technisehe, minerals/iurefreie Milchsiture Methylorangepapier intensiv rot f/~rbt. Um das widersprechende Verhalten des Vegetalins nnd der Miichsgure aufzuklgren, wurde nun eine Probe reiner Milchsfi.ure bis zum Versehwinden der gegen Methylorange saueren Reaktion neutralisiert und dabei gefunden, daiS die Rotf~rbung des Methylorange viel eher aufhSrte als die des Laekmuspapiers. Es ergab sich hieraus, daiS bei tier partiellen 1Neutralisation ein milchsaures Salz gebildet women war und daf~ dieses die Rotf/irbung des Methylorange dureh freie Milehs/~ure verhinderte, Da nun das Vegetalin neben freier Milchs~mre viet milchsaure Ss]ze enthglt~ so erkI~ren sieh die oben angegebenen, anscheinend widersprechenden Tatsachen in einfachster Weise. Verf. berichtigt daher seine oben erw/ihnte Behauptung dahin, daiS die Rotfarbung yon Methylorange dutch eine technische MilehsKure die Anwesenheit yon freier Minerals/~ure weder ausschlieist noeh beweist. Im Anschlusse hieran teilt Verf. noch eine einfaehe 1V[ethode zur Erkennung yon freier Schwefels/iure in Milchs~ure mit. Nach dieser schfittelt man 1 Tell Milchs~ure mit 5 Teil6n Alkohol von 95 °/0 im Reagensglase. l~ach ~/&-stiindigem Stehen filtriert man e~wa 5 - - 1 0 cem yon der Fltissigkeit ab und setzt zu dem klaren Filtrat einige Tropfen einer mit Salzs/iure anges~uerten LSsung yon Ch]orcalcium. Entsteht hierbei nicht sofort oder nach ganz s. Band. I 1. ~November 1904.J R e f e r a t e . -- Allgem. analyt. )~Iethodenu. Apparate. 559 kurzer Zeit eine Triibung, so ist Schwefels/iure in der Milchs~iure nicht vorhanden. Die Methode griindet sieh auf die UnlSslichkeit der meisten Sulfate in starkem A1kohol. Auch Magnesiumsulfat, welches dureh Benutzung harten magnesiahaltigen Wassers oder magnesiahaltiger Kreide bei der Herstellung tier Milchs{~ure in das fertige Produkt fibergehen kann, soll, trotz der verhiiltnismii/~ig leichten LSsliehkeit des Magnesiumsulfats in Alkohol, die Priifung auf freie Schwefelsiiure nach dieser Methode= nicht stSren. A. Oelker. M. D i t t r i c h : ~ b e r F i i t r i e r e n und Veraschen von sehleimige~ Niederschl~gen. (Bet. Deutsch. Chem. Gcsellseh. 1904, 87, 1 8 4 0 - - 1 8 4 2 . ) - GelatinSse ~iederschlfige wie Eisenhydroxyd u. s. w. laufen bekanntllch zufolge des Uberganges in den kolloidalen Zustand h~ufig triibe durch das Filter. Eine teilweise Abhitfe kann durch SchiitteIn diesel" Niederschl~ige mit Papierbrei erzielt werden. E s werden so gut filtrierende ~TiederscM~ge erhalten, die leicht auszuwaschen und zu veraschen sind. Der auf diese Weise erhaltene Glfihrfickstand bildet aui~erdem ein feines Pulver und nicht eine zusammenhiingende Masse, was den weiteren Vorteil bietet, dal~ dasselbe sich, wenn nStig, leicht aufschliel]en l~]~t. J. Mayrhofer. A. D~miehel: E l e k t r i s c h e r V e r a s e h u n g s o f e n von tteraeus. (Bull. Assoc. Chlm. Suer. et Distill. 1904, 21, 1137.) - - Verf. beschreibt den von t t e r a e u s konstruierteu elektrisch geheizten Ofen, welcher der Itauptsache nach aus einem m i t Platinfolie spiralfSrmig umwiekelten Porzellanrohr besteht. Die Platinfolie, dutch welche der Strom geschiekt wlrd, fibertr~igt die W~rme auf das Porzellan. Bei 110 Volt und 5 Ampc~re erreieht man in 5 - - 1 0 Minuten 1200 °. Fiir Zuckerveraschung sind Schalen yon besonderer Form hergestellt. Mittels Vorsehaltwiderstandes li~l~t sich die Temperatur leicht regulieren. Die Veraschung geht schneller vor sich als in den gewShnlichen MuffelSfen und die Asche wird locker und weil~. G. Sonntag. Arturo P e l l i z z a : A p p a r a t zur kontinuierlichen Extraktion yon LSsungen. (Chem.-Ztg. 190¢, 28, 186.) - - Das Extraktionsgefiil~ gleicht dem bekannten S o x h 1 e t ' schen Apparat, dessen Heberrohr V (Fig. 7) als Dampfsteigrohr dient. Um das Zuriickhebern tier im Apparate befindlichen Fliissigkeit zu ¥erhindern, ist dasselbe nach oben verliingert, sodal~ dessen Scheitelpunkt unter allen Umstiinden hSher liegt Ms der Fliiss!.gkeitsspiegel im Extraktionsrohr. Der dureh das Rohr aufsteigende Atherdampf tritt am Boden des doppelwandigen, mit Wasserkfihlung versehenen Extraktionsrohres E ein, steigt in der zu extrahierenden Fliissigkeit in die HShe ~nd sammelt sich an der Oberfliiche an, yon wo aus der mit der gelSsten Substanz beladene ]~ther durch ein gerades offenes Rohr, welches durch den Boden des Extraktors fiihrt, in das SiedekSlbchen P zuriickgelangt u. s.w. Ein aufgesetzter Kiihler R vervollstandigt die Kondensation des Athers, ein am Boden des Extraktors angebrachter Ginshahn r gestattet Entleerung des Apparates und Kontrolle der Extraktion. J. Mayrhofer. f[_ Emm. Pozzi-Escot: E i n k l e i n e r A p p a r a t f i i r W a s s e r Fig. 7. dampfdestillation. (Bull. Assoc. Chim. Sucr. et Distill. 1904, 21, 1139--1140.) - - Der leicht zusammenstellbare Apparat besteht aus einem Destillierkolben yon etwa 30 ccm Inhalt mit langem Hals und Ansatzrohr; im Innern desselben ist etwa in der Mitte des tIalses ein Glasrohr angeschmolzen, das sich nach auSen 5ffnet und fast bis zum Boden reieht. Dieser Kolben wird mittels eines Stopfens luhdicht in den weiten Itals einer zur Hfi.lfte mit Wasser gefiillten Koehflasche ein- 560 1~e fer a t e. -- Forense Chemie. [Zeitsehr, f. Un~erst~chung Ld. Nshr.-u. Genul~mittel. gesetzt. Wird das Wasser ins Sieden gebracht, so mul~ der Wasserdampf seinen Weg durch das innere Rohr des eingesetzten Destillierko~bens in die zu destillierende Flfissigkeit, welehe slsdann schon auf die Temperatur des s]edenden Wassers erhitzt ist, und yon da in den Kfihler nehmen. Der Apparat ist einfach und bedarf w~hrend der Destillation keiner Uberwaehung. G. Sonntag. F o r e n s e Chemie. R. Maggiora: E r p r o b u n g d e r n e u e n H o d i f i k a t i o n des bioehemls e h e n A r s e n i k n a c h w e i s e s n a e h Gosio. (CentrbI. Bakteriol. II. Abt. 1903, 11, 237--238.) - - Verf. hat unter Anwendung der neueren yon G o s i o empfohlenen Modifikation (Ablagerung des arsenikhaltigen Materials in frischen Kulturen yon Penicillium brevieaule auf Kartoffeln) 51 ArsenikprKparate untersucht und auch bei sehr kleinen Mengen stets die Reaktion erhalten. Tellur gibt ebenfalls den Knoblauchgeruch; doch ist eine Verwechselung mit Arson wegen seines seltenen Vorkommens wohl ausgeschlossen. .,4. Spieckermann. S. R. T r o t m a n : Die elektrolytische Bestimmung yon Arson. (Joum. Soc. Chem. Ind. 1904, 23, 177--179.) - - An Hand einer Abbildung wild ein Apparat beschrieben, den Verf. schon seit zwei Jahren zur elektrolytischen Bestimmung yon Arson benutzt hat und der die Erkennung yon 0,000002 g Arsentrioxyd gestattet. Ein Glaseylinder, dessen untere 0ffnung mit Pergamentpapier fiberbunden ist, tr•gt oben elnen KautsehukstSpsel und unterhalb dessen eine GasableimngsrShre, an die sieh TroekenrShre und Gl'fihrShre anschtie~en. Dureh den K ~ t schukstSpsel geht die RShre eines Trop~triehters zum Einffitlen des Untersuebungsmaterials, sowie ein die Platinkathode tragender Leitungsdraht. Der Cylinder steht in einem Glasgef~l~ und ist yon der ringfSrmigen Anode umgeben. Das Ganze steht in einem weiteren, mit Wasser geffillten Kfihlgef~,l:3. Dem Elektrolyten werden einige Tropfen ZinksutfatlSsung zugesetzt. Zur ~Vasserzersetzung dient Stm'kstrom, der durch einen Lampenwlderstand reguliert wird. Das Untersuchungsmaterial kann ohne ZerstSmng der organischen Substanz in die Zersetzungszelle gebracht werden. C. x~/Iai. Julian L Baker: E i n e Z u s a m m e n f a s s u n g der Beriehte und l~itteilungen der kSnigl.'Kommission ffir Arsenvergiftung. (Journ. Soe. Chem. Ind. 1904, 23, 159~174.) - - Verf. gibt eine fibersiehtllche Zusammenstellung der bis Ende 1903 von der zum Studium der Massenvergii~tung dutch arsenhaltlges Bier 1900 eingesetzten Kommission hemusgegebenen Berichte und Vorschl~ge. In sieben Abteilungen werden Mitteihngen fiber die klinischen Beobachtungen u. s. w. wahrend der Vergiftung, die Verfahren zum l~aehweise des Arsens in l~ahmngsmitteln u. s. w., die MSglichkeiten der Verunreinigung yon l~ahrungsstoffen mit Arsen, die Vorsichtsmagregeln zum Fernhalten des Arsens yon diesen, die Uberwaehung der Reinheit in bezug auf Arson u. s. w. gemacht, fiber deren wesentliche Einze]heiten an dieser Stette sehon wiederholt berichtet women ist. Was die Verfahren zum l~'aehweis kleiner Arsenmengen betrifft, so gibt die Kommission der Arsenbestimmung auf elektrolytischem Wege den Vorzug vet dem Verfahren nach M a r s h - B e r z e l i u s. Die Vorzfige des zuerst yon B l o x a m (Journ. Chem. Soe. 1861, 13, 12 und 338) angegebenen Verfahrens sind die Vermeidung yon Zink und Sgure, die Einfachheit tier Ausffihrung, Erlangung gleicher Ergebnisse durch versehiedene AnaIytiker durch Anwendung bestimmter Stromstgrken, MSglichkeit der direkten Prilfung von Bier, Malz u. s. w. ohne ZerstSrung der organischen Substanz, Gleiehmgl3igkeit der erhaltenen Arsenspiegel und dadurch bedingte grSl~ere Genauigkeit b e i d e r Vergleichung mit Normalspiegeln und die gleichzeitige Ausffihrbarkei~ mehrerer Bestimmungen im selben Stromkreis. Die I~Iaehteile des Verfahrens sind die hSheren An]agekosten und die AbhKngigkeit yore Vorhandensein einer geeigneten elektrisehen Stromquelle. C. Mai.
https://openalex.org/W4320929058	https://sciresol.s3.us-east-2.amazonaws.com/srs-j/JCBS/pdf/VOL3/Issue-4/Brief%20communication.pdf	English	null	Evaluation of plasma fibrinogen and plasma fibrin degradation product (FDP) in Preeclampsia	Journal of clinical and biomedical sciences	2,013	cc-by	2,405	Introduction J Clin Biomed Sci 2013; 3(4):201-03 2013 Journal of Clinical and Biomedical Sciences. All rights reserved. Corresponding Author Sonal Sogani, Department of Biochemistry, Mahatma Gandhi Memorial Medical College, Indore, Madhya Pradesh. India. E mail : sonal.sogani246@gmail.com. Preeclampsia is a systemic disease character- ised by hypertension, oedema and proteinuria. Hae- matological, genetic and immunological factors play role in preeclampsia aetiopathogenesis. Fibrin deposit in vascular and endothelial area of many organs and placenta is the well-known feature of this disease (1). Preeclampsia is a major cause of maternal morbidity and mortality in the world. It develops in 5-8% of hu- man pregnancies (2). Normal pregnancy is associated with impres- sive changes in the haemostatic mechanism and is a hypercoagulable state associated with increase in many coagulation factors. Coagulation and fibrinolytic systems undergo major alteration associated with re- duced fibrinolytic activity and increased levels of fi- brinogen and FDP in normal pregnancy which has been reported in many studies(3,4). It has been hypoth- esised that preeclampsia is a generalised intravascular inflammatory response, which occurs in normal pregnancy too, but is more exaggerated in preeclampsia (5). Endothelial cell dysfunction and inflammation are considered to have a role in the pathophysiology of preeclampsia (6). Mediators of an inflammatory response like plasma fibrinogen are altered in preeclamptic women. A systemic inflam- matory response involves both the immune system and clotting and fibrinolytic system (7) . An excess of FDP with diminished or normal systemic fibrinolytic activity suggests that local intravascular fibrin depo- sition and fibrinolysis occur in preeclampsia. The high level of FDP is associated with the pathogenesis of the defective haemostasis in preeclampsia (8). In- creased fibrinolytic activity in preeclampsia patients has been reported earlier, resulting in increased lev- els of FDP (9,10) . So this study intended to estimate the plasma fibrinogen and plasma fibrin degradation product (FDP) levels in preeclamptic pregnant wom- en and compare them with normal pregnant women Quick access Code pregnancy too, but is more exaggerated in preeclampsia (5). Endothelial cell dysfunction and inflammation are considered to have a role in the pathophysiology of preeclampsia (6). Mediators of an inflammatory response like plasma fibrinogen are altered in preeclamptic women. A systemic inflam- matory response involves both the immune system and clotting and fibrinolytic system (7) . An excess of FDP with diminished or normal systemic fibrinolytic activity suggests that local intravascular fibrin depo- sition and fibrinolysis occur in preeclampsia. Corresponding Author Sonal Sogani, Department of Biochemistry, Mahatma Gandhi Memorial Medical College, Indore, Madhya Pradesh. India. E mail : sonal.sogani246@gmail.com. Sonal Sogani, Purnima Dey Sarakar Department of Biochemistry, Mahatma Gandhi Memorial Medical College, Indore, Madhya Pradesh. Indi Received: 05th October –2013 Accepted : 25th November - 2013 Published: 30th December - 2013 Abstract Background: Preeclampsia is one of the commonest complications of pregnancy. It is associated with a state of hypercoagulability. The present study aimed to estimate the plasma fibrinogen and plasma FDP levels in preeclampsia com- pared to normal pregnancies. Materials and Methods: This is a case-control hospital based study carried in the Department of Biochemistry M.G.M. Medical College and associated M.Y. Hospital, Indore (M.P., India). Normal pregnant women (n=36) and women with preeclampsia (n=64) in their third trimester were included in the study. Preeclamptic group was classified in to mild (n=42) and severe (n=22) preeclampsia. Plasma fibrinogen and FDP levels were analysed, and compared between the groups. Results: Preeclampsia and normal pregnancy groups were comparable for age and body mass index but preeclampsia group had higher blood pressures and less period of gestation (p<0.0001). The levels of plasma fibrinogen (654.5±131.74 vs. 491.52±81.7 mg/dL) and plasma FDP (10.96±2.32 vs. 5.54±0.8 µg/L) were higher in the preeclampsia group as compared to normal pregnancy (p<0.0001). Elevations in fibrinogen and FDP levels were more marked for severe preeclampsia group than mild preeclampsia group. Conclusion: Preeclampsia is associated with high fibrinogen and FDP levels as compared to normal pregnancies. Severe preeclampsia patients have greater elevations as compared to mild preeclampsia patients. Key words: Fibrin degradation products, Fibrinogen, pre-eclampsia. Evaluation of plasma fibrinogen and plasma fibrin degradation product (FDP) in Preeclampsia. Sonal Sogani, Purnima Dey Sarakar 201 J Clin Biomed Sci 2013; 3(4):201-03 2013 Journal of Clinical and Biomedical Sciences. All rights reserved. Corresponding Author Sonal Sogani, Department of Biochemistry, Mahatma Gandhi Memorial Medical College, Indore, Madhya Pradesh. India. E mail : sonal.sogani246@gmail.com. Quick access Cod Materials and Methods Maternal age and body mass index (BMI) were comparable in the preeclampsia and normal pregnancy groups (Table 1). Gestational age was low- er while systolic and diastolic blood pressures were significantly (p<0.0001) higher in preeclamptic pa- tients as compared to normal pregnant women (Table 1). Average values of fibrinogen and FDP were higher for the preeclamptic pregnant patients as compared to normal pregnancy group (p<0.0001), although the normal pregnant group also showed elevated levels of fibrinogen than the normal range (200-400 mg/dL). Fibrinogen (727.95 vs 616.02 mg/dL, p<0.05) and FDP levels were significantly higher in severe preeclampsia group than in the mild preeclampsia group (12.28 vs. 10.27 µg/L, p<0.001). This case control study was conducted in the Department of Biochemistry M.G.M. Medical College and associated M.Y. Hospital, Indore, Madhya Pra- desh, India. The subjects were pregnant women clini- cally diagnosed as preeclampsia during third tri- mester (28-40 weeks) with the age 18-35 years visit- ing obstetrics OPD and wards of MY Hospital, Indore. Sixty-four consecutive preeclampsia patients were included in the study. Preeclampsia patients were classified in to mild preeclampsia (n=42) and severe preeclampsia (n=22) women. As a control group 36 pregnant women without evidence of any illness were taken. The normal pregnant women were also in the third trimester (28-40 weeks) of their preg- nancy with the age 18-35 years. Inclusion criteria for women included in the study were: should not be using any kind of oral contraceptives, anticoagulant drugs, should be non-smokers and non-alcoholics. Exclusion criteria were: past history of diabetes, sys- temic or endocrine disorder, chronic infection, chronic renal disease, previous history of hyperten- sion, women in active labour, were excluded from the study. Table:1- Clinico-biochemical features in the normal and preeclampsia groups Disc ssion Parameters Normal pregnant women (n=36) Preeclamptic pregnant women (n=64) Mild preeclamptic pregnant women (n=42) Severe preeclamptic pregnant women (n=22) Age (years) 23±2.62 23.14±2.97 22.90±2.96 23.59±2.98 BMI (kg/m2) 24.17±1.91 24.66±1.84 24.87±1.76 24.26±2.07 Gestational age (weeks) 39.13±2.82 36.68±1.78 37.19±1.65 35.72±1.54 Systolic blood pressure (mm of Hg) 114.72±6.96 148.10±15.78 139.52±5.38 164.5±16.17 Diastolic blood pressure (mm of Hg) 74.44±5.03 100.15±12.11 94.04±7.26 111.81±10.9 7 Plasma fibrinogen (mg/dL) 491.52±81.7 654.5±131.74 616.02±124. 65 727.95±118. Plasma fibrinogen in preeclampsia Plasma fibrinogen in preeclampsia 202 in their third trimester. in their third trimester. in their third trimester. Materials and Methods 91 Plasma FDP (µg/L) 5.54±0.8 10.96±2.32 10.27±2.39 12.28±1.57# Preeclampsia was diagnosed according to American College of Obstetrics and Gynecology (ACOG) criteria: a blood pressure higher than 140/90 mm Hg and proteinuria more than 300 mg/24hour were observed on at least two occasions more than 6 hours apart after the 20th week of preg- nancy. Preeclamspia was classified as severe if dias- tolic blood pressure increased to at least 110 mmHg, proteinuria >5000 mg per day and the presence of headache, visual disturbances, epigastric pain, oligu- ria, elevated liver function tests, elevated renal func- tion tests, thrombocytopenia. Introduction The high level of FDP is associated with the pathogenesis of the defective haemostasis in preeclampsia (8). In- creased fibrinolytic activity in preeclampsia patients has been reported earlier, resulting in increased lev- els of FDP (9,10) . So this study intended to estimate the plasma fibrinogen and plasma fibrin degradation product (FDP) levels in preeclamptic pregnant wom- en and compare them with normal pregnant women Preeclampsia is a systemic disease character- ised by hypertension, oedema and proteinuria. Hae- matological, genetic and immunological factors play role in preeclampsia aetiopathogenesis. Fibrin deposit in vascular and endothelial area of many organs and placenta is the well-known feature of this disease (1). Preeclampsia is a major cause of maternal morbidity and mortality in the world. It develops in 5-8% of hu- man pregnancies (2). Normal pregnancy is associated with impres- sive changes in the haemostatic mechanism and is a hypercoagulable state associated with increase in many coagulation factors. Coagulation and fibrinolytic systems undergo major alteration associated with re- duced fibrinolytic activity and increased levels of fi- brinogen and FDP in normal pregnancy which has been reported in many studies(3,4). It has been hypoth- esised that preeclampsia is a generalised intravascular inflammatory response, which occurs in normal J Clin Biomed Sci 2013; 3(4):201-03 2013 Journal of Clinical and Biomedical Sciences. All rights reserved. Corresponding Author Sonal Sogani, Department of Biochemistry, Mahatma Gandhi Memorial Medical College, Indore, Madhya Pradesh. India. E mail : sonal.sogani246@gmail.com. Quick access Code 2013 Journal of Clinical and Biomedical Sciences. All rights reserved. J Clin Biomed Sci 2013; 3(4):201-03 J Clin Biomed Sci 2013; 3(4):201-03 J Clin Biomed Sci 2013; 3(4):201-03 Discussion In the present study, the plasma fibrinogen and FDP levels were significantly higher in preeclampsia, more so in the severe preeclampsia women as compared to mild preeclampsia. Enhanced coagulation-fibrinolysis and coagulopathy are well known features of preeclampsia (11). Fibrinogens are the major coagulation heterogeneous protein and it is symmetrical glycoprotein of 340 KD molecular weight. The results of our study showed that in preeclampsia the level of plasma fibrinogen was higher as compared to normal pregnancy and recon- firms the results from earlier studies(12,13).The in- crease of fibrinogen in preeclampsia results from the exaggerated inflammatory response and subsequent endothelial dysfunction and activation which are cur- rently believed to be the key pathophysiological mechanism in preeclampsia (12). The increase in fi- brinogen in normal pregnancy is also due to the in- flammatory responses which explain that fibrinogen Blood samples for plasma fibrinogen and FDP was collected into test tubes containing 1 ml of 3.8% sodium-citrate. Centrifugation of these speci- mens was done for ten minutes at room temperature and at 2500xg. Plasma fibrinogen was estimated by Clauss method using Start analyser -a compact 4- channel coagulation instrument. The levels of FDP were investigated using latex immunoturbidimetric method based on the principle of ELISA. The results were expressed as mean ± SD and groups were com- pared using ANOVA. Statistical analysis was carried out by using SPSS software, version 20. The level of significance was set at < 0.05. J Clin Biomed Sci 2013; 3(4):201-03 Plasma fibrinogen in preeclampsia 203 is the acute phase reactant-marker of inflammation, in both normal pregnancy and preeclamptic pregnan- cy. The increase of fibrinogen in normal pregnancy may also be due to utilisation in uteroplacental circu- lation, enhanced synthesis and also due to hormonal changes like oestrogen synthesis (14). 7. Teran E, Escudero C, Moya W, Flores M, Vallance P, Lopez-Jaramillo P. Elevated CRP and proin- flammatory cytokines in Andean women with preeclampsia.Int J Gynecol Obstet 2001;75:243- 249. 8. Toshihiko Terao, Masahiro Maki, Tsuyomu Ike- noue, Kaoru Gotoh, Makoto Murata et al .The re- lationship between Clinical Signs and Hypercoag- ulable State in Toxemia of Pregnancy. Gynecol Obstet Invest 1991; 31:74-85. Fibrin degradation products (FDP) are com- ponents of the blood produced by clot degeneration. These are the substances left behind when clots dis- solve in the blood. These are produced by the action of plasmin (proteolytic enzymes) on deposited fibrin. Discussion Excess FDP can cause severe haemostatic defects (15). High plasma FDP levels in our preeclamptic patients are in agreement with the finding of others (16,17). In this study, raised plasma FDP levels indicate increase in fibrinolytic activity and concurs with the previous finding which showed an intravascular coagulation disturbance observed in patients with preeclampsia (17,18). However, more research in this field is war- ranted. 9. Rakoczi I, Tallian F, Bagdany S, Gati I: Platelet life span in normal pregnancy and preeclampsia as determined by a non-radioisotopes technique. Throm Res 1979; 15:553. 10. Pritchard JA, Cunningham FG, Mason RA. Coagu- lation changes in eclampsia: Their frequency and pathogenesis. Am J Obstet Gynecol. Thromb Res 1976; 124:855. 11. Cadroy Y, Grandjean H, Pichon J, Desprats R,et al. Evaluation of six markers of haemostatic system in normal pregnancy and pregnancy complicated by hypertension or preeclampsia.Br J Obstet Gy- necol 1993;100:416-20. Our study concluded that increase in plasma fibrinogen explains it to be an acute phase reactant indicating the exaggerated inflammatory responses in preeclampsia and the endothelial activation which are believe to be the pathophysiological mechanism in preeclampsia. Also the elevated FDP indicates the increased intravascular coagulation in preeclampsia and increased in fibrinolytic activity. Both are thus the complementary predictors explaining the severi- ty of the disease. 12. Manten GT, Sikkema JM, Franx A, Hameeteman TM, Visser GH, de Groot PG, and Voorbij HA. In- creased high molecular weight fibrinogen in preeclampsia. Thromb Res.2003;111(3):143-7. 13. Ustun Y, Engin-Ustun E, Kamaci M. Association of fibrinogen and CRP with severity of preeclamp- sia. Eur J Obstet Gynecol Reprod Biol 2005;121:154-158. References 14. Koos JB, Moore JP. Maternal physiology during pregnancy. In Alan HD, Lauren N editors. Current obstetric and gynecologic diagnosis and treat- ment, 9th ed. New York: Mc Graw Hill Compo- nies; 2003.p.154-62. 1. Stekkinger E, Zandstra M, Peeters LL, Spaander- nen ME. Early-onset preeclampsia and the preva- lence of postpartum metabolic syndrome. Obstet Gynaecol 2009 ; 114(5):1076-84. 2. The world health report 2005: make every moth- er and child count.Geneva: WHO; 2005. 2. The world health report 2005: make every moth- er and child count.Geneva: WHO; 2005. 15. Gaffney PJ, Edgell T, Creighton-Kempsford LJ, Wheeler S, Tarelli E . Fibrin degradation product (FDP) assays: analysis of standardization issues and target antigens in plasma. Br. J. Haematol. 1995;90 (1): 187–94. 3. Tommaso MD, Ferretti C, Conforti D, et al. Hema- tocrit and hemoglobin parameters of hematic viscosity in pregnancy induced hypertension. Minerva Ginecologica 1991; 43(5):237-40. 16. Jambulkar S, Shrikhande A, Shrivastav R, Deshmukh K. Coagulation profile in pregnancy induced hypertension Indian J Hematol Blood Transfus.2001;19(1):3-5. 4. Benneth B, Moore NR, Cruickshank DJ, et al. Plas- minogen activator inhibitors (PAI-1) and (PAI-2) in normal pregnancies preeclampsia and hyditi- form mole. BJOG 1993; 100:370-74. 17. Namavar Jahromi B, Rafiee SH. Coagulation Fac- tors in Severe Preeclampsia. IRCMJ .2009; 11 (3):321-324. 5. Redman CWG, Sacks GP, Sargent IL. Preeclamp- sia: an excessive maternal inflammatory re- sponse to pregnancy .Am J Obstet Gynecol 1999; 180:499-506. 18. Manaj A, Rrugia A, Manoku N. The impact of preeclampsia in pregnancy. J Prenat Med.2011;5 (1):19-22 6. James DK, Steer PJ, Weiner CP, Gonik B, editors. High Risk Pregnancy Management Option.3rd ed. Philadelphia: WB Saunders; 2005. J Clin Biomed Sci 2013; 3(4):201-03
https://openalex.org/W4316495447	https://www.researchsquare.com/article/rs-2468781/latest.pdf	English	null	13C pulse-labeling suggests separate fast and slow dynamics for two amino acid pools in ectomycorrhizal fungi	Research Square (Research Square)	2,023	cc-by	5,931	13C pulse-labeling suggests separate fast and slow dynamics for two amino acid pools in ectomycorrhizal fungi rik A. Hobbie (  Erik.Hobbie@unh.edu ) University of New Hampshire https://orcid.org/0000-0002-1629-6307 Erik A. Hobbie (  Erik.Hobbie@unh.edu ) University of New Hampshire https://orcid.org/0000-0002-1629-6307 University of New Hampshire https://orcid.org/0000-0002-1629-6307 University of New Hampshire https://orcid.org/0000-0002-1629-6307 Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Plant and Soil on July 25th, 2023. See the published version at https://doi.org/10.1007/s11104-023-06125-0. Page 1/14 Results Prior to 13C labeling, amino acid δ13C was higher in Cortinarius than Lactarius, which suggests uptake of older soil-derived amino acids by Cortinarius. 13C labeling in amino acids was 58 ± 3% of that in structural carbohydrates. In stepwise regression, 58% of amino acid carbon tracked the δ13C of the structural carbon which peaked on day 16. The other 42% of amino acid carbon peaked later, on day 30, at 19% of the 13C enrichment of structural carbon, indicating two pools of amino acids. Both structural carbohydrates and the ‘fast’ pool of amino acids derived from recent plant sugars. Abstract Ectomycorrhizal fungi depend on recent photosynthate from their host plants but can also assimilate soil-derived amino acids into fungal protein. The dynamics of different pools of amino acids may differ because their energy cost for acquisition may differ. Methods To study carbon dynamics between Pinus sylvestris and ectomycorrhizal fungi, 13CO2 was traced for 97 days into amino acids and structural carbohydrates of Cortinarius semisanguineus and Lactarius rufus sporocarps. Conclusion It is hypothesized that fast cycling amino acids had simple synthesis pathways such as alanine, aspartic acid and glutamic acid, whereas slow cycling amino acids had multiple enzymatic steps to synthesis, such as lysine, leucine, and isoleucine. This slow pool is more likely than the fast pool to reflect soil- derived amino acids. Studies of amino acid use by ectomycorrhizal fungi should account for the large differences in dynamics and energetic costs between slow and fast cycling amino acids. Introduction Ectomycorrhizal fungi depend on plant-supplied sugars for energy and carbon supply, with up to 30% of primary production diverted to support these symbioses (Ouimette et al. 2019). In boreal forests, trees primarily rely on ectomycorrhizal fungal symbionts for their nitrogen supply (Read et al. 2004), in particular because of the enzymatic capabilities of these fungi to extract organic N from the soil (Clemmensen et al. 2008; Talbot and Treseder 2010). The ability of many ectomycorrhizal fungi to access Page 2/14 Page 2/14 soil-derived organic nitrogen may be particularly important in boreal forests, where N turnover rates are rather slow (Read et al. 2004). ved amino acids indicates that ectomycorrhizal fungi have two potential car Uptake of soil-derived amino acids indicates that ectomycorrhizal fungi have two potential carbon sources, plant sugars from recent photosynthesis and amino acids from soil organic nitrogen. Sporocarps of ectomycorrhizal fungi have been separated into amino acids and structural carbohydrates to study the uptake of these soil-derived amino acids by fungi using natural abundance radiocarbon (14C) (Hobbie et al. 2013). In that study in Finnish boreal forests, Cortinarius fungi took up decades-old organic nitrogen, whereas Lactarius fungi took up younger organic nitrogen. Short-term dynamics of amino acids have been studied by applying 13C-labeled amino acids to soils and following the incorporation of that tracer for days or weeks (Näsholm et al. 2009). An alternative approach is to apply 13C-labeled CO2 to host plants and trace that isotopic label for days or weeks into different pools including ectomycorrhizal roots, ectomycorrhizal sporocarps, or into ectomycorrhizal respiration (Högberg et al. 2010; Högberg et al. 2008; Moyano et al. 2008). Such studies indicate that C fixed through photosynthesis appears in soil-respired CO2 within one to five days (Moyano et al. 2008), appears in ectomycorrhizal sporocarps within six days, and has a half-life of 10 days in sporocarps (Högberg et al. 2010). However, whether the dynamics of fungal amino acids and carbohydrates differ has not been studied. Retention rates of amino acids in soils and in microbial biomass depend on their structure, with microbial retention of aspartic acid and glutamic acid averaging 45%, glycine 70%, and isoleucine, leucine, and lysine averaging 82% (Hobbie and Hobbie 2012). The order follows the length of their catabolic and anabolic pathways. Site description The research site was a young Pinus sylvestris forest in northern Sweden (64°09’ N, 19°05’ E) (Högberg et al. 2008), with trees 14 years old in 2007. Each 50 m2 plot (n = 16) contained 62 ± 9 trees (mean tree height 2.5 ± 0.2 m), with an understory composed of dwarf shrubs such as Calluna vulgaris L. and Vaccinium vitis-idaea L. Cladonia lichens covered the ground. The soil is a podzol, with an organic mor layer of 2–3 cm thickness, C:N ratio of 33 ± 1, and a pH of 4.5 ± 0.1. Introduction For example, aspartic acid and glutamic acid readily enter the citric acid cycle after deamination to oxaloacetate and α-ketoglutarate, respectively, while additional transformations are necessary for the turnover of leucine, isoleucine, and lysine (Crawford et al. 1974). Patterns of biosynthesis follow similar patterns (Barton et al., 2010), with one synthesis step required for alanine, glutamic acid, and aspartic acid, whereas the eight amino acids considered essential for mammalian physiology average eight biosynthetic steps. In the current study, the transfer times or residence times of carbon in different fungal pools were inferred. The dynamics of amino acids and of structural carbon were specifically examined within sporocarps of the dominant species of ectomycorrhizal fungi, Cortinarius semisanguineus (Fr.) Gillet and Lactarius rufus (Scop.) Fr., in a 13C pulse-chase experiment from Pinus sylvestris L. stands in northern Sweden initiated by a research group at the Swedish Agricultural University (SLU) in Umeå (Högberg et al., 2010). To do this, amino acids and structural carbon were isolated from sporocarps collected by these researchers from six to 97 days after pulse-labeling with 13CO2. Since amino acids are directly incorporated by ectomycorrhizal fungi (Abuzinadah and Read 1989) and some amino acids are preferentially retained relative to sugars, the hypotheses were that (1) the incorporation of soil-derived amino acids into the fungal amino acid pool will lead to initially lower 13C labeling in the fungal amino Page 3/14 acid pool than in bulk sporocarp material and in structural carbon; (2) 13C labeling in amino acids should decrease more slowly than that of other pools because of preferential retention by fungi of amino acids relative to carbohydrates. acid pool than in bulk sporocarp material and in structural carbon; (2) 13C labeling in amino acids should decrease more slowly than that of other pools because of preferential retention by fungi of amino acids relative to carbohydrates. Pulse 13CO2 labeling For pulse 13CO2 labeling, two closed, octagonal chambers that were 5 m tall and 50 m2 in surface area were used (Högberg et al. 2010). To study carbon dynamics in the plant-soil-fungal system over the growing season, 13C-labeled CO2 was added to eight 50 m2 plots in June 2007 and to a separate eight plots in August 2007. Four of the plots were fertilized by broadcasting pellets of calcium nitrate at a dose of 100 kg N ha− 1 two weeks before 13CO2 labeling of the N addition plots in June 2007. This N addition did not affect 13CO2 uptake (Högberg et al., 2010). An additional four plots served as natural abundance plots. The temperature inside the chambers was controlled and set to track the ambient air temperature outside. 25 liters of 99 atom% 13CO2 were released into each chamber (~ 50 g), which resulted in 21.9 ± 0.5 atom% 13C (compared with 1.1 atom% 13C in ambient CO2) and 531 ± 7 ppm CO2 (compared with c. 380 ppm in ambient air) at the start of labeling in 2007. The final CO2 concentration was 317 ± 11 ppm. The CO2 concentration inside the chambers was monitored using infrared gas analyzers. These were used to follow the overall photosynthetic draw-down of CO2 inside the chambers. To measure CO2 concentration and 13CO2 uptake, air samples were taken for isotope ratio mass spectrometry analysis from inside the chamber directly after the release of 13CO2, in the middle of the labeling period, and immediately before its end. Two chambers were labelled in parallel with a time lag of 0.5 h between the start of the first and the second labeling, with a labeling duration between 1.5 and 3.5 h. The duration of labeling was adjusted so that 13C uptake across plots was similar. In August 2007, the ecosystem 13C uptake, calculated as the difference between the initial and final contents of 13CO2 in the chamber air of control plots, was 10.3 ± 0.5 g 13C. The soil temperature at 5 cm depth at the time of 13CO2 labeling was 11.5°C. Chemical separation of structural carbon and amino acids from bulk sporocarps Chemical separation techniques previously developed to isolate structural carbon and amino acids were used (Hobbie et al. 2013). Finely ground samples of sporocarps were first extracted three times with hexane (30 min at 70°C) to remove non-polar compounds, and then by three extractions with 80% ethanol (30 min at 100°C) to remove soluble polar compounds, such as carbohydrates and organic acids. The remaining solid was hydrolyzed with 6 M hydrochloric acid at 110°C for 24 h. The hydrolysate was filtered and solubilized compounds were purified to retrieve the amino acid fraction using cation- exchange chromatography. The amino acids were eluted and then dried down to represent the protein fraction. The remaining, insoluble material was extracted at 100°C for 2 h with 0.2 M sodium hydroxide to remove residual proteins and the remaining solid material was considered the structural fraction. After separation, samples were analyzed for 13C using a Costech 4010 Elemental Analyzer coupled to a Thermo Delta Plus XP isotope ratio mass spectrometer at the University of New Hampshire. Isotopic signatures are reported relative to the standard VPDB for carbon with the delta notation, δ13C (‰), equivalent to (13C:12Csample/13C:12Cstandard – 1) × 1000 (‰) (Coplen 2011). Laboratory standards differing greatly in C/N were concurrently run. Standard deviations of laboratory standards for δ13C averaged less than 0.2‰. Sporocarp harvest Page 4/14 Page 4/14 In 2007, all fungal sporocarps in the plots from both the June and August 13CO2 labeling campaigns were collected by the research group at SLU; the peak fruiting months were August and September. The 13C analyses of bulk sporocarps were reported in Högberg et al. (2010). Here, sporocarp subsamples were provided by the SLU research group of the two dominant species of these sporocarps, Cortinarius semisanguineus (65% of sporocarps) and Lactarius rufus (17% of sporocarps). These sporocarps were used to isolate fungal amino acids and structural carbohydrates for subsequent 13C analyses. Statistical analyses δ13Cstructural was assumed to reflect the temporal dynamics of photosynthates provided by 13C-labeled trees to ectomycorrhizal fungi. To allow for potentially complex temporal dynamics, stepwise multiple regression models of 13Cbulk and δ13Camino acids were analyzed with a fourth-order polynomial of the number of days after the 13C pulse (that is, days, days2, days3, and days4). A fourth-order polynomial was chosen since the decay of the 13C pulse may be non-linear. Statisticians recommend that models should have no more terms than the square-root of the sample numbers of the dependent variable (18 in this case). Nitrogen treatment (added or not), δ13Cstructural, and genus (Lactarius or Cortinarius) were also included as independent variables. Data were analyzed statistically using JMP 13 Pro (SAS Institute, Middleton, Massachusetts, USA). Models that minimized Aikike’s Information Criteria were selected, with a correction for sample size (AICc). Results Page 5/14 Page 5/14 Sporocarps collected from the natural abundance plots provided control δ13C signatures for bulk, amino acid, and structural pools (Table 1). Across both fungal taxa, bulk carbon averaged -25.84 ± 0.13‰ (n = 11), amino acids − 22.82 ± 0.23‰ (n = 11), and structural carbon − 24.23 ± 0.46‰ (n = 10). Amino acids therefore were 1.38 ± 0.35‰ higher in δ13C than structural carbon (n = 10). Cortinarius had significantly higher δ13C signatures than Lactarius in amino acids (p = 0.049) but not in structural carbohydrates (p = 0.602) or sporocarps (p = 0.272). Sporocarps collected from the natural abundance plots provided control δ13C signatures for bulk, amino acid, and structural pools (Table 1). Across both fungal taxa, bulk carbon averaged -25.84 ± 0.13‰ (n = 11), amino acids − 22.82 ± 0.23‰ (n = 11), and structural carbon − 24.23 ± 0.46‰ (n = 10). Amino acids therefore were 1.38 ± 0.35‰ higher in δ13C than structural carbon (n = 10). Cortinarius had significantly higher δ13C signatures than Lactarius in amino acids (p = 0.049) but not in structural carbohydrates (p = 0.602) or sporocarps (p = 0.272). The 13C-labeled CO2 added in August was first detected in a Lactarius sporocarp collected 6 days after pulse labeling. Sporocarp, amino acid, and structural δ13C signatures peaked on day 16 at 173‰, 117‰ and 198‰, respectively (Fig. 1a). In contrast, a previous study reported that phloem sap δ13C at 0.3 m height peaked after 24 hours (Högberg et al. 2008), at a δ13C value of 140‰ (Fig. 1b). By day 28, 13C labeling in structural carbohydrates had declined to less than 10% of the peak on day 16. The two sporocarps collected on that day had higher δ13C signatures in amino acids than in structural carbon (Fig. 2), as did five sporocarps collected in the June pulse-labeling plots 72 to 97 days after labeling, although three sporocarps collected 93 days after the June labeling had δ13C signatures near background levels. In simple linear regressions, the slopes between the δ13C of structural carbon (x) and either amino acids (y1) or bulk carbon (y2) were 61 ± 3% and 90 ± 3%, respectively (Fig. 3). Discussion A 13C pulse chase experiment and isotopic analysis on biochemical fractions of fungal sporocarps was used to investigate carbon dynamics in two ectomycorrhizal species. Fungal structural compounds and bulk sporocarps reached similar labeling levels as phloem sap (Fig. 1b), suggesting plant C as the primary source for these fungi. In contrast, the maximum 13C labeling of the amino acid pool reached only ~ 60% of that of the structural carbohydrate pool (Fig. 3, Table 2) and the δ13C signature of the amino acid pool declined more slowly than the structural pool after the 13C pulse. A month after labeling, the amino acid pool had retained more 13C label than the structural carbohydrate pool (Fig. 2) despite its lower maximum labeling level. From these data, it appears that roughly 60% of amino acid carbon was derived from recent plant photosynthesis and 40% from carbon of longer residence time (either plant- derived or soil-derived). A partitioning of amino acid carbon into distinct pools that differ in their turnover rate can be plausibly explained by examining the biosynthetic pathways of different amino acids. Amino acids that are derived from glycolytic or citric acid cycle intermediates in only a few steps can be frequently resynthesized and have a higher turnover rate compared to amino acids with carbon skeletons not directly derived from glycolytic or citric acid cycle intermediates (Hobbie and Hobbie 2012). For example, amino acids such as glutamic acid (synthesized in one enzymatic step from the citric acid cycle intermediate 2-oxoglutarate), aspartic acid (synthesized in one enzymatic step from the citric acid cycle intermediate oxaloacetate), and glycine (synthesized in four steps from the glycolytic intermediate 3-phosphoglycerate), have more rapid turnover rates in microbes than those with more complex biosynthethic pathways (Hobbie and Hobbie 2012), such as lysine (eight enzymatic steps from the citric acid cycle intermediate 2- oxoglutarate), leucine (four enzymatic steps after valine synthesis), and isoleucine (five enzymatic steps after threonine synthesis) (Barton et al. 2010). Assuming that C in ‘fast’ amino acid pool has the same origin of recent plant photosynthates as the structural carbon pool, the regression analysis indicates that the 13C of this ‘slow’ pool of amino acids peaked later (30 days post-labeling at 18‰) and then gradually declined to a δ13C value of -12‰ by day 93 (Fig. 4). Results In the stepwise multiple regression of amino acid δ13C, polynomial terms of the days since 13C labeling (days, days-squared, days-cubed, and days to the fourth power) were included to account for the variable signature over time of amino acid carbon not derived from recent plant photosynthesis (Table 2). Minimum value of AICc was with a four- term model consisting of structural carbohydrates and three temporal terms. Neither genus nor N addition were significant factors. In the stepwise regression, 58 ± 3% of amino acid carbon was derived from the same pool as the structural carbon, which was designated as the fast amino acid pool. Accordingly, 42% of amino acid carbon was from a separate pool of slower dynamics. The daily δ13C of this ‘slow amino acid pool’ could be approximated by summing the three temporal regression terms (days, days-cubed, and days to the fourth power) and dividing this quantity by 0.42 (Appendix 1). The equation can be summarized as the following. δ13CAA = 0.58 × δ13CfastAA + 0.42 × δ13CslowAA (1) The fast amino acid pool and the structural carbon pool were assumed to have the same origin of recent plant photosynthates, and therefore: The fast amino acid pool and the structural carbon pool were assumed to have the same origin of recent plant photosynthates, and therefore: δ13CfastAA = δ13Cstructural C + constant (2) Accordingly, substituting (2) into (1) leads to: Accordingly, substituting (2) into (1) leads to: δ13CAA = 0.58 × δ13Cstructural C + 0.42 × δ13CslowAA + constant (3) δ13CAA = 0.58 × δ13Cstructural C + 0.42 × δ13CslowAA + constant (3) Page 6/14 Page 6/14 Page 6/14 The calculated δ13C of the slow amino acid pool peaked 30 days post-labeling at 18‰ and then gradually declined to a δ13C value of -12‰ by day 93 (Fig. 4). The calculated δ13C of the slow amino acid pool peaked 30 days post-labeling at 18‰ and then gradually declined to a δ13C value of -12‰ by day 93 (Fig. 4). Discussion Estimated peak labeling levels of this ‘slow’ amino acid pool are only ~ 18% of the 13C labeling in structural carbon, with an estimated 41‰ enrichment relative to control values, compared to maximum 13C enrichment of structural carbon of the ectomycorrhizal fungi of 222‰ higher than controls. A partitioning of amino acid carbon into distinct pools that differ in their turnover rate can be plausibly explained by examining the biosynthetic pathways of different amino acids. Amino acids that are derived from glycolytic or citric acid cycle intermediates in only a few steps can be frequently resynthesized and have a higher turnover rate compared to amino acids with carbon skeletons not directly derived from glycolytic or citric acid cycle intermediates (Hobbie and Hobbie 2012). For example, amino acids such as glutamic acid (synthesized in one enzymatic step from the citric acid cycle intermediate 2-oxoglutarate), aspartic acid (synthesized in one enzymatic step from the citric acid cycle intermediate oxaloacetate), and glycine (synthesized in four steps from the glycolytic intermediate 3-phosphoglycerate), have more rapid turnover rates in microbes than those with more complex biosynthethic pathways (Hobbie and Hobbie 2012), such as lysine (eight enzymatic steps from the citric acid cycle intermediate 2- To further assess the plausibility of two pools of amino acids which differ in turnover time, information on the typical amino acid composition of ectomycorrhizal mushrooms was used to examine the possible identity of the amino acids in these two different pools. In Table 3, information on the complexity of the biosynthesis of different amino acids is listed (Barton et al. 2010), together with relative contributions of different amino acids to sporocarps of four commercially cultivated fungal species (Mattila et al. 2002). Page 7/14 Page 7/14 The nine amino acids with four or less enzymatic steps to final synthesis (average, 2.0 steps) comprised 47% of sporocarp amino acid carbon, whereas the eleven amino acids with six or more enzymatic steps (average, 9.0 steps) comprised 53% of amino acid carbon in sporocarps. Limited data on catabolic metabolism of these amino acids in microbes agreed with less turnover (and greater retention) of more biosynthetically complex amino acids (Hobbie and Hobbie 2012). Discussion Turnover (assessed as conversion to CO2) averaged 55% for glutamic acid and aspartic acid (one enzymatic step to synthesis), averaged 30% for glycine (four enzymatic steps), and averaged 18% for leucine, lysine, and isoleucine (respectively, seven, ten, and eleven enzymatic steps to final synthesis). The results from this labeling experiment cannot reveak if some of the ‘slow’ amino acids are derived from current-year photosynthates or from uptake from soil organic nitrogen. However, in prior work with radiocarbon, both Cortinarius and Lactarius amino acids were partially derived from soil organic N uptake. Cortinarius amino acids from northern Finland were significantly older than Lactarius amino acids (Hobbie et al. 2013). Given the increase in age and δ13C signatures from surface litter horizons to deeper horizons, this plausibly accounts for the 0.9‰ higher δ13C signatures of Cortinarius amino acids than Lactarius amino acids (Table 1). For example, δ13C signatures of bulk soil increased by about 2‰ from the S to the H horizon at two nearby research sites of Norrliden and Rosinedalsheden (Hobbie & Hasselquist, 2018). A hypothesis of how amino acids and carbohydrates cycle in this ectomycorrhizal system is summarized in Fig. 5. After assimilation of added 13CO2 by Pinus sylvestris into plant sugars, these are transported belowground within a few days and converted into fungal sugars and amino acids. These sugars and amino acids are used to construct the developing sporocarp. Easily synthesized amino acids (such as aspartic acid or glutamic acid) turn over quickly (‘fast’ amino acids) and will have essentially the same A hypothesis of how amino acids and carbohydrates cycle in this ectomycorrhizal system is summarized in Fig. 5. After assimilation of added 13CO2 by Pinus sylvestris into plant sugars, these are transported belowground within a few days and converted into fungal sugars and amino acids. These sugars and amino acids are used to construct the developing sporocarp. Easily synthesized amino acids (such as aspartic acid or glutamic acid) turn over quickly (‘fast’ amino acids) and will have essentially the same 13C labeling level as fungal sugars used to construct the structural carbohydrates that make up most of sporocarp biomass. Amino acids with longer biosynthetic pathways also turn over more slowly (‘slow’ amino acids) and will lag the 13C labeling of fungal carbohydrates and the ‘fast’ amino acid pool. Discussion Because of the slow turnover of some amino acids, they are more likely than the ‘fast’ amino acids to be preserved after mobilization from soil organic nitrogen or after biosynthesis from plant-derived sugars. These ‘slow’ amino acids in sporocarps accounted for the lower δ13C signatures of fungal amino acids than fungal structural carbon during the first few weeks after the pulse-chase addition of 13CO2, and presumably accounted for the higher δ13C of fungal amino acids four weeks and 70–100 days after 13CO2 addition. The 13C tracer data cannot be used by itself in this study to indicate amino acid uptake by ectomycorrhizal fungi. However, the natural abundance δ13C data on amino acids, with Cortinarius higher in δ13C than Lactarius (Table 1), does suggest that soil-derived amino acids differing in δ13C are taken up by these two taxa. If ‘fast’ and ‘slow’ amino acids are taken up from the soil in similar proportions to their abundance in soil protein but turnover rates in fungal biomass differ as suggested, then mass balance Page 8/14 considerations would dictate that a greater proportion of ‘slow’ amino acids than ‘fast’ amino acids must be derived from soil amino acids (Fig. 5). Studies of amino acid uptake by plants in soil have primarily focused on metabolically simple amino acids, such as glycine, aspartic acid, alanine, and glutamic acid (Chapin et al. 1993; Gallet-Budynek et al. 2009; Kielland 1994; Näsholm et al. 1998; Näsholm et al. 2000). One study investigated the uptake and catabolism of 13C-labeled glycine, arginine, and a mixture of extracted peptides from cyanobacteria (Persson et al. 2003). In that study, respiratory losses of 13CO2 were intermediate for the peptide mix, highest in the glycine treatment, and lowest in the arginine treatment. This corresponded to the relative rates of predicted respiration rates based on the division of amino acids into ‘fast’ cycling (e.g., glycine), ‘slow` cycling (e.g., arginine) amino acid pools, and a combination of these two amino acid types in peptides. By comparing the dynamics of the structural carbon pool and the amino acid pool here, the presence of ‘fast’ and ‘slow’ cycling pools of amino acids were inferred; these pools conformed to literature information on the turnover rates and biosynthetic pathways of amino acids (Barton et al., 2010; Hobbie and Hobbie, 2012). Discussion The higher energetic cost of synthesizing these complex amino acids should make both soil uptake and retention of these amino acids a favored strategy for many fungi. The proposed dichotomy in amino acid behavior suggests that additional effort should be devoted to comparing the turnover and uptake patterns of amino acids with their biosynthetic and catabolic pathways. Statements and Declarations Funding: This work was supported by U.S. National Science Foundation grants DEB-0743348 and OPP- 0612598. Competing interests: The author has no relevant financial or non-financial interests to disclose. Competing interests: The author has no relevant financial or non-financial interests to disclose. Author contributions: Not relevant. Data availability: The data analyzed in the current study are available as Online Resource 1. Data availability: The data analyzed in the current study are available as Online Resource 1. Acknowledgments This work was supported by U.S. National Science Foundation grants DEB-0743348 and OPP-0612598. I thank Mona Högberg for supplying sporocarp samples, Andy Ouimette and Zach McEvoy for laboratory assistance, and Rebecca Hood-Nowotny, Thomas Larsen, Katja Rinne-Garmston, and Wolfgang Wanek for comments on the manuscript. References Page 9/14 Page 9/14 1. Abuzinadah R, Read D (1989) The role of proteins in the nitrogen nutrition of ectomycorrhizal plants: IV. The utilization of peptides by birch (Betula pendula L.) infected with different mycorrhizal fungi. New Phytol 112:55–60 2. Barton MD, Delneri D, Oliver SG, Rattray M, Bergman CM (2010) Evolutionary systems biology of amino acid biosynthetic cost in yeast.PloS One5 2. Barton MD, Delneri D, Oliver SG, Rattray M, Bergman CM (2010) Evolutionary systems biology of amino acid biosynthetic cost in yeast.PloS One5 3. Chapin CT, Moilanen L, Kielland K (1993) Preferential use of organic nitrogen for growth by a non- mycorrhizal arctic sedge. Nature 361:150–153 3. Chapin CT, Moilanen L, Kielland K (1993) Preferential use of organic nitrogen for growth by a non- mycorrhizal arctic sedge. Nature 361:150–153 4. Clemmensen KE, Sorensen PL, Michelsen A, Jonasson S, Strom L (2008) Site-dependent N uptake from N-form mixtures by arctic plants, soil microbes and ectomycorrhizal fungi. Oecologia 155:771– 783 4. Clemmensen KE, Sorensen PL, Michelsen A, Jonasson S, Strom L (2008) Site-dependent N uptake from N-form mixtures by arctic plants, soil microbes and ectomycorrhizal fungi. Oecologia 155:771– 783 5. Coplen TB (2011) Guidelines and recommended terms for expression of stable-isotope‐ratio and gas‐ratio measurement results. Rapid Commun Mass Spectrom 25:2538–2560 5. Coplen TB (2011) Guidelines and recommended terms for expression of stable-isotope‐ratio and gas‐ratio measurement results. Rapid Commun Mass Spectrom 25:2538–2560 6. Crawford CC, Hobbie J, Webb K (1974) The utilization of dissolved free amino acids by estuarine microorganisms. Ecology 55:551–563 6. Crawford CC, Hobbie J, Webb K (1974) The utilization of dissolved free amino acids by estuarine microorganisms. Ecology 55:551–563 7. Gallet-Budynek A, Brzostek E, Finzi AC et al (2009) Intact amino acid uptake by northern hardwood and conifer trees. Oecologia 160: 129–138 7. Gallet-Budynek A, Brzostek E, Finzi AC et al (2009) Intact amino acid uptake by northern hardwood and conifer trees. Oecologia 160: 129–138 8. Hobbie EA, Ouimette AP, Schuur EA, Kierstead D, Trappe JM, Bendiksen K, Ohenoja E (2013) Radiocarbon evidence for the mining of organic nitrogen from soil by mycorrhizal fungi. Biogeochemistry 114:381–389 8. Hobbie EA, Ouimette AP, Schuur EA, Kierstead D, Trappe JM, Bendiksen K, Ohenoja E (2013) Radiocarbon evidence for the mining of organic nitrogen from soil by mycorrhizal fungi. Biogeochemistry 114:381–389 9. References Persson J, Hogberg P, Ekblad A, Hogberg MN, Nordgren A, Nasholm T (2003) Nitrogen acquisition from inorganic and organic sources by boreal forest plants in the field. Oecologia 137:252–257 20. Read DJ, Leake JR, Perez-Moreno J (2004) Mycorrhizal fungi as drivers of ecosystem processes in heathland and boreal forest biomes. Can J Botany-Revue Canadienne De Botanique 82:1243–1263 20. Read DJ, Leake JR, Perez-Moreno J (2004) Mycorrhizal fungi as drivers of ecosystem processes in heathland and boreal forest biomes. Can J Botany-Revue Canadienne De Botanique 82:1243–1263 21. Talbot JM, Treseder KK (2010) Controls over mycorrhizal uptake of organic nitrogen. Pedobiologia 53:169–179. doi: 10.1016/j.pedobi.2009.12.001 21. Talbot JM, Treseder KK (2010) Controls over mycorrhizal uptake of organic nitrogen. Pedobiologia 53:169–179. doi: 10.1016/j.pedobi.2009.12.001 References Hobbie JE, Hobbie EA (2012) Amino acid cycling in plankton and soil microbes studied with radioisotopes: measured amino acids in soil do not reflect bioavailability. Biogeochemistry 107:339– 360 9. Hobbie JE, Hobbie EA (2012) Amino acid cycling in plankton and soil microbes studied with radioisotopes: measured amino acids in soil do not reflect bioavailability. Biogeochemistry 107:339– 360 10. Högberg MN, Briones MJ, Keel SG, Metcalfe DB, Campbell C, Midwood AJ, Thornton B, Hurry V, Linder S, Näsholm T (2010) Quantification of effects of season and nitrogen supply on tree below-ground carbon transfer to ectomycorrhizal fungi and other soil organisms in a boreal pine forest. New Phytol 187:485–493 11. Högberg P, Högberg M, Göttlicher S, Betson N, Keel S, Metcalfe D, Campbell C, Schindlbacher A, Hurry V, Lundmark T (2008) High temporal resolution tracing of photosynthate carbon from the tree canopy to forest soil microorganisms. New Phytol 177:220–228 12. Kielland K (1994) Amino acid absorption by arctic plants: implications for plant nutrition and nitrogen cycling. Ecology 75:2373–2383 13. Mattila P, Salo-Väänänen P, Könkö K, Aro H, Jalava T (2002) Basic composition and amino acid contents of mushrooms cultivated in Finland. J Agric Food Chem 50:6419–6422 14. Moyano FE, Kutsch WL, Rebmann C (2008) Soil respiration fluxes in relation to photosynthetic activity in broad-leaf and needle-leaf forest stands. Agric For Meteorol 148:135–143 15. Näsholm T, Ekblad A, Nordin A, Giesler R, Högberg M, Högberg P (1998) Boreal forest plants take up organic nitrogen. Nature 392:914–916 Page 10/14 Page 10/14 16. Näsholm T, Huss-Danell K, Högberg P (2000) Uptake of organic nitrogen in the field by four agriculturally important plant species. Ecology 81:1155–1161 17. Näsholm T, Kielland K, Ganeteg U (2009) Uptake of organic nitrogen by plants. New Phytol 182:31– 48 18. Ouimette AP, Ollinger SV, Lepine LC, Stephens RB, Rowe RJ, Vadeboncoeur MA, Tumber-Davila SJ, Hobbie EA (2019) Accounting for carbon flux to mycorrhizal fungi may resolve discrepancies in forest carbon budgets. Ecosystems: 1–15, doi.org/10.1007/s10021-10019-00440-10023 18. Ouimette AP, Ollinger SV, Lepine LC, Stephens RB, Rowe RJ, Vadeboncoeur MA, Tumber-Davila SJ, Hobbie EA (2019) Accounting for carbon flux to mycorrhizal fungi may resolve discrepancies in forest carbon budgets. Ecosystems: 1–15, doi.org/10.1007/s10021-10019-00440-10023 19. Persson J, Hogberg P, Ekblad A, Hogberg MN, Nordgren A, Nasholm T (2003) Nitrogen acquisition from inorganic and organic sources by boreal forest plants in the field. Oecologia 137:252–257 19. Figures Figure 1 δ13C signatures of bulk ectomycorrhizal sporocarps, structural carbon, and amino acid pools after 13C labeling. The number of days since 13CO2 labeling is given on the x-axis. Pools are given as bulk biomass (▼), structural carbon (●) and amino acid carbon (○). b. In contrast, the δ13C of phloem sap at 0.3 m Tables Tables 1-3 is available in the Supplementary Files section. Figure 1 δ13C signatures of bulk ectomycorrhizal sporocarps, structural carbon, and amino acid pools after 13C labeling. The number of days since 13CO2 labeling is given on the x-axis. Pools are given as bulk biomass (▼), structural carbon (●) and amino acid carbon (○). b. In contrast, the δ13C of phloem sap at 0.3 m δ13C signatures of bulk ectomycorrhizal sporocarps, structural carbon, and amino acid pools after 13C labeling. The number of days since 13CO2 labeling is given on the x-axis. Pools are given as bulk biomass (▼), structural carbon (●) and amino acid carbon (○). b. In contrast, the δ13C of phloem sap at 0.3 m Page 11/14 Page 11/14 Page 11/14 height peaked at 140‰ only 24 hours after 13C labeling and then declined rapidly (data from Högberg et al., 2008) height peaked at 140‰ only 24 hours after 13C labeling and then declined rapidly (data from Högberg et al., 2008) Figure 2 The 13C enrichment in ectomycorrhizal sporocarps of the structural carbon pool relative to the amino acids pool over time. Days since 13CO2 labeling is given on the x-axis. Signatures above or below the line indicate greater 13C labeling in the structural or amino acids pool, respectively Figure 3 δ13C signatures of structural carbon correlate with that of amino acids and with bulk carbon. δ13Camino acids = 0.611 ± 0.030 × δ13Cstructural – 1.64 ± 2.41‰. Adjusted r2 = 0.961, n = 18, p < 0.0001. δ13Cbulk = 0.901 ± 0.030 × δ13Cstructural – 0.21 ± 1.43‰. Adjusted r2 = 0.994, n = 18, p < 0.0001 δ13C signatures of structural carbon correlate with that of amino acids and with bulk carbon. δ13Camino acids = 0.611 ± 0.030 × δ13Cstructural – 1.64 ± 2.41‰. Adjusted r2 = 0.961, n = 18, p < 0.0001. δ13Cbulk = 0.901 ± 0.030 × δ13Cstructural – 0.21 ± 1.43‰. Adjusted r2 = 0.994, n = 18, p < 0.0001 Figure 2 The 13C enrichment in ectomycorrhizal sporocarps of the structural carbon pool relative to the amino acids pool over time. Days since 13CO2 labeling is given on the x-axis. Signatures above or below the line indicate greater 13C labeling in the structural or amino acids pool, respectively Page 12/14 Page 12/14 Figure 5 Schematic of the movement of primary metabolites (sugars) and amino acids with pulse-labeling in a plant-fungal system, starting with the assimilation of 13CO2 into sugars; sAA or fAA indicate ‘slow’ or ‘fast’ turnover amino acids. The thickness of the arrows indicates flux rates, with high fluxes of fungal sugars into fast fungal amino acids (fAAs), low fluxes of fungal sugars into slow amino acids, high catabolic fluxes of fast fAAs to CO2, and high fluxes of soil amino acids to slow fAAs. Greater recycling of intact amino acids in slow fAAs than in fast fAAs is indicated by sizes of double-arrowed circle Schematic of the movement of primary metabolites (sugars) and amino acids with pulse-labeling in a plant-fungal system, starting with the assimilation of 13CO2 into sugars; sAA or fAA indicate ‘slow’ or ‘fast’ turnover amino acids. The thickness of the arrows indicates flux rates, with high fluxes of fungal sugars into fast fungal amino acids (fAAs), low fluxes of fungal sugars into slow amino acids, high catabolic fluxes of fast fAAs to CO2, and high fluxes of soil amino acids to slow fAAs. Greater recycling of intact amino acids in slow fAAs than in fast fAAs is indicated by sizes of double-arrowed circle Figure 4 Modeled δ13C signatures since time of 13CO2 pulse-labeling of the slow amino acid pool (open circles) compared with the structural carbohydrate signal (filled circles) as a proxy for the fast amino acid δ13C Page 13/14 Page 13/14 Figure 5 Table13.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 14/14
https://openalex.org/W4249281180	https://wellcomeopenresearch.org/articles/2-84/v1/pdf	English	null	The integration site of the APP transgene in the J20 mouse model of Alzheimer’s disease	Wellcome open research	2,017	cc-by	9,467	The integration site of the APP transgene in the J20 mouse model of Alzheimer’s disease [version 1; peer review: 1 approved, 2 approved with reservations] Justin L. Tosh 1, Matthew Rickman1, Ellie Rhymes1, Frances E. Norona1, Emma Clayton1, Lennart Mucke 2, Adrian M. Isaacs1,3, Elizabeth M.C. Fisher 1, Frances K. Wiseman1 1Department of Neurodegenerative Disease, Institute of Neurology, University College London, London, WC1N 3BG, UK 2Gladstone Institute of Neurological Disease and University of California, San Francisco, CA, 4158, USA 3UK Dementia Research Institute, University College London, London, WC1E 6BT, UK Open Peer Review Approval Status 1 2 3 version 2 (revision) 10 Oct 2018 view version 1 13 Sep 2017 view view view Open Peer Review Approval Status 1 2 3 version 2 (revision) 10 Oct 2018 view version 1 13 Sep 2017 view view view Karen H. Ashe, University of Minnesota, Minneapolis, USA 1. Takaomi C. Saido, RIKEN Brain Science Institute, Saitama, Japan 2. Guillaume Pavlovic , University of Strasbourg, Illkirch, France PHENOMIN-ICS, Strasbourg, France 3. Any reports and responses or comments on the article can be found at the end of the article. First published: 13 Sep 2017, 2:84 https://doi.org/10.12688/wellcomeopenres.12237.1 Latest published: 10 Oct 2018, 2:84 https://doi.org/10.12688/wellcomeopenres.12237.2 v1 First published: 13 Sep 2017, 2:84 https://doi.org/10.12688/wellcomeopenres.12237.1 Latest published: 10 Oct 2018, 2:84 https://doi.org/10.12688/wellcomeopenres.12237.2 v1 Abstract Background: Transgenic animal models are a widely used and powerful tool to investigate human disease and develop therapeutic interventions. Making a transgenic mouse involves random integration of exogenous DNA into the host genome that can have the effect of disrupting endogenous gene expression. The J20 mouse model of Alzheimer’s disease (AD) is a transgenic overexpresser of human APP with familial AD mutations and has been extensively utilised in preclinical studies and our aim was to determine the genomic location of the J20 transgene insertion. Results: We demonstrate that the J20 transgene construct has inserted within the genetic locus of endogenous mouse gene Zbtb20 on chromosome 16 in an array, disrupting expression of mRNA from this gene in adult hippocampal tissue, while expression of Zbtb20 protein remains unchanged. We note that the endogenous mouse App gene also lies on chromosome 16, although 42 Mb from the Zbtb20 locus. Conclusions: These data will be useful for future studies utilising this popular model of AD, particularly those investigating gene interactions between the J20 APP transgene and other genes present on Mmu16 in the mouse. RESEARCH ARTICLE The integration site of the APP transgene in the J20 mouse model of Alzheimer’s disease [version 1; peer review: 1 approved, 2 approved with reservations] Justin L. Tosh 1, Matthew Rickman1, Ellie Rhymes1, Frances E. Norona1, Emma Clayton1, Lennart Mucke 2, Adrian M. Isaacs1,3, Elizabeth M.C. Fisher 1, Frances K. Wiseman1 1Department of Neurodegenerative Disease, Institute of Neurology, University College London, London, WC1N 3BG, UK 2Gladstone Institute of Neurological Disease and University of California, San Francisco, CA, 4158, USA 3UK Dementia Research Institute, University College London, London, WC1E 6BT, UK First published: 13 Sep 2017, 2:84 https://doi.org/10.12688/wellcomeopenres.12237.1 Latest published: 10 Oct 2018, 2:84 https://doi.org/10.12688/wellcomeopenres.12237.2 v1 Abstract Background: Transgenic animal models are a widely used and powerful tool to investigate human disease and develop therapeutic interventions. Making a transgenic mouse involves random integration of exogenous DNA into the host genome that can have the effect of disrupting endogenous gene expression. The J20 mouse model of Alzheimer’s disease (AD) is a transgenic overexpresser of human APP with familial AD mutations and has been extensively utilised in preclinical studies and our aim was to determine the genomic location of the J20 transgene insertion. Methods: We used a combination of breeding strategy and Targeted Locus Amplification with deep sequencing to identify the insertion site of the J20 transgene array. To assess RNA and protein expression of Zbtb20, we used qRT-PCR and Western Blotting. Results: We demonstrate that the J20 transgene construct has inserted within the genetic locus of endogenous mouse gene Zbtb20 on chromosome 16 in an array, disrupting expression of mRNA from this gene in adult hippocampal tissue, while expression of Zbtb20 protein remains unchanged. We note that the endogenous mouse App gene also lies on chromosome 16, although 42 Mb from the Zbtb20 locus. Conclusions: These data will be useful for future studies utilising this Open Peer Review Approval Status 1 2 3 version 2 (revision) 10 Oct 2018 view version 1 13 Sep 2017 view view view Karen H. Ashe, University of Minnesota, Minneapolis, USA 1. Takaomi C. Saido, RIKEN Brain Science Institute, Saitama, Japan 2. Guillaume Pavlovic , University of Strasbourg, Illkirch, France PHENOMIN-ICS, Strasbourg, France 3. Any reports and responses or comments on the article can be found at the end of the article. Wellcome Open Research 2017, 2:84 Last updated: 23 MAR 2022 RESEARCH ARTICLE The integration site of the APP transgene in the J20 mouse model of Alzheimer’s disease [version 1; peer review: 1 approved, 2 approved with reservations] Justin L. Tosh 1, Matthew Rickman1, Ellie Rhymes1, Frances E. Norona1, Emma Clayton1, Lennart Mucke 2, Adrian M. Isaacs1,3, Elizabeth M.C. Fisher 1, Frances K. Wiseman1 1Department of Neurodegenerative Disease, Institute of Neurology, University College London, London, WC1N 3BG, UK 2Gladstone Institute of Neurological Disease and University of California, San Francisco, CA, 4158, USA 3UK Dementia Research Institute, University College London, London, WC1E 6BT, UK First published: 13 Sep 2017, 2:84 https://doi.org/10.12688/wellcomeopenres.12237.1 Latest published: 10 Oct 2018, 2:84 https://doi.org/10.12688/wellcomeopenres.12237.2 v1 Abstract Background: Transgenic animal models are a widely used and powerful tool to investigate human disease and develop therapeutic interventions. Making a transgenic mouse involves random integration of exogenous DNA into the host genome that can have the effect of disrupting endogenous gene expression. The J20 mouse model of Alzheimer’s disease (AD) is a transgenic overexpresser of human APP with familial AD mutations and has been extensively utilised in preclinical studies and our aim was to determine the genomic location of the J20 transgene insertion. Methods: We used a combination of breeding strategy and Targeted Locus Amplification with deep sequencing to identify the insertion site of the J20 transgene array. To assess RNA and protein expression of Zbtb20, we used qRT-PCR and Western Blotting. Results: We demonstrate that the J20 transgene construct has inserted within the genetic locus of endogenous mouse gene Zbtb20 on chromosome 16 in an array, disrupting expression of mRNA from this gene in adult hippocampal tissue, while expression of Zbtb20 protein remains unchanged. We note that the endogenous mouse App gene also lies on chromosome 16, although 42 Mb from the Zbtb20 locus. Conclusions: These data will be useful for future studies utilising this Open Peer Review Approval Status 1 2 3 version 2 (revision) 10 Oct 2018 view version 1 13 Sep 2017 view view view Karen H. Ashe, University of Minnesota, Minneapolis, USA 1. Takaomi C. Saido, RIKEN Brain Science Institute, Saitama, Japan 2. Guillaume Pavlovic , University of Strasbourg, Illkirch, France PHENOMIN-ICS, Strasbourg, France 3. Any reports and responses or comments on the article can be found at the end of the article. Wellcome Open Research 2017, 2:84 Last updated: 23 MAR 2022 Wellcome Open Research 2017, 2:84 Last updated: 23 MAR 2022 RESEARCH ARTICLE RESEARCH ARTICLE Transgenic, mouse model, Alzheimer’s disease, APP, Zbtb20, J20, Amyloid Precursor Protein Keywords Transgenic, mouse model, Alzheimer’s disease, APP, Zbtb20, J20, Amyloid Precursor Protein Page 1 of 13 Wellcome Open Research 2017, 2:84 Last updated: 23 MAR 2022 Corresponding authors: Elizabeth M.C. Fisher (elizabeth.fisher@ucl.ac.uk), Frances K. Wiseman (f.wiseman@prion.ucl.ac.uk) Author roles: Tosh JL: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing; Rickman M: Investigation; Rhymes E: Investigation, Writing – Review & Editing; Norona FE: Investigation, Writing – Review & Editing; Clayton E: Conceptualization, Investigation, Writing – Review & Editing; Mucke L: Validation, Writing – Review & Editing; Isaacs AM: Conceptualization, Writing – Review & Editing; Fisher EMC: Conceptualization, Funding Acquisition, Methodology, Project Administration, Resources, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing; Wiseman FK: Conceptualization, Funding Acquisition, Methodology, Project Administration, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Competing interests: The authors declare no competing interests. Grant information: This work was supported by the Wellcome Trust [098330], Strategic Award to The London Down Syndrome (LonDownS) Consortium; the Alzheimer’s Society [192(ALZSOC-PhD-2013-001)], awarded to FKW and EMCF; the Medical Research Council [MR/J004022/1], to AMI; and Alzheimer’s Research UK [ARUK-PPG2014B-5] to AMI. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2017 Tosh JL 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 work is properly cited. How to cite this article: Tosh JL, Rickman M, Rhymes E et al. The integration site of the APP transgene in the J20 mouse model of Alzheimer’s disease [version 1; peer review: 1 approved, 2 approved with reservations] Wellcome Open Research 2017, 2:84 https://doi.org/10.12688/wellcomeopenres.12237.1 First published: 13 Sep 2017, 2:84 https://doi.org/10.12688/wellcomeopenres.12237.1 Corresponding authors: Elizabeth M.C. Fisher (elizabeth.fisher@ucl.ac.uk), Frances K. Animal welfare Mice were housed in controlled conditions in accordance with guidelines from the UK Medical Research Council in Respon- sibility in Use of Animals for Medical Research (1993). Two female J20 positive animals were killed at 6 months of age to pro- vide splenic material for the TLA study. Furthermore, 3 month hippocampal tissue was collected from J20 animals: N=5, 3 male, 2 female. C57BL/6J controls: N=5, 3 male, 2 female. All used for qRT-PCR and western blotting. All experiments were conducted under license from the UK Home Office and with Local Ethical Review approval. Tg(PDGFB-APPSwInd)20Lms/2Mmjax ani- mals (J20) were obtained from The Jackson laboratory (stock no. 034836) and maintained on a C57BL/6J background in our animal facility. Chmp2b knockout animals were already available in our animal facility (Ghazi-Noori et al., 2012). Mice had access to a mouse house with bedding material and wood chips. All animals had continual access to water and RM1 (Special Diet Services) (stock animals) or RM3 (Special Diet Services) (breeding animals) chow ad libitum. Mice were housed in individually ventilated cages in a specific pathogen free facility with a 12 hour light/dark cycle. The model was developed by Mucke and colleagues using the PDGF- APPSw,Ind transgene construct described previously (Games et al., 1995; Rockenstein et al., 1995), which includes a human APP mini- gene, carrying the familial AD-linked 717V-F (Indiana) mutation (Murrell et al., 1991) and 670/671KM-NL (Swedish) double mutation (Mullan et al., 1992). The transgene construct was designed so that the APP mini-gene included genomic sequence for APP introns 6–8, allowing expression of hAPP695, hAPP751 and hAPP770 isoforms. The PDGF-APPSw,Ind transgene expression is driven in neurons throughout the brain by the human platelet-derived growth factor β chain (PDGFβ) promoter (Harris et al., 2010; Sasahara et al., 1991). The J20 mouse is an important model: currently, 125 articles have been catalogued in the Mouse Genome Database bibliography (Blake et al., 2017), which report genotypic and/or phenotypic data from this mouse. This strain has been used for several classical genetic studies to determine the interaction of genes of interest with the APP transgene including a seminal report of the importance of Tau to Aß-associated neuronal dysfunction (Roberson et al., 2007). Moreover, this model has been used to elucidate the role of Aß in synaptic dysfunction (Palop & Mucke, 2016; Palop et al., 2007; Sanchez et al., 2012). Keywords Page 2 of 13 Wellcome Open Research 2017, 2:84 Last updated: 23 MAR 2022 Wellcome Open Research 2017, 2:84 Last updated: 23 MAR 2022 Wellcome Open Research 2017, 2:84 Last updated: 23 MAR 2022 Introduction site by Targeted Locus Amplification (TLA) with deep sequencing. We discuss how integration of the transgene affects expression of the flanking loci. The Tg(PDGFB-APPSwInd)20Lms (MGI:3057148, here referred to as ‘J20’) mouse model is a transgenic animal that overexpresses mutant human APP protein (amyloid precursor protein), and is widely used as a model of amyloid deposition and pathogenesis in the study of Alzheimer’s disease (AD). J20 mice recapitulate many AD-like phenotypes, including synaptic loss, amyloid plaque depo- sition and cognitive impairment (Hong et al., 2016; Mucke et al., 2000; Palop & Mucke, 2016). Animal welfare Genotyping of J20 and Chmp2b knockout mice DNA was extracted from tail tip or ear biopsy by the HOTSHOT method (Truett et al., 2000). The presence of the J20 human APP transgene was tested by PCR using primers (Eurofins) APP-F: 5’-GTGAGTTTGTAAGTGAT- GCC-3’ APP-R: 5’-TCTTCTTCTTCCACCTCAGC-3’, control primers ContF: 5’-CAAATGTTGCTTGTCTGGTG-3’ ContR: 5’-GTCAGTCGAGTGCACAGTTT-3). Copy number of the human APP transgene was validated by quantitative PCR using a Taqman Fast machine (Applied Biosystems) with the following primers and probes: hAPPF: 5’-TGGGTTCAAACAAAGGTGCAA-3’ hAPPR: 5’-GATGAAGATCACTGTCGCTATGAC-3’ hAPP- probe: FAM-CATTGGACTCATGGTGGGCGGTG-3’ qContF: 5’-CACGTGGGCTCCAGCATT-3’ qContR: 5’-TCACCAGT- CATTTCTGCCTTTG-3’ qContProbe: VIC-CCAATGGTCG- GGCACTGCTCAA-3’. Transgenic mice are conventionally generated by direct injection of linear foreign DNA into the pronucleus of fertilised zygotes. Once inside the cell, these linear fragments undergo circularisation and concatemer formation before integrating into the host genome as a tandem array (Bishop & Smith, 1989). In principle, transgenes insert randomly into the host genome; however, ∼45% of integration sites lie within host gene regions (∼13.2 exonic, 31.6% intronic), potentially as a result of increased accessibility of transcriptionally active DNA (Yan et al., 2013). Integration of a transgene array into coding sequences can induce new mutations (for example, haploin- sufficiency) (Haruyama et al., 2009), and so it is important to know the site of integration for a transgene array in a mouse model. The Chmp2b knockout locus was detected by PCR with the follow- ing primers: Int2_F: 5’-CCATTGCCACTTGGATGTAA-3’ Int2_R: 5’-GACGCACTTTAAGGTCACAGC-3’ KO_R: 5’- TCTCTGT- GCAAGAAGCATGAA-3’. PCR products were separated by agar- ose gel electrophoresis and visualised using a BioRad Gel Doc XR UV transilluminator. A recent study suggested an association of a heterozygous dele- tion of the CHMP2B gene (charged multivesicular body protein 2B) with early-onset Alzheimer’s disease (Hooli et al., 2014). Interestingly, mutations in CHMP2B are a rare genetic cause of Frontotemporal dementia (Skibinski et al., 2005). We have previously reported generation of a Chmp2b knockout mouse (Ghazi-Noori et al., 2012). To determine if Chmp2b deletion modu- lates APP/Aß biology in vivo, we attempted to cross our Chmp2b knockout with the J20 mouse, to study potential double mutant progeny. The Chmp2b locus lies on mouse chromosome 16. Keywords Wiseman (f.wiseman@prion.ucl.ac.uk) Author roles: Tosh JL: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing; Rickman M: Investigation; Rhymes E: Investigation, Writing – Review & Editing; Norona FE: Investigation, Writing – Review & Editing; Clayton E: Conceptualization, Investigation, Writing – Review & Editing; Mucke L: Validation, Writing – Review & Editing; Isaacs AM: Conceptualization, Writing – Review & Editing; Fisher EMC: Conceptualization, Funding Acquisition, Methodology, Project Administration, Resources, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing; Wiseman FK: Conceptualization, Funding Acquisition, Methodology, Project Administration, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Competing interests: The authors declare no competing interests. Grant information: This work was supported by the Wellcome Trust [098330], Strategic Award to The London Down Syndrome (LonDownS) Consortium; the Alzheimer’s Society [192(ALZSOC-PhD-2013-001)], awarded to FKW and EMCF; the Medical Research Council [MR/J004022/1], to AMI; and Alzheimer’s Research UK [ARUK-PPG2014B-5] to AMI. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2017 Tosh JL 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 work is properly cited. How to cite this article: Tosh JL, Rickman M, Rhymes E et al. The integration site of the APP transgene in the J20 mouse model of Alzheimer’s disease [version 1; peer review: 1 approved, 2 approved with reservations] Wellcome Open Research 2017, 2:84 https://doi.org/10.12688/wellcomeopenres.12237.1 First published: 13 Sep 2017, 2:84 https://doi.org/10.12688/wellcomeopenres.12237.1 nding authors: Elizabeth M.C. Fisher (elizabeth.fisher@ucl.ac.uk), Frances K. Wiseman (f.wiseman@prion.ucl.ac.uk Author roles: Tosh JL: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing; Rickman M: Investigation; Rhymes E: Investigation, Writing – Review & Editing; Norona FE: Investigation, Writing – Review & Editing; Clayton E: Conceptualization, Investigation, Writing – Review & Editing; Mucke L: Validation, Writing – Review & Editing; Isaacs AM: Conceptualization, Writing – Review & Editing; Fisher EMC: Conceptualization, Funding Acquisition, Methodology, Project Administration, Resources, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing; Wiseman FK: Conceptualization, Funding Acquisition, Methodology, Project Administration, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Competing interests: The authors declare no competing interests. Extraction of spleen cells from J20 animalsi Extraction of spleen cells from J20 animalsi Mice were sacrificed by rising concentration of CO2 and confirmed by dislocation of the neck and the spleen dissected and kept on ice. Splenocytes were then dissociated through a 40µm mesh filter and the cells collected by centrifugation at 4°C at 500 x g for 5 minutes. The supernatant was discarded and the pellet re-suspended in 1 ml red blood cell lysis buffer (4.13g NH4Cl, 0.5g KHCO3, 193.5µl Here, we present the outcome of these genetic cross experiments and the resulting mapping of the J20 transgene array integration Page 3 of 13 Wellcome Open Research 2017, 2:84 Last updated: 23 MAR 2022 Wellcome Open Research 2017, 2:84 Last updated: 23 MAR 2022 Wellcome Open Research 2017, 2:84 Last updated: 23 MAR 2022 determined using Bradford assay (Bio-Rad). Samples from indi- vidual animals were run separately and not pooled. determined using Bradford assay (Bio-Rad). Samples from indi- vidual animals were run separately and not pooled. determined using Bradford assay (Bio-Rad). Samples from indi- vidual animals were run separately and not pooled. 0.5M EDTA dissolved in 500ml H2O) for three minutes at room temperature to lyse splenic erythrocytes. To terminate the lysis reaction, 0.5ml phosphate buffered saline (PBS) was added and the splenocytes were collected again by centrifugation at 500 × g for 5 minutes. The supernatant was discarded and the pellet re-suspended in 0.5ml PBS before a final centrifugation step for 2 minutes. The supernatant was discarded and the pellet was re-suspended in 1ml freezing buffer (PBS with 10% fetal calf serum and 10% dimethyl sulphoxide). Samples were stored at -80°C before preparation for TLA processing. 0.5M EDTA dissolved in 500ml H2O) for three minutes at room temperature to lyse splenic erythrocytes. To terminate the lysis reaction, 0.5ml phosphate buffered saline (PBS) was added and the splenocytes were collected again by centrifugation at 500 × g for 5 minutes. The supernatant was discarded and the pellet re-suspended in 0.5ml PBS before a final centrifugation step for 2 minutes. The supernatant was discarded and the pellet was re-suspended in 1ml freezing buffer (PBS with 10% fetal calf serum and 10% dimethyl sulphoxide). Samples were stored at -80°C before preparation for TLA processing. Equal amounts of hippocampal brain proteins were denatured in LDS sample buffer (ThermoFisher) and β-Mercaptoethanol for 10 minutes at 100°C, prior to separation by SDS polyacrylamide gel electrophoresis in 4–12% pre-cast gels (ThermoFisher). Targeted Locus Amplification Processing of samples for TLA was performed by Cergentis B.V. (Utrecht, The Netherlands), as previously described (de Vree et al., 2014). A primer pair targeted to the APP transgene sequence was used to perform the TLA. Sequences of the PCR primers are (5’ to 3’): 1917_APP_F GAAACTCATCTTCACTGGCA; 1698_APP_R GGGTAGACTTCTTGGCAATA. PCR products were purified and library prepped using the Illumina NexteraXT protocol and sequenced on an Illumina Miniseq sequencer. Sequence alignment and analysis of TLA TLA reads were mapped using BWA-SW, which is a Smith- Waterman alignment tool. This allows for partial mapping, which is optimally suited for identifying break-spanning reads. The mouse Mm10 genome assembly version was used for mapping. Visu- alisation and interpretation of the data were performed using the Integrative Genomics Viewer (IGV) from the Broad Institute (Robinson et al., 2011). Extraction of spleen cells from J20 animalsi Sepa- rated proteins were transferred to 0.2µm nitrocellulose membrane and blocked in 5% milk/phosphate buffered saline (with 0.05% Tween 20, PBST) for one hour at room temperature. The membrane was then cut horizontally at the 49KDa band (SeeBlue Plus II pro- tein ladder, Invitrogen) and the lower half was incubated in mouse monoclonal antibody to β-Actin (A5441, Sigma-Aldrich) diluted 1:200,000 in 1% bovine serum albumin (BSA)/PBST overnight at 4°C. The upper half was incubated overnight with rabbit polyclo- nal primary antibody against ZBTB20 (23987-1-AP, ProteinTech) diluted 1:1000 in 1% BSA/PBST. After washing with PBST the upper and lower membranes were incubated with HRP-conjugated secondary rabbit and mouse antibodies, respectively, diluted in 1% BSA/PBST for 1 hour at room temperature. SuperSignal™ West Pico Chemiluminescent Substrate and X-ray film was used to visualise bands, Image J 1.49c software (NIH) was used to analyse band intensity. Graphpad prism 5 (Graphpad Software, Inc.) was used to perform statistical analyses and plot graphs. Mouse breeding strategy To investigate the role of Chmp2b in the pathogenesis of AD, we attempted to generate a homozygous knockout of Chmp2b in the Tg(PDGFB-APPSwInd)20Lms (J20) APP transgenic mouse strain. To accomplish this we set up matings over two genera- tions (Figure 1a), firstly between Chmp2b-/- and hemizygous J20 mice (referred for simplicity as TgAPPJ20/-, where ‘J20’ denotes the presence of the transgene). This cross produced Chmp2b+/-;TgAPPJ20/- and Chmp2b+/-;TgAPP-/-progeny. In the second stage, the Chmp2b+/-;TgAPPJ20/- offspring were crossed to Chmp2b-/- null animals. This cross was predicted to produce four progeny genotypes at equal Mendelian ratios (0.25): RNA extraction and quantitative reverse transcription PCR RNA was extracted from whole hippocampus from J20 animals and age and litter matched controls. Total RNA was extracted using the Qiagen miRNeasy kit and reverse transcribed using the Applied Biosystems High-Capacity RNA-to-cDNA™ Kit. Quantitative RT-PCR was carried out on the Zbtb20 gene tran- script using a predesigned PrimeTime® probe-based qPCR assay (assay ID: Mm.PT.58.41805451, Integrated DNA Technologies [IDT]) targeted to exons 8–9 (RefSeq transcript NM_181058) with a FAM probe. TaqMan reactions were run with Taqman Universal Master Mix 2 on a 7500 Fast machine (Applied Biosystems) using standard cycling conditions. Transcript levels were normalised against Applied Biosystems mouse Actb (Assay ID: 4352933E) and Integrated DNA Technologies B2m (assay ID: Mm.PT.39a.22214835) endogenous controls in independent experiments and the results averaged geometrically. Both controls contained VIC probes. (i) homozygous for Chmp2b knockout, without the APP transgene (Chmp2b-/-;TgAPP-/-), (ii) homozygous for Chmp2b knockout, hemizygous for APP transgene (Chmp2b-/-;TgAPPJ20/-), (iii) hemizygous for Chmp2b knockout, hemizygous for APP transgene (Chmp2b+/-;TgAPPJ20/-), (iv) hemizygous for Chmp2b knockout, without the APP transgene (Chmp2b+/-;TgAPP-/-). (iv) hemizygous for Chmp2b knockout, without the APP transgene (Chmp2b+/-;TgAPP-/-). (iv) hemizygous for Chmp2b knockout, without the APP transgene (Chmp2b+/-;TgAPP-/-). The second stage cross resulted in 76 progeny, but the observed ratio of genotypes significantly differed from the expected ratio (Figure 1b). Strikingly no Chmp2b-/-;TgAPPJ20/- progeny were produced. This suggests that either Chmp2b-/-;TgAPPJ20 may not be viable or the TgAPP allele might be on the same chromosome as Chmp2b - mouse chromosome 16 (Mmu16), preventing typical Mendelian segregation. Assessment of ZBTB20 expression Assessment of ZBTB20 expression The integration site co-ordinates localize the J20 transgene inser- tion and deletion entirely within intron 1 of the gene Zinc-finger and BTB domain containing 20 (Zbtb20, transcript NM_001285805.1) on Mmu16. Zbtb20 is a member of the BTB/POZ family of tran- scriptional repressors and functions primarily as a transcriptional repressor (Xie et al., 2008); it is important for hippocampal devel- opment and function, the site of greatest Aβ deposition in aging J20 animals (Mucke et al., 2000). Moreover, missense mutations in this gene are associated with Primrose syndrome, a cause of intellectual disability with autism (Cordeddu et al., 2014; Mattioli et al., 2016). Additionally, haploinsufficiency of the gene has been suggested as an important factor in del3q13.31 syndrome, a cause of develop- mental delay and intellectual disability (Rasmussen et al., 2014). To determine whether transgene insertion has affected Zbtb20 tran- scription in J20 animals in hippocampal tissue, we investigated mRNA and protein levels of ZBTB20 in wildtype and J20 animals. To determine the cause of the absence of Chmp2b-/-;TgAPPJ20 off- spring, we mapped the site of the J20 TgAPP transgene insertion, and so sequenced flanking regions around the insertion site by TLA. Sequence analysis (Figure 2a) showed that the TgAPP insertion site lies on Mmu16. Targeted reads mapped the integration break- points to genomic co-ordinates Mmu16: 43,127,050 (3’ end of the transgene array) and Mmu16: 43,127,512 (5’ end of the transgene array). In addition, sequencing around the insertion site revealed a 41.17kb deletion in the mouse genome between chr16:43,085,979 and chr16:43,127,149 (Figure 2b). Thus the lack of Chmp2b-/- ;TgAPPJ20 offspring is the result of the insertion of TgAPPJ20 on Mmu16, explaining the absence of Chmp2b-/-;TgAPPJ20 progeny from the stage 2 cross. TLA also allowed us to assess transgene sequence integrity. We found three SNPs (hAPP transgene sequence: 624 G > A, 979 G > A, 10649 G > A) and four indels (TG: 185 G > +1T, TG: 1168 G > +8GGCGGGAC, TG: 1423 C > +1G, TG: 5932 C > -1T) within the integrated transgene construct; however, all were silent muta- tions or found within intronic sequence. Furthermore, TLA analysis showed at least one transgene copy is truncated at the 3’ end (TG: 12088), ablating the ampicillin cassette, and is fused to the 5’ of another transgene copy to form a concatemer (TG:12088 fused to TG:3). ue preparation and western blotting for ZBTB20 For analysis of ZBTB20 in hippocampus, J20 and age/sex-matched wildtype littermate controls were dissected under ice-cold PBS before homogenisation in radioimmunoprecipitation buffer (150mM sodium chloride, 50mM Tris, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate) with Protease Inhibitor Cocktail Set 1 (Merck). Total protein concentration was Page 4 of 13 Page 4 of 13 Wellcome Open Research 2017, 2:84 Last updated: 23 MAR 2022 Figure 1. Breeding scheme and observed ratios of offspring in a two-generation cross between Chmp2b-/- and J20+/- animals. (A) Two- generation breeding scheme to generate Chmp2b-/-; J20+/- animals. (B) Observed ratios of offspring genotype differed significantly from the expected ratio χ2(3, N = 90.21) = 0.58, p < 0.0001. Breeding scheme and observed ratios of offspring in a two-generation cross between Chmp2b-/- and J20+/- animals. (A) Two- Figure 1. Breeding scheme and observed ratios of offspring in a two-generation cross between Chmp2b-/- and J20+/- animals. (A) Two- generation breeding scheme to generate Chmp2b-/-; J20+/- animals. (B) Observed ratios of offspring genotype differed significantly from the expected ratio χ2(3, N = 90.21) = 0.58, p < 0.0001. Targeted locus Amplification analysis of J20 transgene insertion Assessment of ZBTB20 expression Assessment of ZBTB20 expression Firstly, a predesigned RT-qPCR assay was chosen from IDT to overlap the exon 8–9 boundary within the protein coding region of the Zbtb20 transcript, downstream of the transgene insertion site. Importantly this junction is present in all predicted RefSeq protein coding transcript isoforms. We detected significantly less transcript in J20 animals compared to wildtype using this assay (Figure 3a). To determine whether reduction in Zbtb20 transcript results in a reduction of ZBTB20 protein in J20 animals, we assayed total Page 5 of 13 Wellcome Open Research 2017, 2:84 Last updated: 23 MAR 2022 Figure 2. Insertion site of the J20 APP transgene (Tg) on mouse chromosome 16. (A) Representation of Mmu16 showing insertion site of the J20 Tg array. Also shown are the positions of endogenous mouse App and Chmp2b. (B) TLA applied to the APPSwInd transgene in the J20 mouse. Read mapping to the mouse (mm10) genome assembly shows the exact Tg insertion site (red arrows) with associated genomic deletion (yellow bar). Gene annotation is shown below labelling a small portion of intron 1 of the Zbtb20 gene. (C) TLA reads mapped to the APP Tg sequence. Complete coverage was achieved, showing that head to tail concatemerization of the Tg has occurred at position 12088 ablating the ampicillin cassette of the plasmid construct. Coloured lines represent SNPs found in the Tg sequence compared to the published sequence. A linearised map of the plasmid construct is shown below. Figure 2. Insertion site of the J20 APP transgene (Tg) on mouse chromosome 16. (A) Representation of Mmu16 showing insertion site of the J20 Tg array. Also shown are the positions of endogenous mouse App and Chmp2b. (B) TLA applied to the APPSwInd transgene in the J20 mouse. Read mapping to the mouse (mm10) genome assembly shows the exact Tg insertion site (red arrows) with associated genomic deletion (yellow bar). Gene annotation is shown below labelling a small portion of intron 1 of the Zbtb20 gene. (C) TLA reads mapped to the APP Tg sequence. Complete coverage was achieved, showing that head to tail concatemerization of the Tg has occurred at position 12088 ablating the ampicillin cassette of the plasmid construct. Coloured lines represent SNPs found in the Tg sequence compared to the published sequence. A linearised map of the plasmid construct is shown below. site of the J20 APP transgene (Tg) on mouse chromosome 16. Assessment of ZBTB20 expression (A) Representation of Mmu16 showing insertio Figure 3. Expression of Zbtb20 mRNA and protein in 3 month old wildtype and J20+/- hippocampus. (A) Zbtb20 mRNA expression is significantly reduced in J20 hippocampus compared to wildtype (WT), detected by quantitative RT-PCR using primers spanning exons 8–9 (unpaired t-test, n = 5 wildtype/6 J20. P = 0.0046). (B) ZBTB20 protein levels are unchanged in J20 hippocampus compared to wildtype animals (unpaired t-test, n= 5 wildtype/4 J20 in hippocampus. p = 0.91). P 6 f 13 Figure 3. Expression of Zbtb20 mRNA and protein in 3 month old wildtype and J20+/- hippocampus. (A) Zbtb20 mRNA expression is significantly reduced in J20 hippocampus compared to wildtype (WT), detected by quantitative RT-PCR using primers spanning exons 8–9 (unpaired t-test, n = 5 wildtype/6 J20. P = 0.0046). (B) ZBTB20 protein levels are unchanged in J20 hippocampus compared to wildtype animals (unpaired t-test, n= 5 wildtype/4 J20 in hippocampus. p = 0.91). Page 6 of 13 Page 6 of 13 Wellcome Open Research 2017, 2:84 Last updated: 23 MAR 2022 hippocampal protein by western blot for ZBTB20 with an affinity purified polyclonal antibody, and observed that ZBTB20 protein is unaltered in adult J20 hippocampal tissue. We also validated this negative result with an additional antibody to ZBTB20 (data not shown). Thus, any perturbations in transcript abundance of Zbtb20 in J20 hippocampus at three months of age do not translate to defi- cits in ZBTB20 protein. found within the transgene construct’s three intronic regions and are unlikely to affect the J20 transgene. TLA analysis and in-house copy number qPCR (data not shown) indicate that this transgene has integrated multiple times in an array on Mmu16. Multiple transgene insertion may be liable to recombination events, caus- ing loss of some copies of the transgene, resulting in delayed onset of phenotype in affected animals. This potential confound can be allayed by undertaking a genomic copy-number qPCR to confirm copy-number consistency between individuals. The Jackson Laboratory have published their assay for general use. Data availabilityi Aligned BAM files from the TLA, uncropped western blot for Figure 3B, hippocampal qPCR data and western blot data for Zbtb20 are available on OSF http://doi.org/10.17605/OSF. IO/4UGZF (Tosh, 2017). de Vree PJ, de Wit E, Yilmaz M, et al.: Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping. Nat Biotechnol. 2014; 32(10): 1–9. PubMed Abstract \| Publisher Full Text Games D, Adams D, Alessandrini R, et al.: Alzheimer-type neuropathology in transgenic mice overexpressing V717F beta-amyloid precursor protein. Nature. 1995; 373(6514): 523–527. PubMed Abstract \| Publisher Full Text Ghazi-Noori S, Froud KE, Mizielinska S, et al.: Progressive neuronal inclusion formation and axonal degeneration in CHMP2B mutant transgenic mice. Brain. Discussion Using TLA we have located the insertion site of the J20 APP transgene on Mmu16 within intron 1 of the Zbtb20 gene. We have also found a 41kb deletion of intronic sequence flanking the insertion site. Due to the key role of Zbtb20 in hippocampal cell differentiation and function, we determined its expression in hip- pocampal tissue, and found that while Zbtb20 transcript expression is reduced in J20 hippocampus, protein expression is not. In a similar study investigating the transgene insertion site of the R6/2 Huntingdon’s disease model mouse, Jacobsen et al. discovered that the HTT exon 1 transgene inserted within intron 7 of the Gm12695 gene, causing an almost 30 fold increase of expression of this gene compared to wildtype in cortical tissue irrespective of CAG-repeat length polymorphisms, showing that the genomic aber- ration caused by foreign DNA insertion within intronic sequence can alter gene regulation (Jacobsen et al., 2017). Notably, disrup- tion of the expression of nearby genes may also occur in gene- targeted systems. For example, two of the four lines of mice that are null for the Prion protein gene Prnp, exhibited late onset ataxia and neurodegeneration -- this was subsequently found to be the result of aberrant upregulation of a gene (Prnd) downstream of Prnp, which occurred as a result of induced exon skipping (Moore et al., 1999). In the J20 model, hippocampal Zbtb20 transcription is perturbed without concomitant protein reduction. This is perhaps not as surprising as it seems; recent data indicate mRNA levels explain only around 40% of variability in protein levels (Wilhelm et al., 2014), with protein abundance being primarily dependent on translational control (Schwanhäusser et al., 2011; Schwanhäusser et al., 2013). Genetic engineering can result in unintended disruption of the genome, which can confound interpretation of phenotype if the genomic alterations cause changes in protein expression. Trans- genic models have been and continue to be powerful tools for biomedical research, but knowledge of the insertion site and the local effects on gene expression will inform phenotyping studies. Bishop JO, Smith P: Mechanism of chromosomal integration of microinjected DNA. Mol Biol Med. 1989; 6(4): 283–298. PubMed Abstract Blake JA, Eppig JT, Kadin JA, et al.: Mouse Genome Database (MGD)-2017: community knowledge resource for the laboratory mouse. Nucleic Acids Res. 2017; 45(D1): D723–D729. PubMed Abstract \| Publisher Full Text \| Free Full Text Cordeddu V, Redeker B, Stellacci E, et al.: Mutations in ZBTB20 cause Primrose syndrome. Nat Genet. 2014; 46(8): 815–817. PubMed Abstract \| Publisher Full Text Grant information This work was supported by the Wellcome Trust [098330], Stra- tegic Award to The London Down Syndrome (LonDownS) Con- sortium; the Alzheimer’s Society [192(ALZSOC-PhD-2013-001)], awarded to FKW and EMCF; the Medical Research Council [MR/J004022/1], to AMI; and Alzheimer’s Research UK [ARUK- PPG2014B-5] to AMI. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. It may be important to assess expression in multiple neuronal cell types throughout development in the J20 model, considering that ectopic expression of Zbtb20 in the subiculum and post-subiculum results in aberrant CA1 type development in those regions with associated CA1-specific markers (Nielsen et al., 2010). The transgene sequence itself is intact in the J20, excluding several single nucleotide mutations; however, these are either silent or Competing interests The authors declare no competing interests. Competing interests The authors declare no competing interests. Acknowledgements We thank the Medical Research Council Prion Unit Biological Services Facility for animal husbandry and tissue collection serv- ices and Dr Rachele Saccon for use of edited mouse diagrams (Figure 1A). PubMed Abstract Mullan M, Crawford F, Axelman K, et al.: A pathogenic mutation for probable Alzheimer’s disease in the APP gene at the N-terminus of beta-amyloid. Nat Genet. 1992; 1(5): 345–347. PubMed Abstract \| Publisher Full Text Tosh JL: J20 Transgene Mapping. 2017. Tosh JL: J20 Transgene Mapping. 2017. References Page 7 of 13 Wellcome Open Research 2017, 2:84 Last updated: 23 MAR 2022 of Alzheimer’s disease. Neuron. 2007; 55(5): 697–711. of Alzheimer’s disease. Neuron. 2007; 55(5): 697–711. 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Mattioli F, Piton A, Gérard B, et al.: Novel de novo mutations in ZBTB20 in Primrose syndrome with congenital hypothyroidism. Am J Med Genet Part A. 2016; 170(6): 1626–1629. ( ) PubMed Abstract \| Publisher Full Text Open Peer Review Current Peer Review Status: © 2017 Pavlovic G. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. PubMed Abstract Wilhelm M, Schlegl J, Hahne H, et al.: Mass-spectrometry-based draft of the human proteome. Nature. 2014; 509(7502): 582–587. Nielsen JV, Blom JB, Noraberg J, et al.: Zbtb20-Induced CA1 Pyramidal Neuron Development and Area Enlargement in the Cerebral Midline Cortex of Mice. Cereb Cortex. 2010; 20(8): 1904–1914. 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PubMed Abstract \| Publisher Full Text Yan BW, Zhao YF, Cao WG, et al.: Mechanism of random integration of foreign DNA in transgenic mice. Transgenic Res. 2013; 22(5): 983–992. Palop JJ, Chin J, Roberson ED, et al.: Aberrant excitatory neuronal activity and compensatory remodeling of inhibitory hippocampal circuits in mouse models Palop JJ, Chin J, Roberson ED, et al.: Aberrant excitatory neuronal activity and compensatory remodeling of inhibitory hippocampal circuits in mouse models Palop JJ, Chin J, Roberson ED, et al.: Aberrant excitatory neuronal activity and compensatory remodeling of inhibitory hippocampal circuits in mouse models Page 8 of 13 Page 8 of 13 Wellcome Open Research 2017, 2:84 Last updated: 23 MAR 2022 Guillaume Pavlovic We have updated the figure legend to reflect the reviewers comments (error bars are SEM) and have also sign-posted the link to the supplementary data in the legend for Figure 3; where all primary data including uncropped blots can be viewed, and note that the analysis of our western blots complied with best practice ( http://journals.plos.org/plosone/s/submission-guidelines). Guillaume Pavlovic Taylor SC, Berkelman T, Yadav G, Hammond M: A defined methodology for reliable quantification of Western blot data.Mol Biotechnol. 2013; 55 (3): 217-26 PubMed Abstract \| Publisher Full Text 3. Ghosh R, Gilda JE, Gomes AV: The necessity of and strategies for improving confidence in the accuracy of western blots.Expert Rev Proteomics. 2014; 11 (5): 549-60 PubMed Abstract \| Publisher Full Text References 1. Taylor SC, Posch A: The design of a quantitative western blot experiment.Biomed Res Int. 2014; 2014: 361590 PubMed Abstract \| Publisher Full Text 2. Taylor SC, Berkelman T, Yadav G, Hammond M: A defined methodology for reliable quantification of Western blot data.Mol Biotechnol. 2013; 55 (3): 217-26 PubMed Abstract \| Publisher Full Text 3. Ghosh R, Gilda JE, Gomes AV: The necessity of and strategies for improving confidence in the accuracy of western blots.Expert Rev Proteomics. 2014; 11 (5): 549-60 PubMed Abstract \| Publisher Full Text 1. Taylor SC, Posch A: The design of a quantitative western blot experiment.Biomed Res Int. 2014; 2014: 361590 PubMed Abstract \| Publisher Full Text 2. Taylor SC, Berkelman T, Yadav G, Hammond M: A defined methodology for reliable quantification of Western blot data.Mol Biotechnol. 2013; 55 (3): 217-26 PubMed Abstract \| Publisher Full Text 3. Ghosh R, Gilda JE, Gomes AV: The necessity of and strategies for improving confidence in the accuracy of western blots.Expert Rev Proteomics. 2014; 11 (5): 549-60 PubMed Abstract \| Publisher Full Text Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Author Response 23 Sep 2018 Justin Tosh, University College London, London, UK The authors thank Dr Pavlovic for his insightful comments. Guillaume Pavlovic 1 Institut Clinique de la Souris (ICS), CNRS, INSERM, University of Strasbourg, Illkirch, France 2 PHENOMIN-ICS, Strasbourg, France The authors have found non mendalian ratio in cross between Chrmp2b-/- and J20+/- animals. They found that J20 transgene is integrated ~22 Mb of the Chrmp2b gene in the Zbtb20 gene. The report is well written, easy to understand and relevant. However, the Western Blot data does not allow to conclude that Zbtb20 protein expression remains unchanged. Comments: Comments: - Figure 3 Unpaired t-test: your statistical analysis hypothesize normal distribution of the data. Using a non parametric test will be more correct if you do not provide the normality test results. Please indicate what are the error bars (SEM, SD or 95% Cl)? - Assessment of ZBTB20 expression: Western Blot analysis The western blot results provided in the paper do not prove that the “ZBTB20 protein is unaltered in adult J20 hippocampal tissue”. Knock-out controls and molecular size markers are missing to prove that the showed band is not an unspecific signal. Moreover, the lack of appropriate controls (i.e. different quantity of protein extract) do not allow to validate the use of Figure 2B as a quantitative Western Blot. See why in the publications 1 to 3 Finally, as recommended in PLOS ONE for example ( Moreover, the lack of appropriate controls (i.e. different quantity of protein extract) do not allow to validate the use of Figure 2B as a quantitative Western Blot. See why in the publications 1 to 3 Finally, as recommended in PLOS ONE for example ( http://journals.plos.org/plosone/s/submission-guidelines), the author should provide: Original uncropped and unadjusted blots and gels, including molecular size markers, should be provided in either the figures or the supplementary files. ○ The image should include all relevant controls, and controls should be run on the same blot or gel as the samples. ○ All blots and gels that support results reported in the manuscript should be provided. Please provide ZBTB20 additional antibody results (which are data not shown in the paper) as additional files. Add reference of this additional antibody in material and methods. ○ Page 9 of 13 Wellcome Open Research 2017, 2:84 Last updated: 23 MAR 2022 References 1. Taylor SC, Posch A: The design of a quantitative western blot experiment.Biomed Res Int. 2014; 2014: 361590 PubMed Abstract \| Publisher Full Text 2. https://doi.org/10.21956/wellcomeopenres.13248.r25970 https://doi.org/10.21956/wellcomeopenres.13248.r25970 © 2017 Saido T. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. References 1. Tonchev AB, Tuoc TC, Rosenthal EH, Studer M, et al.: Zbtb20 modulates the sequential generation of neuronal layers in developing cortex.Mol Brain. 2016; 9 (1): 65 PubMed Abstract \| Publisher Full Text 2. Liu G, Zhou L, Zhang H, Chen R, et al.: Regulation of hepatic lipogenesis by the zinc finger protein Zbtb20.Nat Commun. 2017; 8: 14824 PubMed Abstract \| Publisher Full Text Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, Saitama, Japan Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, Saitama, Japan Gene expression is regulated by promoters, enhancers, silencers, etc. in a tissue-specific manner. For instance, Zbtb20 plays an essential role in hepatic de novo lipogenesis, so it is necessary to examine the mRNA and protein levels of Zbtb20 in all the tissues in addition to hippocampus including the liver because the transgene is inserted into every corresponding chromosome. In particular, it is surprising that the authors avoided examining developed and developing cortex because Zbtb20 modulates the sequential generation of neuronal layers in developing cortex. It would also be quite convincing if there was a negative control in Figure 3B (and in additional Western blot data) using tissues from Zbtb20-KO mice. One comment on the Western blot data, the quantities of protein are not sufficently normalized. If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Author Response 23 Sep 2018 Justin Tosh, University College London, London, UK The authors thank Dr Pavlovic for his insightful comments. We have updated the figure legend to reflect the reviewers comments (error bars are SEM) and have also sign-posted the link to the supplementary data in the legend for Figure 3; where all primary data including uncropped blots can be viewed, and note that the analysis of our western blots complied with best practice ( http://journals.plos.org/plosone/s/submission-guidelines). Page 10 of 13 Wellcome Open Research 2017, 2:84 Last updated: 23 MAR 2022 In our qPCR and western blot experiments we could not test for, or assume normality of data distribution, so an alternative non-parametric test was used instead (Mann-Whitney U). Competing Interests: No competing interests were disclosed. Competing Interests: No competing interests were disclosed. Reviewer Report 25 September 2017 Author Response 23 Sep 2018 Justin Tosh, University College London, London, UK The authors thank Dr Saido for his insightful comments. We agree the transgene is inserted into Zbtb20 in every cell of the body, and therefore may affect multiple organs. However it is beyond the scope of this initial characterization of the genomic location of the APP transgene in the J20 animal model to study transcription of Zbtb20 in every organ of the mouse. We wished to communicate the issue of disruption of the Zbtb20 locus to the scientific community immediately so that further detailed studies can be undertaken. We note that have not been able to source tissue from either Zbtb20-/- or Zbtb20-/+ mice, and that Zbtb20-/- mice have poor postnatal survival (Sutherland 2009). Thus it is not possible at this time to verify the results presented in Figure 3B using an appropriate negative control (adult hippocampus from a Zbtb20-/-). Therefore we agree that until we can undertake this important validation of the experiment, we cannot state categorically that the ZBTB20 protein level is unchanged in the J20 model system and have edited our paper accordingly. Competing Interests: No competing interests were disclosed. Reviewer Report 19 September 2017 Reviewer Report 19 September 2017 I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Page 11 of 13 Wellcome Open Research 2017, 2:84 Last updated: 23 MAR 2022 Partly Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Neuroscience I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Author Response 23 Sep 2018 Justin Tosh, University College London, London, UK The authors thank Dr Saido for his insightful comments. We agree the transgene is inserted into Zbtb20 in every cell of the body, and therefore may affect multiple organs. However it is beyond the scope of this initial characterization of the genomic location of the APP transgene in the J20 animal model to study transcription of Zbtb20 in every organ of the mouse. We wished to communicate the issue of disruption of the Zbtb20 locus to the scientific community immediately so that further detailed studies can be undertaken. We note that have not been able to source tissue from either Zbtb20-/- or Zbtb20-/+ mice, and that Zbtb20-/- mice have poor postnatal survival (Sutherland 2009). Thus it is not possible at this time to verify the results presented in Figure 3B using an appropriate negative control (adult hippocampus from a Zbtb20-/-). Therefore we agree that until we can undertake this important validation of the experiment, we cannot state categorically that the ZBTB20 protein level is unchanged in the J20 model system and have edited our paper accordingly. Competing Interests: No competing interests were disclosed. Partly Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Neuroscience I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Karen H. Ashe Department of Neurology, University of Minnesota, Minneapolis, MN, 55455, USA The authors have shown that an unexpected ratio of genotypes that occurred when J20 mice were bred to Chmp2b KO mice was due to the insertion of the J20 transgene array near the Chmp2b gene, resultig in linkage disequilibrium. The report is interesting, relevant and well-done. The only correction I recommend is to change Figure 1 so that the colors in the rectangles match the colors of the mice (e.g., make the orange rectangle red and the red rectangle orange). Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Neurobiology of Alzheimer's disease I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Author Response 23 Sep 2018 Justin Tosh, University College London, London, UK The authors thank Professor Ashe for her insightful comments. In Version 2 of the article we have altered Figure 1 to improve colour clarity as suggested. Competing Interests: No competing interests were disclosed. Page 13 of 13 https://doi.org/10.21956/wellcomeopenres.13248.r25971 https://doi.org/10.21956/wellcomeopenres.13248.r25971 © 2017 Ashe K. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Page 12 of 13 Wellcome Open Research 2017, 2:84 Last updated: 23 MAR 2022 Are the conclusions drawn adequately supported by the results? Yes Justin Tosh, University College London, London, UK The authors thank Professor Ashe for her insightful comments. In Version 2 of the article we have altered Figure 1 to improve colour clarity as suggested. The authors thank Professor Ashe for her insightful comments. In Version 2 of the article we have altered Figure 1 to improve colour clarity as suggested. Page 13 of 13
https://openalex.org/W4311125389	https://bmcbioinformatics.biomedcentral.com/counter/pdf/10.1186/s12859-022-05093-z	English	null	DiviK: divisive intelligent K-means for hands-free unsupervised clustering in big biological data	BMC bioinformatics	2,022	cc-by	12,388	© 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 mate- rial. 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/publi cdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 https://doi.org/10.1186/s12859-022-05093-z Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 https://doi.org/10.1186/s12859-022-05093-z BMC Bioinformatics Open Access Grzegorz Mrukwa1,2 and Joanna Polanska1* Correspondence: Joanna.Polanska@polsl.pl 1 Department of Data Science and Engineering, Silesian University of Technology, Akademicka 16, 44‑100 Gliwice, Poland 2 Netguru, Małe Garbary 9, 61‑756 Poznań, Poland DiviK: divisive intelligent K‑means for hands‑free unsupervised clustering in big biological data Grzegorz Mrukwa1,2 and Joanna Polanska1 Abstract Background: Investigating molecular heterogeneity provides insights into tumour origin and metabolomics. The increasing amount of data gathered makes manual analyses infeasible—therefore, automated unsupervised learning approaches are utilised for discovering tissue heterogeneity. However, automated analyses require experience setting the algorithms’ hyperparameters and expert knowledge about the analysed biological processes. Moreover, feature engineering is needed to obtain valu- able results because of the numerous features measured. Results: We propose DiviK: a scalable stepwise algorithm with local data-driven feature space adaptation for segmenting high-dimensional datasets. The algorithm is compared to the optional solutions (regular k-means, spatial and spectral approaches) combined with different feature engineering techniques (None, PCA, EXIMS, UMAP, Neural Ions). Three quality indices: Dice Index, Rand Index and EXIMS score, focusing on the overall composition of the clustering, coverage of the tumour region and spatial cluster consistency, are used to assess the quality of unsupervised analyses. Algorithms were validated on mass spectrometry imaging (MSI) datasets—2D human cancer tis- sue samples and 3D mouse kidney images. DiviK algorithm performed the best among the four clustering algorithms compared (overall quality score 1.24, 0.58 and 162 for d(0, 0, 0), d(1, 1, 1) and the sum of ranks, respectively), with spectral clustering being mostly second. Feature engineering techniques impact the overall clustering results less than the algorithms themselves (partial η2 effect size: 0.141 versus 0.345, Kendall’s concordance index: 0.424 versus 0.138 for d(0, 0, 0)). Conclusions: DiviK could be the default choice in the exploration of MSI data. Thanks to its unique, GMM-based local optimisation of the feature space and deglomerative schema, DiviK results do not strongly depend on the feature engineering technique applied and can reveal the hidden structure in a tissue sample. Additionally, DiviK shows high scalability, and it can process at once the big omics data with more than 1.5 mln instances and a few thousand features. Finally, due to its simplicity, DiviK is easily generalisable to an even more flexible framework. Therefore, it is helpful for other -omics data (as single cell spatial transcriptomic) or tabular data in general (including medical images after appropriate embedding). A generic implementation is freely available under Apache 2.0 license at https://github.com/gmrukwa/divik. *Correspondence: Joanna.Polanska@polsl.pl 1 Department of Data Science and Engineering, Silesian University of Technology, Akademicka 16, 44‑100 Gliwice, Poland 2 Netguru, Małe Garbary 9, 61‑756 Poznań, Poland Background Mass Spectrometry Imaging (MSI) is widely used for discovering molecular profiles, as it provides unparalleled insight into the metabolomics of tissue samples [1–3]. Applied to a tumour specimen, MSI allows investigating heterogeneities that could indicate functional differences across tissue regions, as well as varying cancer sub- types that require dedicated treatment [4–10]. Technically, MSI is an excellent example of big biological data, with the following characteristics: • Volume: the spatial resolution of 5 −100µm leads to 10,000—4,000,000 spectra potentially acquired from a 1cm2 tissue sample with Time-of-Flight (ToF) spec- trometers and becomes an even more severe problem for 3D MSI data [11]; • Velocity: the last three years have brought more than 4500 MSI datasets uploaded to the METASPACE database [12, 13]; • Variety: a dataset may consist of more than 200,000 mass channels (features) per single spectrum (observation), representing proteins, peptides, and/or metabo- lites; • Veracity: the acquisition methods and various biological phenomena introduce heavy duplication of information, thus capturing the most relevant nuances becomes complicated with dominating high-level patterns amplified [14]. On the other hand, feature importance may be diverse across separate regions of interest. Efficient analyses of thousands of MSI spectra usually require careful feature space adaptation, irrespective of the extensive preprocessing pipeline [15–18]. In a fully unsupervised setup, there are the following groups of methods: filtering, linear (e.g. Principal Components Analysis), and non-linear (e.g. Universal Manifold Approxi- mation and Projection) [19]. Filtering removes features according to some threshold (often constant). Linear methods produce a new set of features, a linear combination of the input features. Non-linear methods aim to find a low-dimensional representa- tion that best approximates distances between pairs of points. All these methods are applied once globally to the entire dataset.i Many clustering methods are known, and one can divide them into a few significant groups based on their approach: centroid-based (e.g. k-means), connectivity-based (e.g. hierarchical clustering), distribution-based (e.g. Gaussian Mixture Modeling), and density-based (e.g. graph-cuts clustering). In addition, sometimes, a method is proposed that exercises more than a single property of the data (e.g. both connectivity and centroids) and such a method is called hybrid clustering. The clustering quality can be captured via metrics like Dunn’s index [20] or Adjusted Rand Index [21]. These were introduced for low-dimensional data cluster- ing, but studies [22, 23] compare their usefulness for high-dimensional data and sub- space clustering. © 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 mate- rial. 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/publi cdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 24 Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Keywords: Machine learning, Unsupervised clustering, Feature engineering, Local feature space adaptation, High-dimensional data analysis, Omics, Mass spectrometry imaging, Tumour heterogeneity Background Hundreds of features that clustering algorithms process are already Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Page 3 of 24 seen as a high-dimensionality problem, but it is still at least an order of magnitude less severe than in MSI data analyses. Therefore domain-related work must be inves- tigated [18]. The earliest solutions for Mass Spectrometry Imaging take advantage of Principal Components Analysis (PCA), and agglomerative clustering [24]. PCA transforms the data, and components explaining 70% of the variance are selected. The authors use the Euclidean metric to capture spectra similarity and Ward linkage to provide the most meaningful results. The approach is semi-supervised, as it requires the manual setting of the number of clusters based on histological examination. Another method features high-dimensional data clustering (HDDC) [25]—a hybrid approach based on the Gaussian Mixture Model (GMM). The fitting process yields locally relevant features, so the domain is independently adapted for each estimated component. The observations’ similarity is considered Euclidean metric in some sub- spaces of the original feature space. For MSI data, a combination of HDDC with edge-preserving denoising of m/z-images is applied [26]. Oppositely to smoothing the clustering results, the authors propose an approach oriented on m/z images, so the denoising provides better quality data for the clustering method. The idea of denoising is extended [27]. The Fast Map algorithm adapts the domain, and K-means clustering fol- lows, which is more straightforward than HDDC. The denoising stage contains the main advancements, which we will omit here. The overlap of clusters and actual structures is the primary quality metric. However, both HDDC and K-means approaches [26, 27] also compare the visual perception of cluster consistency. Region consistency is the main idea behind EXIMS [28]—a modern PCA-oriented feature engineering approach. First, histogram equalisation enhances the contrast of a molecular image. Then, the algorithm proposes a scoring method to assess whether structures exist in the enhanced molecular image. The random distribution of counts over the image leads to a low score value. Next, typical structure types like regions, curves, gradients and islets are recognised with a grey level co-occurrence matrix, resulting in a higher score value. However, the definition of the score yields unbounded values. Thus there is no clear threshold that can help discern informative peaks. Finally, the informative peaks are PCA-transformed and clustered with the fuzzy C-means algorithm. Background K-means and spectral clustering are further investigated in a two-step scenario to speed up computations [29]. In the first step of clustering, both methods consider sev- eral random subsets of input data independently, and then cluster representatives are grouped to form the final clusters. Algorithms use cosine distance to measure spectrum similarity. Authors manually perform hyperparameter selection (i.e. selecting the num- ber of eigenvectors of the connectivity graph) to provide the best results (visually). Both methods isolate 7 clusters per prior settings. Feature engineering techniques also address the increased dataset volume. For example, Hierarchical Stochastic Neighbor Embedding (HSNE) has been claimed to be superior to classical non-linear techniques [30]. Reduced feature space is con- structed hierarchically, with the approach Overview-First, Details-on-Demand. Firstly, characteristic points of the dataset (landmarks) are embedded to provide an overview. Then, the algorithm creates a new embedding for local neighbourhoods. Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Page 4 of 24 Finally, each point is assigned the likelihood of being represented by a specific land- mark. The authors discuss using maps of the likelihood for molecular segmentation given a landmark. However, they propose clusters of landmarks manually. Another scalable feature engineering example is the Universal Manifold Approxi- mation and Projection (UMAP) algorithm. It is more scalable than t-SNE and pre- serves more of the global structure [31]. Moreover, no restriction is made on the embedding dimensions. Thus, it may be used as a more general feature engineering method in MSI than the visualizing-only capabilities of t-SNE [32]. The recent years introduced a new category of unsupervised segmentation approaches based on deep learning. They form groups that correspond to dual inter- pretation of MSI data: • Spectrum oriented: neural network creates a low-dimensional embedding of spec- tra [33–35] that is further analysed with classical clustering methods or visualised.i • Ion-image oriented: molecular images are analysed to find similarities in molecule distribution and enhance biologically relevant information. The neural approach extends the idea from EXIMS [28] and classical community detection algorithms [36]. It mimics a human recognising detailed molecular patterns and clusters the ion images based on spatial expressions [37]. This way, new molecular features are obtained, simultaneously representing whole groups of ion images. These neu- ral ion images are subject to a classical unsupervised analysis pipeline, including pixel-wise approaches. Background The unsupervised analyses of MSI data via clustering outlined above follow a single schema: some domain adaptation is applied globally, and a basic clustering algorithm is applied. This approach has several drawbacks. • PCA applied to the whole dataset can capture the most variance on a high level, but at the same time, it discards all nuances. These nuances might be crucial for hierarchical analyses. Other domain adaptation methods may partially resolve the issue, but the disproportion between low- and high-level detail is significant with thousands of dimensions. • Non-hierarchical approaches (e.g. one-step, two-step) cannot provide insights into the internal structures unless the molecular differences dominate the whole dataset. • Only hierarchical approaches describe the relation of subgroups as a clustering tree that captures context like parent, child, or sibling. This context provides addi- tional insight into molecular diversity. For example, the parent-child relation of clusters could describe different functional subregions of the tumour region, while sibling clusters could represent other types of tumours. • Agglomerative or bisecting deglomerative hierarchical algorithms provide limited information about siblings since only two objects are merged or divided on each level. Therefore, many siblings are mistreated as an artificial parent-child relation. Such mistreatment requires an additional post-processing step. Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Page 5 of 24 We propose a hybrid framework that directly answers the above drawbacks: Divisive intelligent K-means (DiviK). It is a stepwise clustering procedure with feature engineer- ing applied locally at each step (see Fig. 1). We implemented local feature engineering as filtering based on GMM decomposition of the feature variance across the subregion. The clustering algorithm implemented in our approach is a standard K-means algorithm with a tailored procedure for initial condition selection. One can easily substitute GMM filtering and K-means with other algorithms. Information criteria decide the number of clusters in the optimised feature space. The decision on the cluster number is two-stage: due to its numerical complexity, we use GAP at the first stage when assessing the data’s Fig. 1 The proposed hierarchical framework for unsupervised clustering of MSI data with local feature domain adaptation. For each ROI, an analysis was performed on whether it contains any implicit structures. If structures were present, molecular segmentation was continued recursively for each cluster/subcluster Fig. 1 The proposed hierarchical framework for unsupervised clustering of MSI data with local feature domain adaptation. Feature space optimisation We propose to use a feature filtering procedure based on GMM decomposition [38, 39]. First, we average abundance and calculate abundance variance across the analysed sub- cluster for each peak. Then, empirical distributions of average abundances and abun- dance variances across all peaks, presented on the logarithmic scale (upper panel of Fig. 2), are decomposed into Gaussian mixtures, as shown in the middle panel of Fig. 2. We use the maximum conditional probability criterion to classify a particular peak into one of the mixture components. The crossing points between Gaussian components serve as the thresholds. We discarded the GMM components containing less than 1% of peaks to avoid numeric artefacts. We filter out all the peaks with the average abundance below the first crossing point of the GM model. For the abundance variance, we persisted only the peaks with the var- iance above the topmost crossing point, assuming it results in no less than 1% of the available features, as is shown in the bottom right panel of Fig. 2. If the 1% condition is not fulfilled, the second left or the second topmost threshold is used. i As we aim for a hands-free pipeline, Bayesian Information Criterion is used to select the optimal number of GMM components for both the model of average abundance dis- tribution for global noise filtering and the model of local abundance variance distribu- tion for discrimination of locally the most informative features. Implementation Three main components of the DiviK algorithm: feature engineering and filtering tech- nique, stop condition definition, and clustering algorithm, constitute its framework. All of these can be easily changed to adapt to the specificity of the analysed data. Background For each ROI, an analysis was performed on whether it contains any implicit structures. If structures were present, molecular segmentation was continued recursively for each cluster/subcluster Fig. 1 The proposed hierarchical framework for unsupervised clustering of MSI data with local feature domain adaptation. For each ROI, an analysis was performed on whether it contains any implicit structures. If structures were present, molecular segmentation was continued recursively for each cluster/subcluster Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Page 6 of 24 homogeneity. We use the approximate Dunn’s index to select the best local partition in the next step. The strength of the proposed solution lies in optimising the feature space locally for data clustering according to the variability of the analysed subregion. DiviK considers separate regions independently on each step, starting from the entire input dataset fea- ture space. It provides biologically relevant results and sustains scalability for large data- sets, contrary to sophisticated approaches. DiviK addresses the drawbacks mentioned above in the following way: • It has a hierarchical nature to provide the most comprehensive insight. • During the analysis, local domain adaptation is performed separately for each clus- ter/subcluster to avoid the excessive influence of dominating global patterns. l • Before the cluster/subcluster analysis, a check is performed on whether there is evi- dence that diverse groups are present in the cluster/subcluster. Stop condition We calculate a conditional probability for each Gaussian component for each value of the selected characteristic. Then we apply the maximum classification rule, which leads to the interpretation that the crossing points of the neighbouring GMM components become filtering thresholds. We remove all the peaks represented by the first GMM component for the average abundance. For the abundance variance, we persist only the peaks represented by the topmost GMM components, but not less than 1% of all the peaks Fig. 2 Data-driven MSI peaks selection based on a histogram decomposition. Filtering considers each MSI peak’s average abundance (left panel) and abundance variance (right panel). The histograms of these two characteristics are decomposed into a GMM. Components represent sets of features similar concerning selected characteristics, i.e., average abundance or abundance variance across a cluster. We calculate a conditional probability for each Gaussian component for each value of the selected characteristic. Then we apply the maximum classification rule, which leads to the interpretation that the crossing points of the neighbouring GMM components become filtering thresholds. We remove all the peaks represented by the first GMM component for the average abundance. For the abundance variance, we persist only the peaks represented by the topmost GMM components, but not less than 1% of all the peaks segmentation. These take into account cluster separability, compactness or probabilistic measures. However, only a few allow for a comparison between a multi-cluster partition and no further partition (one cluster only). For the K-means algorithm, the GAP statistic [40] is one of the options. It relates a data-specific partition to partitions obtained via the same method over random datasets within the same bounds (see Fig. 3). We stop split- ting of particular ROI if GAP statistic suggests region homogeneity. Stop condition Having a feature space optimised for cluster discovery, we must assess whether there are any clusters. There exist many indices that can validate the quality of unsupervised Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Page 7 of 24 100 1000 10k 100k 1M 100 10k 1M 100M 10B 1T 100T average peak-related molecule abundance within the cluster peak-related molecule abundance variance within the cluster 100 1000 10k 100k 1M 100 10k 1M 100M 10B 1T 100T molecule average abundance modeling molecule abundance variation modeling 100 1000 10k 100k 1M discarded molecules 100 10k 1M 100M 10B 1T 100T discarded molecules low average abundance molecules ﬁltering selection of highly variate molecules Fig. 2 Data-driven MSI peaks selection based on a histogram decomposition. Filtering considers each MSI peak’s average abundance (left panel) and abundance variance (right panel). The histograms of these two characteristics are decomposed into a GMM. Components represent sets of features similar concerning selected characteristics, i.e., average abundance or abundance variance across a cluster. We calculate a conditional probability for each Gaussian component for each value of the selected characteristic. Then we apply the maximum classification rule, which leads to the interpretation that the crossing points of the neighbouring GMM components become filtering thresholds. We remove all the peaks represented by the first GMM component for the average abundance. For the abundance variance, we persist only the peaks represented by the topmost GMM components, but not less than 1% of all the peaks 100 10k 1M 100M 10B 1T 100T peak-related molecule abundance variance within the cluster 100 1000 10k 100k 1M average peak-related molecule abundance within the cluster 100 1000 10k 100k 1M molecule average abundance modeling 100 10k 1M 100M 10B 1T 100T molecule abundance variation modeling g g 100 1000 10k 100k 1M discarded molecules low average abundance molecules ﬁltering 100 10k 1M 100M 10B 1T 100T discarded molecules selection of highly variate molecules 100 10k 1M 100M 10B 1T 100T selection of highly variate molecules 100 10k 1M 100M 10B 1T 100T selection of highly variate molecules Fig. 2 Data-driven MSI peaks selection based on a histogram decomposition. Filtering considers each MSI peak’s average abundance (left panel) and abundance variance (right panel). The histograms of these two characteristics are decomposed into a GMM. Components represent sets of features similar concerning selected characteristics, i.e., average abundance or abundance variance across a cluster. Clustering In this work, we focus only on the K-means-based implementation due to its promising initial results [9, 41] and low computational complexity. With a cluster molecular diver- sity confirmed, we can proceed with the clustering. For MSI data analysis, we adjusted the distance metric [41] and the initialisation method. The K-means algorithm requires the number of clusters to be specified before the anal- ysis occurs. Since the actual number of molecularly homogeneous regions is unknown, Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Page 8 of 24 data draw N random uniform data sets group uniform data sets compute dispersions in random samples labels centroids compute dispersions in real data dispersion ratio Fig. 3 Flowchart of GAP statistic computation used in the two-trial scenario. Spectra belonging to an ROI are artificially clustered into a single cluster and then into two clusters. The GAP statistic was compared for both cases. If spectra form a single group, the GAP statistic tends to be greater for the artificial division. Otherwise, it tends to be greater for two clusters (but not necessarily optimal) compute dispersions in random samples compute dispersions in real data Fig. 3 Flowchart of GAP statistic computation used in the two-trial scenario. Spectra belonging to an ROI are artificially clustered into a single cluster and then into two clusters. The GAP statistic was compared for both cases. If spectra form a single group, the GAP statistic tends to be greater for the artificial division. Otherwise, it tends to be greater for two clusters (but not necessarily optimal) we use unsupervised quality metrics to guide the computations. The current implemen- tation includes Dunn’s index [20] and GAP statistic [40]. Initialisation method Since the DiviK algorithm aims to identify biologically meaningful heterogeneity in the data, we decided to implement the deterministic initialisation procedure (see Fig. 4). Although there are plenty of well-known algorithms for random initialisation, the deter- ministic approaches are usually customised to the data structure, allowing to achieve the clustering goal. As mentioned above, in the case of MSI data, we seek the most hetero- geneous tissue regions independently of the cluster imbalance. To address the expected redundancy in MSI data and decrease the computational time, we compress the data by the KD-tree algorithm (with the compression factor equal to 0.01 for 2D data and 0.001 for 3D data). The obtained “boxes of spectra” are represented by their centroids and weighting factors related to the number of spectra assigned to the particular box. The next step requires computing the distances from the global dataset centroid to the box-specific centroids. We approximate the distances’ cumulative distribution function (cdf) and choose the centroid containing the k-th percentile. It serves as the first initial seed for the clustering algorithm. The procedure mentioned above repeats to obtain the second seed, but we substitute the global centroid with the first seed and the box con- taining that seed is not considered in the calculations. Having two initial seeds, we can search for the third one (if required). We estimate the cdf of the distance between the box-specific centroids and the line defined by already chosen two seeds to find the box containing k-th percentile. The centroid of that box is considered the third seed. One can identify the subsequent seeds similarly, substituting the line by the hyperplane defined by the already found seeds. We perform the initial seed setting in the already locally optimised feature space. The comprehensive study on the initialisation procedures (not shown in the manuscript) demonstrated the power and robustness of our approach when compared to the commonly used initialisation algorithms. Scalability y DiviK is an original stepwise deglomerative algorithm that provides scalability for big data in several ways: Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Page 9 of 24 data build KD-Tree leaf centroids leaf weights (number of observations) ∅ build linear model ﬁnd leaf containing N th percentile residual set of initial seeds are there K elements? return set of initial seeds compute distances from leaf centroids to their nearest initial seed ﬁnd leaf containing N th percentile of distances initialize yes no insert centroid check insert centroid Fig. 4 Flowchart of K-means initialization procedure. We find a moderately outlying KD-Tree leaf centroid as the first seed and follow an iterative deterministic algorithm similar to the K-means++ algorithm. KD-Tree keeps the order of computational complexity low, while the linear model makes it more predictable than the K-means++ algorithm data build KD-Tree leaf weights (number of observations) leaf centroids are there K elements? Fig. 4 Flowchart of K-means initialization procedure. We find a moderately outlying KD-Tree leaf centroid as the first seed and follow an iterative deterministic algorithm similar to the K-means++ algorithm. KD-Tree keeps the order of computational complexity low, while the linear model makes it more predictable than the K-means++ algorithm • It uses a locally optimised K-means algorithm iteratively. K-means algorithm is known as one of the few highly scalable clustering algorithms. • Filtration-based feature engineering, used in the local optimisation of the feature space step, requires conducting neither PCA nor UMAP. The Gaussian Mixture Model approach supports threshold identification necessary for the feature filtra- tion (see Fig. 2). It is computationally efficient, as it requires only computing the Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Page 10 of 24 within-cluster mean and the variance of each feature, followed by an automated data-driven threshold selection. within-cluster mean and the variance of each feature, followed by an automated data-driven threshold selection. • To ensure computational complexity is linear with the number of samples, we devel- oped a procedure of deterministic initialisation based on a KD-Tree algorithm. • The automatic selection of the number of clusters in the K-means algorithm is com- putationally expensive by default. Therefore, we split the decision process into two steps. First, we use the sampled distribution of GAP statistics to decide if the cluster is heterogeneous (requires further splitting) or homogenous (the splitting process is finished). Results In order to compare the effectiveness of the DiviK framework and the existing approaches, we evaluated the tool against two MSI datasets of different characteristics: • Oral Squamous Cell Carcinoma (OSCC). It consists of two individual slices collected from different patients suffering from OSCC, annotated by an experienced patholo- gist; • Mouse kidney. It consists of 75 serial slices from a single 3D-allocated volume from a mouse kidney without annotation. We compared DiviK to the combinations of state-of-the-art MSI data clustering and global feature engineering methods. We selected K-means, spectral and spatial cluster- ing [27] as representatives of clustering algorithms validated for MSI. All were used in a fully unsupervised setting with the number of clusters selected using the GAP statistic. For global feature engineering, we used one of the following options: no global feature engineering, PCA with the knee-based selection of the number of components [43], PCA on top of EXIMS-preselected features [28], UMAP [31], and neural ion images obtained with Xception network [37]. All the experiments were carried out in the Polyaxon environment [44]. Technology DiviK was written mainly in Python, and it is distributed cross-platform under the Apache 2.0 license through Python Package Index (https://pypi.org/project/divik/) and Docker Hub (https://hub.docker.com/r/gmrukwa/divik). It is designed to be simple, effi- cient, accessible to a non-expert, and highly reusable. Thus, DiviK is accessible in Python directly and via a command-line interface (CLI). The Python API follows the scikit-learn [42] design patterns and similar package organisation conventions to provide reusable building blocks. The CLI allows the construction of highly-flexible processing pipelines owing to its injection-based configuration system. Scalability In the case of a heterogeneous cluster, we use Dunn’s Index to decide on the number of subclusters, which keeps the process scalable. Oral squamous cell carcinoma They were selected sequentially to optimise the Dice index. Secondly, all ambiguous assignments were resolved via optimising the Rand Index to form the normalised labels Fig. 6 Cluster label normalisation procedure. Red = false negative; yellow = false positive; grey = true positive tumour. In the first step, a binary decision was made on whether the label should belong to one of the pathologist-defined regions. Clusters were sorted by the percentage of their area covered by the ROI. They were selected sequentially to optimise the Dice index. Secondly, all ambiguous assignments were resolved via optimising the Rand Index to form the normalised labels Fig. 6 Cluster label normalisation procedure. Red = false negative; yellow = false positive; grey = true positive tumour. In the first step, a binary decision was made on whether the label should belong to one of the pathologist-defined regions. Clusters were sorted by the percentage of their area covered by the ROI. They were selected sequentially to optimise the Dice index. Secondly, all ambiguous assignments were resolved via optimising the Rand Index to form the normalised labels set maximising Dice index for a given ROI, zero otherwise. If only one pair (C, ROIi) is assigned 1 for a given cluster C, the assignment is considered final. Otherwise, we evaluate each pair (assignment scenario) in terms of the Rand index. We used this dataset to compare ROI reconstruction capabilities across different clus- tering algorithms and feature engineering techniques. With clusters reassigned to the pathologist-defined ROIs, we considered the following quality metrics: • Rand Index—to capture global multi-ROI reconstruction capabilities; • Dice Index—to capture tumour reconstruction capabilities; • EXIMS score—to capture the spatial consistency of the clusters. The EXIMS score is a measure with unbounded values, useful in comparative analyses, but its magnitude is hard to interpret. Therefore we scaled and clipped the values so that the highest multi-cluster result was 1. DiviK sweeps for up to 10 clusters on each segmentation level. It operates with cor- relation distance, as researchers have already proven it to work well for MSI data [29, 32]. The minimal number of local features required to preserve is 1%. The minimal size of the cluster was set to 200 spectra. The leaf size of KD-Tree was 1%, and the algo- rithm started from the leaf containing the 99th percentile of the distance. Oral squamous cell carcinoma The OSCC dataset [41] is a two-dimensional MALDI-ToF MSI dataset. The origi- nal dataset consists of 45,738 raw spectra with 109,568 mass channels. After Page 11 of 24 Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Fig. 5 Microscope image of raw HNC samples (upper panel) and samples annotated by a pathologist (lower panel). Regions of interest (ROI): red = tumour; cyan = healthy epithelium; magenta = other healthy tissue. Due to medical relevance, we focused mostly on tumour and epithelium tissue—the origin of OSCC Fig. 5 Microscope image of raw HNC samples (upper panel) and samples annotated by a pathologist (lower panel). Regions of interest (ROI): red = tumour; cyan = healthy epithelium; magenta = other healthy tissue. Due to medical relevance, we focused mostly on tumour and epithelium tissue—the origin of OSCC pre-processing and spectra modelling, 3714 peaks/features were obtained. The spectra are provided with annotations of the tissue type, allowing for distinguishing regions of interest (ROI) of the tumour, epithelium, and healthy areas (Fig. 5). There- fore, the OSCC dataset can be a benchmark for classification and clustering prob- lems. There are diverse sources of variability, including: • inter-patient sample heterogeneity; • tissue type heterogeneity; • intra-tissue heterogeneity. We selected a subset consisting of two patient samples with top tissue variety (19,874 spectra and 3714 peaks in total) and segmented the subset using all the combinations of the methods discussed in the previous section. The obtained clusters of spectra usually consisted of spectra originally annotated to different tissue subtypes, so the annotation translation from spectrum to cluster level is required. The developed reannotation procedure maximises the multi-cluster Dice similarity index related to the primary tissue subtypes (Region of Interests ROI) as presented in Fig. 6. First, we sort clusters according to the percentage of their area covered by the consecutive ROIs. Then, we select clusters sequentially to maximise the Dice index for a given ROI collectively. We assign one for each (cluster, ROI) pair if the cluster is included in the Page 12 of 24 Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Fig. 6 Cluster label normalisation procedure. Red = false negative; yellow = false positive; grey = true positive tumour. In the first step, a binary decision was made on whether the label should belong to one of the pathologist-defined regions. Clusters were sorted by the percentage of their area covered by the ROI. Oral squamous cell carcinoma We sampled ten times 1000 spectra each to compute Dunn’s and GAP indices to keep the computa- tional complexity of quality estimation bounded. Standalone K-means clustering swept for up to 50 clusters, with the same criteria for computing the GAP index as DiviK and the correlation distance. Spatial clustering was launched with a radius of 7. Spectral clus- tering was used with cosine metrics during the embedding. The embedding generated Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Page 13 of 24 the number of components equal to 1% of the number of global features (information capacity equivalent to DiviK filtering). UMAP was run with 30 neighbours, correlation distance, 500 epochs and a negative sample rate of 70 to obtain three components. We used the Xception network with a patch size of 71 × 71 pixels and an upsample rate of 4 to ensure a patch edge of 1.775mm. We presented the visualisation of the performance of the DiviK algorithm and remain- ing combinations in Fig. 7. Dimensions of the presented cube are the quality indices mentioned at the beginning of this section, namely Dice Index, Rand Index, and EXIMS Score. The exact values of quality metrics are available in Table 1. In Fig. 8 one can find a normalised map of the clusters obtained in the process. We compare the combina- tions of global feature engineering methods and clustering algorithms exhaustively. The proposed DiviK approach uses no global feature engineering method, yet we conducted such experiments and presented their results for comparison. Sample spatial ion heat- maps, correlated with the pathologist’s tissue subtypes and the clusters found by DiviK, are presented in Fig. 9. The averaged results for the used clustering methods can be seen in Table 2 whereas Table 3 presents results obtained with the used global feature engi- neering methods for d-based aggregations. Tables 4 and 5 present the rank-based sum- marisation results. Mouse kidney 3Dh The mouse kidney dataset [45] is a three-dimensional MALDI-ToF MSI dataset. There- fore, the dataset has a high volume and can be a benchmark for the algorithm’s scal- ability. The sources of variability are limited to a single source specimen. It consists of 75 sections from the central part of a mouse kidney, 1,362,830 spectra in total, and 7680 Fig. 7 Quality indices of ROI composability by the obtained clusters. The overall quality is defined as the vector’s length limited by the quality indices’ values and the origin of the coordinate system. The length of the vector is displayed next to the points representing the top results. The arrow indicates the best result Fig. 7 Quality indices of ROI composability by the obtained clusters. The overall quality is defined as the vector’s length limited by the quality indices’ values and the origin of the coordinate system. The length of the vector is displayed next to the points representing the top results. Mouse kidney 3Dh The arrow indicates the best result Page 14 of 24 Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Table 1 Values of quality indices measured for OSCC data Top three values of a quality index among pairwise combinations of clustering methods and feature engineering procedures are presented in bold italics Clustering algorithm Global feature engineering method Adjusted Rand index Dice index Relative EXIMS score Adjusted Rand index rank Dice index rank Relative EXIMS score rank Sum of ranks Overall quality d(0, 0, 0) Overall quality d(1, 1, 1) Spectral UMAP 0.2792 0.4844 0.5891 17 16 20 53 0.8122 0.9768 Spatial UMAP 0.0000 0.0000 1.0000 19.5 19 2.5 41 1.0000 1.4142 Spectral Xception 0.0000 0.0000 1.0000 19.5 19 2.5 41 1.0000 1.4142 K-means Knee PCA 0.2723 0.0000 1.0000 18 19 2.5 39.5 1.0364 1.2368 K-means Xception 0.3098 0.4577 0.9197 16 17 8 41 1.0730 0.8814 K-means EXIMS PCA 0.4827 0.5129 0.8323 12 14 9 35 1.0903 0.7300 Spectral EXIMS PCA 0.5447 0.7418 0.6449 8 8 18 34 1.1237 0.6325 K-means none 0.3364 0.5043 0.9712 15 15 6 36 1.1449 0.8288 K-means UMAP 0.5231 0.7238 0.7225 10 10 13 33 1.1487 0.6170 Spatial Knee PCA 0.4985 0.7065 0.7639 11 11 10 32 1.1537 0.6272 DiviK EXIMS PCA 0.6082 0.7765 0.6383 4 5 19 28 1.1749 0.5782 Spectral none 0.5906 0.7966 0.6520 5 4 17 26 1.1868 0.5745 DiviK Knee PCA 0.5567 0.7540 0.7289 7 7 12 26 1.1873 0.5749 DiviK Xception 0.4203 0.6429 0.9395 14 13 7 34 1.2136 0.6835 Spatial none 0.5617 0.7720 0.7587 6 6 11 23 1.2195 0.5498 DiviK UMAP 0.6534 0.8369 0.6568 2 3 16 21 1.2485 0.5143 Spatial Xception 0.6517 0.8465 0.6851 3 2 15 20 1.2691 0.4940 Spectral Knee PCA 0.4594 0.6897 0.9891 13 12 5 30 1.2904 0.6235 Spatial EXIMS PCA 0.7035 0.8672 0.6977 1 1 14 16 1.3167 0.4438 DiviK none 0.5433 0.7372 1.0000 9 9 2.5 20.5 1.3560 0.5269 Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Page 15 of 24 Table 2 Average quality indices for clustering methods with OSCC data Standard deviation is presented in brackets. Mouse kidney 3Dh Top value of a quality index among clustering methods is presented in bold italics Clustering algorithm Adjusted Rand index Dice index Relative EXIMS score Overall quality d(0, 0, 0) Overall quality d(1, 1, 1) Spectral 0.375 (0.241) 0.543 (0.325) 0.775 (0.202) 1.083 (0.184) 0.844 (0.357) K-means 0.385 (0.111) 0.440 (0.266) 0.889 (0.113) 1.099 (0.048) 0.859 (0.234) Spatial 0.483 (0.281) 0.638 (0.363) 0.781 (0.127) 1.192 (0.123) 0.706 (0.402) DiviK 0.556 (0.088) 0.750 (0.071) 0.793(0.167) 1.236 (0.073) 0.576 (0.067) Table 2 Average quality indices for clustering methods with OSCC data Standard deviation is presented in brackets. Top value of a quality index among clustering methods is presented in bold italics K-Means DiviK Spectral clustering Spatial clustering No feature engineering Knee PCA EXIMS UMAP Xception tumor healthy epithelium other tissue Fig. 8 Results of the unsupervised analyses for OSCC data. Clusters were matched to the pathologist regions using the method described. Red = tumour region; cyan = healthy epithelium; grey = other tissue. The top overall quality is marked other tissue Fig. 8 Results of the unsupervised analyses for OSCC data. Clusters were matched to the pathologist regions using the method described. Red = tumour region; cyan = healthy epithelium; grey = other tissue. The top overall quality is marked Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Page 16 of 24 Page 16 of 24 100% 0% mass channel 2175.1 Da 100% 0% mass channel 1142.5 Da mass channel 2175.1 Da Fig. 9 The spatial distribution of the chosen features (mass channels) in OSCC data. Peptides upregulated in tumour (represented by 1142.5 m/z and 2175.1 m/z) are putatively fragments of pyruvate kinase, an enzyme involved in the Warburg effect mass channel 1142.5 Da mass channel 1142.5 Da mass channel 2175.1 Da Fig. 9 The spatial distribution of the chosen features (mass channels) in OSCC data. Peptides upregulated in tumour (represented by 1142.5 m/z and 2175.1 m/z) are putatively fragments of pyruvate kinase, an enzyme involved in the Warburg effect Fig. 9 The spatial distribution of the chosen features (mass channels) in OSCC data. Peptides upregulated in tumour (represented by 1142.5 m/z and 2175.1 m/z) are putatively fragments of pyruvate kinase, an enzyme involved in the Warburg effect Fig. 9 The spatial distribution of the chosen features (mass channels) in OSCC data. Mouse kidney 3Dh Peptides upregulated in tumour (represented by 1142.5 m/z and 2175.1 m/z) are putatively fragments of pyruvate kinase, an enzyme involved in the Warburg effect No feature engineering Knee PCA EXIMS UMAP K-Means DiviK Fig. 10 Results of the unsupervised analyses for 3D mouse kidney data. All 1,362,830 spectra (75 slices) were analysed together with locally adjusted feature space. Different colours represent molecularly different tissue subtypes. We normalised the colours to correspond between experiment scenarios Fig. 10 Results of the unsupervised analyses for 3D mouse kidney data. All 1,362,830 spectra (75 slices) were analysed together with locally adjusted feature space. Different colours represent molecularly different tissue subtypes. We normalised the colours to correspond between experiment scenarios data points per spectrum. The dataset was already subject to Gaussian spectral smooth- ing and baseline reduction with the Top Hat algorithm. We conduct clustering in the same scenarios as previously, except feature engineer- ing based on neural ion images, as it lacks generalisation for 3D data. No expert-defined labels are available for the spectra; thus, one must assess the capabilities of heterogeneity detection visually. Clusters are subject to a similar reannotation procedure as depicted in Fig. 6 to indicate the similarities between results. The minimal number of local features that DiviK is required to preserve is set to 0.5%. We did not split the cluster further if it already was 50,000 spectra or fewer, as this data- set cannot confirm detailed internal tissue diversity. KD-Tree leaf size during the initiali- sation was 0.1%, and the algorithm started from the leaf containing the 95th percentile of the distance. To compute the Dunn’s and GAP indices, we sampled ten times 5000 spectra each to keep the computational complexity of quality estimation bounded. Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Page 17 of 24 In Fig. 10, we present volumes with clusters marked. We matched cluster colours to enable visual comparison, although we used no cluster normalisation procedure, as the ground truth labels are nonexistent. Unfortunately, spectral clustering is not included in this study, as the computational complexity of the basic approach did not allow for enough scalability. On the other hand, a two-step approach [29] did not lead to conver- gence. Additionally, spatial clustering [27] does not consider modified spatial strides in the third dimension. The same clusters are presented in Fig. 11 on each 6th consecutive slice. Fig. 11 Serial slices of the 3D mouse kidney data. Clusters are visualised on each sixth slice. Cluster colours were normalised to match between configurations for easy comparison Oral squamous cell carcinoma As shown in Fig. 8, it is possible to observe varying capabilities for heterogeneity detection depending on the selected methods for clustering and global feature selec- tion. For example, UMAP combined with spatial clustering did not create clusters K-Means DiviK No feature engineering Knee PCA EXIMS PCA UMAP No feature engineering Knee PCA EXIMS PCA UMAP Slice 73 Slice 67 Slice 61 Slice 55 Slice 49 Slice 43 Slice 37 Slice 31 Slice 25 Slice 19 Slice 13 Slice 7 Slice 1 24 Fig 11 Serial slices of the 3D mouse kidney data Clusters are visualised on each sixth slice Cluster colours Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Page 18 of 24 overlapping with biological structures (Dice index 0.00, Rand index 0.00, see Table 1), even though both spatial clustering and UMAP tend to exhibit the potential for cap- turing the necessary detail (see Fig. 8). One can observe similar results for the Xcep- tion-based feature engineering combined with spectral clustering. In contrast, spatial clustering with EXIMS-based global feature engineering approx- imated the tumour region with the highest Dice index and the top ROIs composition expressed via Rand index. Neural ion images with spatial clustering yield the second- highest Dice index and the third-highest Rand index. However, these are not the only criteria used by medical experts during the quality assessment. A visual comparison between clusters (Fig. 8) and pathologist-annotated ROIs (Fig. 5) shows that DiviK yields relatively stable regions regardless of the global fea- ture engineering method. Table 2 seems to support that statement, as the standard deviation of the Dice index and adjusted Rand index is the lowest of all clustering methods. Spectral clustering with the most basic features (no global feature engineer- ing or knee PCA) seems to discover similar regions. Xception-based feature engineering method resulted in much less consistent regions discovered with K-means and DiviK. Two hypotheses seem probable explana- tions for this phenomenon: • Neural ion images amplified noise. Grouping of ion images deduplicates features; however, it increases the proportional representation of the noise-related mass channels compared to the original dataset. The results of spatial clustering seem to support this hypothesis, as its embedded bilateral filter reduces noise. i • The original ion image normalisation based on winsorisation and scaling is not suffi- cient for this dataset. The contrast of the patches analysed by the Xception network is too small to generate useful embedding. Oral squamous cell carcinoma Since the approach proposed by the authors [37] formalises the visual similarity of ion maps, it could probably be useful to apply classical ion image normalisation [46] to enhance the contrast first. The comparison of global feature engineering methods is much less conclusive than the comparison of clustering methods. There does not seem to be any simple rule that could yield outstanding stability or performance against varying clustering methods. While EXIMS PCA and no global feature engineering seem to demonstrate increased tumour reconstruction capabilities (as expressed through the Dice index), at the same time, these yield a medium EXIMS score. Finally, the EXIMS score and the overall quality concept allow us to assess the trade-off between the agreement measures and the visual consistency of clusters. We defined the overall quality in two ways: • overall quality d(0, 0, 0)—it is the length of the vector in 3D space defined by the values of quality indices and the origin of the coordinate system (see Fig. 7). The better the clustering result, the higher the value. • overall quality d(1, 1, 1)—it is the length of the vector in 3D space defined by the values of quality indices and their maximal theoretical value in point (1, 1, 1). The better the clustering result, the lower the value. Page 19 of 24 Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Table 3 Average quality indices for global feature engineering methods with OSCC data Table 3 Average quality indices for global feature engineering methods with OSCC data Standard deviation is presented in brackets. Top value of a quality index among clustering methods is presented in bold italics Global feature engineering method Adjusted Rand index Dice index Relative EXIMS score Overall quality d(0, 0, 0) Overall quality d(1, 1, 1) UMAP 0.364 (0.288) 0.511 (0.371) 0.742 (0.180) 1.052 (0.190) 0.881 (0.407) Xception 0.346 (0.271) 0.487 (0.361) 0.886 (0.138) 1.139 (0.124) 0.868 (0.397) Knee PCA 0.447 (0.123) 0.538 (0.359) 0.871 (0.144) 1.167 (0.105) 0.766 (0.315) EXIMS PCA 0.585 (0.094) 0.725 (0.151) 0.703(0.090) 1.176 (0.100) 0.596 (0.119) None 0.508 (0.116) 0.703 (0.134) 0.846 (0.168) 1.227 (0.091) 0.620 (0.141) Naturally, the most consistent segmentations achieve the top EXIMS score: cases that yield a single cluster (spatial clustering with UMAP) or completely miss one of the ROIs (K-means with PCA). Oral squamous cell carcinoma Since these are not relevant from the medical point of view, we bind their relative EXIMS score at 1.0 for further consideration. The DiviK clustering without a global feature engineering technique achieved the next top EXIMS score. That renders its overall quality d(0, 0, 0) the top one. We obtained the following two best results in terms of overall quality d(0, 0, 0) using Spatial clustering with EXIMS PCA-based feature engineering and Spectral clustering with PCA. At the same time, the overall quality d(1, 1, 1) indicates Spatial clustering with EXIMS PCA- based feature engineering as the top result. Spatial clustering over Xception-gener- ated features and DiviK clustering with UMAP feature engineering yield the next two best scores. Table 1 ranks the individual configurations’ scores (tumour coverage, overall com- position and cluster consistency) for a qualitative comparison. The lower the rank, Table 4 High-level summary of clustering algorithm characteristics Contains sum of ranks for each criterion (lower is better). Top rank of a quality index across clustering methods is presented in bold italics Spectral clustering K-means Spatial clustering DiviK Rand index ranks 62.5 71 40.5 36 Dice index ranks 59 75 39 37 EXIMS score ranks 62.5 38.5 52.5 56.5 Scalability ranks 72.5 32.5 72.5 32.5 Total rank 256.5 217 204.5 162 Table 4 High-level summary of clustering algorithm characteristics Contains sum of ranks for each criterion (lower is better). Top rank of a quality index across clustering methods is presented in bold italics Table 5 High-level summary of feature engineering characteristics Contains sum of ranks for each criterion (lower is better). Top rank of a quality index per clustering method is presented in bold italics UMAP Xception Knee PCA EXIMS PCA None Rand index ranks 48.5 52.5 49 25 35 Dice index ranks 48 51 49 28 34 EXIMS score ranks 51.5 32.5 29.5 60 36.5 Scalability ranks 38 58 38 38 38 Total rank 186 194 165.5 151 143.5 Table 5 High-level summary of feature engineering characteristics Contains sum of ranks for each criterion (lower is better). Top rank of a quality index per clustering method is presented in bold italics Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Page 20 of 24 the better the result. The lowest ranks appear consecutively for Spatial clustering with EXIMS PCA-based feature engineering, Spatial clustering over neural ion images, and DiviK with no feature engineering. Oral squamous cell carcinoma Table 4 continues the ranking for clustering methods. DiviK exhibits the lowest ranks for tumour coverage and overall composi- tion, while K-means exhibits the lowest for cluster consistency. One can find DiviK with no feature engineering method scores within the top three in each of the aggregated overall quality measures. Sums of ranks ranged from 16 to 53. DiviK with no feature engineering was assigned a sum of ranks equal to 20.5, which is around 12% of the observed quality measure range. Overall quality d(0, 0, 0) ranged from 0.8122 to 1.3560, with a top value of 1.3560 for DiviK. Overall quality d(1, 1, 1) ranged from 0.4438 to 1.4142. DiviK with UMAP feature engineering was around 7% of the observed quality measure range. Similarly as for the basic quality measures, Table 2 and 3 summarize also both vari- ants of overall quality. On average, DiviK appears to be the most distant from the origin (mean d(0, 0, 0) = 1.236 ) and the closest to the theoretical maximum of qual- ity measures (mean d(1, 1, 1) = 0.576 ) as its good clustering results do not depend so much on the feature engineering techniques applied. The local subcluster-driven fea- ture space adaptation allows reducing the domain significantly by filtering the less informative and noise-level signals. The second top is the spatial clustering with mean d(0, 0, 0) = 1.192 and mean d(1, 1, 1) = 0.706 . The Cohen’s effect size in differences between these two approaches is close to medium and equals to 0.435 for d(0, 0, 0) and 0.451 for d(1, 1, 1). While the feature engineering techniques for other than DiviK methods are a concern, the first choice should be EXIMS PCA as it outperforms the other techniques tested. In the case of DiviK, no feature engineering is recommended. We conducted a qualitative evaluation of the effect of the feature engineering method and clustering algorithm on the clustering results quality. Table 6 shows the partial η2 effect size and Kendall’s W concordance coefficient on the considered qual- ity indices. The partial η2 effect size values exceed the 0.14 large effect size threshold. The choice of the clustering method influences the Dice and Rand indices more than Table 6 Qualitive evaluation of the effect of the feature engineering method and clustering algorithm. Mouse kidney 3D Published benchmark datasets [45] are oriented explicitly on high-scale computations. The risk of missing the crucial details increases significantly for such a scale. Moreover, it renders many methods useless due to their computational complexity. Therefore, addi- tional modifications are sometimes required [29], yet insufficient. ifi As shown in Fig. 10, the high-level composition of the medical sample is generally con- sistent between the configurations. The most straightforward configuration of K-means clustering with no global feature engineering yields a single cluster for the whole dataset. sistent between the configurations. The most straightforward configuration of K-means clustering with no global feature engineering yields a single cluster for the whole dataset. The structures discovered via K-means and DiviK algorithms look consistent com- pared to other work [30, 35, 47] that has analysed this data. The detected regions share a molecular signature across the 3D volume and exhibit functional differences. The structures discovered via K-means and DiviK algorithms look consistent com- pared to other work [30, 35, 47] that has analysed this data. The detected regions share a molecular signature across the 3D volume and exhibit functional differences. f Algorithms were ranked concerning their scalability limits; Table 4 sums the ranks. K-means and DiviK were the only algorithms that completed computations and were assigned the best ranks. DiviK approach does not require applying PCA or UMAP globally. The GMM-based feature filtration/selection is applied to optimise the feature space locally. Thanks to that approach, the internal structure, dominated by the main clusters, might be revealed in the subsequent divisive steps. We used PCA and UMAP-based pipelines for reference in the comparison study, and they are not a part of the proposed DiviK algorithm. We com- pleted these pipelines using a machine equipped with 48 CPU cores and 256 GB RAM, correspondingly in 23.8 min (PCA) and 198.5 min (UMAP). On the contrary, the GMM-based filtering procedure for the full mouse kidney data- set completes on average within 8.66 seconds (156 ms standard deviation). Due to the deglomerative pipeline of the DiviK algorithm and local optimisation of the feature space, the filtering procedure is repeated per each splitting step, which led in the case of the mouse kidney dataset up to 60 decompositions for different subregions of this tissue sample. It gives, on average, less than 9 min (8.66) in total. Oral squamous cell carcinoma We conducted Kruskal-Wallis test and effect size used in the table is partial η2 Furthermore, we calculated Kendall’s W concordance index Quality measure Feature engineering Clustering Quality measure Feature engineering Clustering Partial η2 Rand index 0.203 0.258 Dice index 0.161 0.293 EXIMS 0.262 0.095 Overall quality d(0, 0, 0) 0.141 0.345 Overall quality d(1, 1, 1) 0.122 0.309 Kendall’s W Concordance Index Rand index 0.328 0.325 Dice index 0.328 0.325 EXIMS 0.136 0.375 Overall quality d(0, 0, 0) 0.424 0.138 Overall quality d(1, 1, 1) 0.472 0.138 Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Page 21 of 24 Page 21 of 24 the feature engineering method. The remaining quality measure (EXIMS score) is, as expected, stronger influenced by the feature engineering method, and the clustering method is less relevant. While an overall quality is considered, choice of clustering algorithm is more important than feature engineering techniques applied. Funding g Grzegorz Mrukwa was awarded the scholarship from NCBiR Grant AIDA No. I029/17-POWR.03.02.00-IP.08-00-DOK/17. This project was financially supported by NCN Grant BITIMS No. UMO-2015/19/B/ST6/01736 for data acquisition and numeri- cal simulations. Availability of data and materials y OSCC dataset is available from Zenodo repository [48]. 3D Mouse Kidney dataset is available from GigaScience Database [49]. y OSCC dataset is available from Zenodo repository [48]. 3D Mouse Kidney dataset is available from GigaScience Database [49]. Author contributions JP and GM designed the DiviK core algorithm. GM implemented the scripts, performed the tests, and developed the package for PyPI submission. JP and GM wrote the manuscript. JP critically revised the work. All authors read and approved the final manuscript. Availability and requirements Availability and requirements Project name: DiviK; Project home page: https://github.com/gmrukwa/divik/; Operating system(s): Linux, Windows, Mac; Programming language: Python ( ≥3.6); License: Apache 2.0; Any restrictions to use by non-academics: none. Conclusionsh The comprehensive comparative study considering several aspects of data exploration demonstrated that DiviK is a tool that provides a reasonable trade-off between the accu- racy of unsupervised heterogeneity discovery and the consistency of obtained clusters. DiviK appears to be scalable and robust enough to cope with both small- and large-scale MSI data. Moreover, it is also insensitive to modifications of the preprocessing pipe- line. Compared with other configurations already proven effective for MSI, we showed that DiviK might be feasible for real-world applications when applied to solve a com- plex multi-dimensional problem. DiviK provides researchers with additional insight into which features were the most important for the differentiation of a specific region, which could be a subject of an even more comprehensive analysis, crucial for doctors to under- stand the underlying phenomena. Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 Page 22 of 24 Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 One could easily extend the DiviK framework to support other kinds of -omics data, with slight adjustments to the similarity measure and local filtering schema. The heterogeneity investigations based on epigenetic, transcriptomic, proteomic and metabolomic profiles require no modifications to the algorithm. Applying the DiviK algorithm to genomics data (GWAS-type) will require the application of a data-specific similarity measure and careful selection of the characteristic for the local feature filtering scheme. Finally, it is worth noting that the DiviK algorithm does not take advantage of any specific characteristics of the MSI data. Therefore, one could apply DiviK as a generic clustering algorithm, useful for grouping images after appropriate embedding (including medical images) or any other kind of tabular data. Abbreviations cdf Cumulative distribution function CLI Command-line interface DiviK Divisive intelligent K-means GMM Gaussian mixture modeling HDDC High-dimensional data clustering MSI Mass spectrometry imaging OSCC Oral squamous cell carcinoma PCA Principal components analysis ROI Region of interest ToF Time-of-flight UMAP Universal manifold approximation and projection Acknowledgements g We would like to thank Piotr Widłak, Monika Pietrowska, Marta Gawin and Mykola Chekan from Maria Skłodowska-Curie National Research Institute of Oncology, Gliwice branch, for providing the necessary biological and MSI data acquisition background. Finally, we would like to thank Katarzyna Frątczak for help in data preprocessing. Availability and requirements Project name: DiviK; Project home page: https://github.com/gmrukwa/divik/; Operating system(s): Linux, Windows, Mac; Programming language: Python ( ≥3.6); License: Apache 2.0; Any restrictions to use by non-academics: none. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Received: 22 December 2020 Accepted: 1 December 2022 Competing interests Page 23 of 24 Mrukwa and Polanska BMC Bioinformatics (2022) 23:538 References Postma E, van den Herik H, van der Maaten L. Dimensionality reduction: a comparative review. J Mach Learn Res. 2009;10(1–41):66–71. y 19. Postma E, van den Herik H, van der Maaten L. Dimensionality reduction: a comparative review. J Mach Learn Res. 2009;10(1–41):66–71. ; 20. Dunn JC. 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https://openalex.org/W2009611791	https://uscholar.univie.ac.at/detail/o:458806.pdf	English	null	Triparental origin of triploid onion, Allium × cornutum (Clementi ex Visiani, 1842), as evidenced by molecular, phylogenetic and cytogenetic analyses	BMC plant biology	2,014	cc-by	11,125	RESEARCH ARTICLE Open Access Fredotović et al. BMC Plant Biology 2014, 14:24 http://www.biomedcentral.com/1471-2229/14/24 Fredotović et al. BMC Plant Biology 2014, 14:24 http://www.biomedcentral.com/1471-2229/14/24 © 2014 Fredotović 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. 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. Triparental origin of triploid onion, Allium × cornutum (Clementi ex Visiani, 1842), as evidenced by molecular, phylogenetic and cytogenetic analyses Željana Fredotović1, Ivica Šamanić1, Hanna Weiss-Schneeweiss2, Juraj Kamenjarin1, Tae-Soo Jang2 and Jasna Puizina1* * Correspondence: puizina@pmfst.hr 1Department of Biology, University of Split, Faculty of Science, Teslina 12, 21000 Split, Croatia Full list of author information is available at the end of the article Abstract Background: Reconstruction of the parental origins of cultivated plants from wild relatives, especially after long periods of domestication, is not a trivial task. However, recent advances in molecular phylogenetics, among other approaches, have proved to be very informative in analyses of the origin and evolution of polyploid genomes. An established minor garden crop, triploid onion Allium × cornutum (Clementi ex Visiani, 1842) (2n = 3x = 24), is widespread in southeastern Asia and Europe. Our previous cytogenetic analyses confirmed its highly heterozygous karyotype and indicated its possible complex triparental genome origin. Allium cepa L. and Allium roylei Stearn were suggested as two putative parental species of A. × cornutum, whereas the third parental species remained hitherto unknown. Results: Here we report the phylogenetic analyses of the internal transcribed spacers ITS1-5.8S-ITS2 of 35S rDNA and the non-transcribed spacer (NTS) region of 5S rDNA of A. × cornutum and its relatives of the section Cepa. Both ITS and NTS sequence data revealed intra-individual variation in triploid onion, and these data clustered into the three main clades, each with high sequence homology to one of three other species of section Cepa: A. cepa, A. roylei, and unexpectedly, the wild Asian species Allium pskemense B. Fedtsh. Allium pskemense is therefore inferred to be the third, so far unknown, putative parental species of triploid onion Allium × cornutum. The 35S and 5S rRNA genes were found to be localised on somatic chromosomes of A. × cornutum and its putative parental species by double fluorescent in situ hybridisation (FISH). The localisation of 35S and 5S rDNA in A. × cornutum chromosomes corresponded to their respective positions in the three putative parental species, A. cepa, A. pskemense, and A. roylei. GISH (genomic in situ hybridisation) using DNA of the three putative parental diploids corroborated the results of the phylogenetic study. Conclusions: The combined molecular, phylogenetic and cytogenetic data obtained in this study provided evidence for a unique triparental origin of triploid onion A. × cornutum with three putative parental species, A. cepa, A. pskemense, and A. roylei. Keywords: Allium × cornutum, Allium cepa, Allium roylei, Allium pskemense, Triparental hybrid, Triploid, Fluorescence in situ hybridisation (FISH), ITS1-5.8S-ITS2, 5S rDNA non-transcribed spacer (NTS), Genomic in situ hybridisation (GISH) * Correspondence: puizina@pmfst.hr 1Department of Biology, University of Split, Faculty of Science, Teslina 12, 21000 Split, Croatia Full list of author information is available at the end of the article Background of inflorescences. Inflorescences of triploid onion are composed of fewer and slightly larger flowers than A. cepa inflorescences. During inflorescence maturation, small reproductive bulbils are formed, and the flowers gradually wilt. Mature inflorescence may contain 20–30 small repro- ductive bulbils. Other reliable characters that allow the distinction between triploid shallot and A. cepa are the morphological features of the underground bulbs, which are elongated and pear-shaped in triploid onions; 10–20 or more individual bulbs usually grow together. The triploid onion leaves are intermediate in shape between semicircular and round, and the stalk that bears the in- florescence is only slightly flattened at the bottom, whereas that of A. cepa is inflated at the base [17]. Polyploidy and hybridisation are regarded as important processes accompanying and contributing to plant diver- sification and speciation. Allopolyploidy, which involves both of these processes, is regarded as a particularly important driving force of plant evolution. Inferring the parental origin of allopolyploids is not a trivial task, particularly after long periods of hybrid domestication. Recent technological advances and the common use of DNA sequence data for phylogenetic reconstructions revolutionised this field and enabled the identification of the parental taxa of many allopolyploids [1]. Most of the established allopolyploids are of biparental origin. There are very few reported cases of triparental polyploids, although well-known examples include common wheat, Triticum aestivum, which is allohexaploid and of triparen- tal origin [2] and tetraploid Damask roses [3]. Most of the known allopolyploids are established on even-ploidy levels with very few consistently odd-ploidy level taxa persisting in nature (e.g., pentaploid Rosa canina) [4,5]). p The karyotype of A. × cornutum consists of 2n = 3x = 24 chromosomes [8,9]. The homology among the chromo- somes is weak and occasional, and it is very difficult, if not impossible, to identify homologous chromosomes [6]. The most common meiotic chromosome associations of A. × cornutum are heterotrivalents, which suggests at least partial homology of the three genomes [14]. Add- itionally, the frequent occurrence of complex multivalents was observed, suggesting that the triploid karyotype might be due to translocations and other chromosomal rear- rangements during evolution [14]. Mapping of the consti- tutive heterochromatin in A. × cornutum chromosomes by Giemsa C-banding demonstrated its hybrid genome structure with only one set of eight chromosomes shown to carry the heterochromatic markers typical of A. cepa [9]. Background Previously, triploid viviparous onions were speculated to be either of an allotriploid (AAB) [8] or segmental allo- triploid (AA’A”) origin [18]. Several independent molecular studies pointed to Allium cepa as one of putative parental species of A. × cornutum (RFLP analysis of the chloroplast DNA and nuclear rDNA [19-21], isozyme analysis [10], and analysis of RAPD molecular markers [12]). Triparental odd-ploidy plant allopolyploids have rarely been found. Previous analyses of established and vegeta- tively propagating triploid Allium × cornutum (Clementi ex Visiani, 1842) suggested that it is one of the rare cases of allotriploid of triparental origin, and two paren- tal taxa were suggested based on cytogenetic analyses of chromosome complements using GISH (genomic in situ hybridisation) [6]. The third parental taxon remained unknown. Triploid onion Allium × cornutum is tradition- ally cultivated in southern and coastal Croatia under the name ‘Ljutika’ (meaning ‘shallot’ in Croatian), and it is very popular as a spice and condiment due to its tasty bulbs and leaves [7]. Similar triploid onions are cultivated as garden crops in southeastern Asia, Europe, and other parts of the world [8-12]. Triploid onion was first de- scribed as Allium cepa L. var. viviparum (Metzg). Alef. [9,13,14]; however, Friesen & Klaas [11] suggested that this name is connected with the other viviparous onion A. × proliferum, and they proposed the use of the name Allium cornutum Clementi ex Visiani [15], which is the only name that is unambiguously connected with triploid onion [10,16]. However, taking into account its hybrid origin, the name was modified to Allium × cornutum [11]. This name was first used by Visiani [15] for a Dalmatian bulbiferous taxon that was observed for the first time on rocks in Dubrovnik [16]. To determine the A. × cornutum origin, genomic in situ hybridisation (GISH) was applied [6,11]. Friesen & Klaas [11] confirmed A. cepa as a parental species of the triploid onion and concluded that the majority of DNA and chromosomes of A. × cornutum originated from A. cepa. Puizina et al. [6] showed that the genomic DNA of A. cepa and A. roylei each only or predominantly labelled only one chromosome set (eight chromosomes with genomic DNA of C – A. cepa and eight with R – A. roylei). The remaining chromosomes of the triploid karyotype were not labelled (or only partially and weakly labelled) by these two genomic probes. * Correspondence: puizina@pmfst.hr 1Department of Biology, University of Split, Faculty of Science, Teslina 12, 21000 Split, Croatia Full list of author information is available at the end of the article Fredotović et al. BMC Plant Biology 2014, 14:24 http://www.biomedcentral.com/1471-2229/14/24 Fredotović et al. BMC Plant Biology 2014, 14:24 http://www.biomedcentral.com/1471-2229/14/24 Page 2 of 14 Molecular phylogenetic analysis of ITS and NTS sequences The length of the ITS1-5.8S-ITS2 region in the four analysed A. ×cornutum individuals (each representing a different population; Table 1) ranged from 627 to 642 bps. The final ITS alignment of 48 sequences (clones) of A. × cornutum had 583 constant characters; 17 were parsimony-uninformative, and 27 were parsimony- informative. In total, 19 distinct ribotypes were found with the most frequent ribotype (denoted by the GenBank accession number KC783412) represented by 24 clones originating from all four individuals (Table 1). The num- ber of variable characters was 18 in the ITS1, four in the 5.8S rRNA gene, and 22 in the ITS2 region (Additional file 1: Figure S1). The sequences of the ITS regions of A. × cornutum were aligned to other ITS sequence data for Allium spe- cies of section Cepa available in GenBank (see Table 2 for the details). Three distinct clades of ITS1-5.8S-ITS2 sequences were identified within the A. × cornutum genome (Figure 1a, Additional file 1: Figure S1). Most of the sequences (41 sequences) formed one large clade, which also included ITS sequences of the wild Asian species A. pskemense and has therefore been designated xas clade P (“pskemense”-type). A second clade, contain- ing six ITS sequences of A. × cornutum, grouped with the sequences of A. roylei and has been designated as clade R (“roylei”-type). One sequence of A. ×cornutum (from an individual from Hvar) showed similarity to A. cepa and A. vavilovii sequences, and hence, the whole clade was designated as clade C (“cepa”-type) (Figure 1a). The seven A. × cornutum ITS sequences consisting of clades R and C had a clearly distinguishable 13 base (CTGTAAA CATACT) insertion in the ITS2 region, which is shared by both A. cepa and A. roylei but absent in A. pskemense (Additional file 1: Figure S1). Another subfamily of rRNA genes encompasses 5S rDNA repeats that are arranged in long tandem arrays in one to several loci in the genome. The 5S rDNA unit consists of a coding region (gene) that is approximately 120 bp in length and a non-transcribed spacer (NTSs), which in plants varies in length and base composition from approximately 100 to more than 700 bp [45,46]. The coding regions of 5S RNA are highly conserved, whereas NTS evolves much more rapidly. Background These GISH results provided the first indication that triploid onion might be of complex triparental origin. In contrast to most flowering alliums in which the leaves start to senesce during flowering, triploid onions are perennials; their leaves remain green throughout the entire year. The plants are sterile and propagate vegeta- tively by underground bulbs and bulbils formed from the inflorescence. Phenotypically, triploid onions closely resemble A. cepa, and it is sometimes difficult to distin- guish between these two types before the development Further progress in the identification of the parental species of A. × cornutum, an ‘enigmatic plant’ [12], was hampered by the lack of information on the phylogenetic relationships between common onion and its wild relatives Fredotović et al. BMC Plant Biology 2014, 14:24 http://www.biomedcentral.com/1471-2229/14/24 Page 3 of 14 (section Cepa of genus Allium). More recently, ITS sequences (internal transcribed spacers 1 and 2 of the 18S-5.8S-26S rDNA) of a large number of common onion relatives originating from Central Asia were depos- ited in GenBank [22-27] together with the sequences of the NTS (non-transcribed spacer) of 5S rDNA [28,29]. genes have also been established in the somatic chromo- somes of A. × cornutum, and the putative parental species were inferred from phylogenetic analyses (A. pskemense, A. roylei, and A. cepa). The triparental origin of A. × cornutum was confirmed using the genomic in situ hy- bridisation (GISH) technique. The newly obtained data are discussed in light of previously published data on genome origin, structure, and evolution of the triploid onion A. × cornutum. ITS sequences have frequently been used as a first- choice marker for inferring the phylogenetic relationships between various wild plant groups and particularly for in- ferring the origins of diploid and polyploid hybrids (e.g., [30-41]). ITS 1 and 2 are parts of the 18S-5.8S-26S nuclear ribosomal DNA, which is present in each eukaryotic gen- ome in high copy number as tandem repeats in one to many loci per haploid genome [42,43]. rDNA units are prone to homogenisation via unequal crossing over and/or gene conversion [29,42]. Parental rDNA copies in hybrid organisms might evolve in various ways: (1) two parental rDNA types can potentially be retained, evolve independ- ently, and provide direct evidence for historical hybridisa- tion with or without polyploidisation (diploid homoploid hybrids vs. Background allopolyploids); (2) only one parental rDNA type might be retained in the genome of a hybrid, which typically can be achieved either by the conversion of all rDNA types towards one parental genome rDNA, or alternatively, rDNA of one parental genome might be removed from the hybrid genome; (3) hybrids might evolve new types of rDNA units that might (or might not) represent combinations of different parental rDNA units (reviewed in [44,45]). Molecular phylogenetic analysis of ITS and NTS sequences × cornutum obtained in this study (Continued) KC794515 Vis_38 KC794516 Vis_40 KC794517 Dubrava_35 KC794518 Vis_23 KC794519 Vis_31 KC794520 Hvar_15 KC794521 Hvar_22 KC794522 Hvar_13 Total: 6 6 - 7 The plant material was collected from four different locations in Croatia: Dubrava, Hvar, Kastela, and Vis. Table 1 GenBank accession numbers for the ITS1-5.8S-ITS2 and NTS-5S rRNA sequences of A. × cornutum obtained in this study (Continued) which summarises the topology and posterior probabilities (PP) from BI plus bootstrap support (BS) from the ML analysis. All the ITS sequences of A. × cornutum clustered into the three clades: P, R and C (Figure 1a). The clades P and R were both strongly supported by both analyses (clade P: 1.0 PP, 98% BS; clade R: 0.98 PP, 97% BS). The clade C containing a single A. × cornutum sequence (Hvar 21) and sequences from A. cepa, A. vavilovii and A. asarense was less strongly supported (1.0 PP; 69% BS). which summarises the topology and posterior probabilities (PP) from BI plus bootstrap support (BS) from the ML analysis. All the ITS sequences of A. × cornutum clustered into the three clades: P, R and C (Figure 1a). The clades P and R were both strongly supported by both analyses (clade P: 1.0 PP, 98% BS; clade R: 0.98 PP, 97% BS). The clade C containing a single A. × cornutum sequence (Hvar 21) and sequences from A. cepa, A. vavilovii and A. asarense was less strongly supported (1.0 PP; 69% BS). g y pp To analyse the variability of non-transcribed spacer (NTS) of the 5S rRNA genes in A. × cornutum, 19 clones were obtained from three A. × cornutum individuals (Table 1). The NTS region showed higher sequence vari- ation than the ITS1 and 2 regions. Nearly all of the cloned sequences were unique. The length of the 5S rDNA unit including the non-transcribed spacer (NTS) region ranged from 339 to 346 bps. The conserved 5S rRNA coding region was excluded from further analyses. The NTS re- gion comprised 224–231 characters, of which 148 were constant and 73 were variable (including 53 parsimony uninformative and 20 parsimony-informative characters). In total, 13 positions in the alignment included gaps (Additional file 2: Figure S2). Phylogenetic analyses of NTS clones of A. × cornutum together with NTS sequences of their potential close relatives retrieved from GenBank (Table 2) supported the grouping of the A. Molecular phylogenetic analysis of ITS and NTS sequences A high rate of base substitution of NTS in some plant groups qualifies this region as highly informative for molecular phylogen- etic analyses and often aids in the identification of parental taxa of diploid and polyploid hybrids [1,46,47]. 5S rDNA does not undergo homogenisation, and unless physically deleted from chromosomes, all types of parental repeats can be detected in hybrids [1,36,37,48]. A lack of efficient homogenisation leads to considerable sequence hetero- geneity among 5S rDNA spacer regions within the indi- vidual arrays, which has been reported in several plant groups [1,49-51]. The presence of different ITS1-5.8S-ITS2 rDNA repeat types within the A. × cornutum genome and their genetic similarity to their putative parental species A. pskemense, A. roylei, and A. cepa was confirmed by phylogenetic ana- lysis. The phylogenetic algorithms Maximum-Likelihood (ML) and Bayesian Inference (BI) resulted in nearly the same tree topology. Only the BI tree is shown (Figure 1a), In this paper, we infer the parental origin of allotriploid Allium × cornutum using molecular phylogenetic analyses of internal transcribed spacers (ITS1-5.8S-ITS2) of 35S rDNA and the non-transcribed spacer (NTS) of the 5S rDNA. The positions of these two classes of ribosomal Fredotović et al. BMC Plant Biology 2014, 14:24 http://www.biomedcentral.com/1471-2229/14/24 Page 4 of 14 which summarises the topology and posterior probabilities (PP) from BI plus bootstrap support (BS) from the ML analysis. All the ITS sequences of A. × cornutum clustered into the three clades: P, R and C (Figure 1a). The clades P and R were both strongly supported by both analyses (clade P: 1.0 PP, 98% BS; clade R: 0.98 PP, 97% BS). The clade C containing a single A. × cornutum sequence (Hvar 21) and sequences from A. cepa, A. vavilovii and A. asarense was less strongly supported (1.0 PP; 69% BS). To analyse the variability of non-transcribed spacer (NTS) of the 5S rRNA genes in A. × cornutum, 19 clones were obtained from three A. × cornutum individuals (Table 1). The NTS region showed higher sequence vari- ation than the ITS1 and 2 regions. Nearly all of the cloned sequences were unique. The length of the 5S rDNA unit including the non-transcribed spacer (NTS) region ranged from 339 to 346 bps. The conserved 5S rRNA coding region was excluded from further analyses. Molecular phylogenetic analysis of ITS and NTS sequences The NTS re- gion comprised 224–231 characters, of which 148 were constant and 73 were variable (including 53 parsimony uninformative and 20 parsimony-informative characters). In total, 13 positions in the alignment included gaps (Additional file 2: Figure S2). Phylogenetic analyses of NTS clones of A. × cornutum together with NTS sequences of their potential close relatives retrieved from GenBank (Table 2) supported the grouping of the A. × cornutum NTS sequences into the three well-supported clades (P, R and C; Figure 1b). The largest clade consisted of 11 sequences and grouped with the NTS sequences of Allium pskemense, A. altaicum and A. × proliferum (88–92% similarity), and this clade has been designated as clade P. The second largest clade (R) consisted of five NTS sequences of A. × cornutum, which grouped with A. roylei NTS regions (89% similarity). The smallest clade (C) consisted of only three NTS sequences of A. × cornutum, which were 99% identical to the NTS sequences of A. cepa and A. vavilovii. The largest clade P, comprising 11 NTS sequences of A. × cornutum, has been Table 1 GenBank accession numbers for the ITS1-5.8S-ITS2 and NTS-5S rRNA sequences of A. × cornutum obtained in this study ITS1-5.8S-ITS2 KC783412 Dubrava_8 Hvar_2 Kastela_1 Vis_5 Dubrava_15 Hvar_3 Kastela_2 Vis_10 Dubrava_16 Hvar_4 Kastela_5 Vis_11 Dubrava_18 Kastela_8 Vis_12 Dubrava_22 Kastela_11 Vis_14 Dubrava_23 Kastela_12 Vis_19 Vis_20 Vis_27 Vis_28 KC783413 Dubrava_14 KC783414 Dubrava_19 Hvar_10 KC783415 Dubrava_23 Kastela_7 KC783416 Dubrava_31 Kastela_6 KC783417 Hvar_1 KC783418 Hvar_8 KC783419 Hvar_12 KC783420 Hvar_17 KC783421 Hvar_18 KC783422 Vis_15 KC783423 Vis_16 KC783424 Vis_22 KC783425 Vis_26 KC783426 Vis_29 KC783427 Dubrava_10 KC783428 Dubrava_12 Dubrava_30 KC783429 Dubrava_13 Hvar_20 Dubrava_17 KC783430 Hvar_21 Total: 15 11 8 14 NTS-5S KC794504 Dubrava_28 KC794505 Vis_30 KC794506 Dubrava_36 KC794507 Hvar_23 KC794508 Hvar_26 KC794509 Hvar_28 KC794510 Dubrava_26 KC794511 Dubrava_38 KC794512 Vis_33 KC794513 Vis_39 KC794514 Dubrava_37 Table 1 GenBank accession numbers for the ITS1-5.8S-ITS2 and NTS-5S rRNA sequences of A. × cornutum obtained in this study (Continued) KC794515 Vis_38 KC794516 Vis_40 KC794517 Dubrava_35 KC794518 Vis_23 KC794519 Vis_31 KC794520 Hvar_15 KC794521 Hvar_22 KC794522 Hvar_13 Total: 6 6 - 7 The plant material was collected from four different locations in Croatia: Dubrava, Hvar, Kastela, and Vis. Table 1 GenBank accession numbers for the ITS1-5.8S-ITS2 and NTS-5S rRNA sequences of A. × cornutum obtained in this study 0 1 2 4 9 0 7 8 Table 1 GenBank accession numbers for the ITS1-5.8S-ITS2 and NTS-5S rRNA sequences of A. Molecular phylogenetic analysis of ITS and NTS sequences × cornutum NTS sequences into the three well-supported clades (P, R and C; Figure 1b). The largest clade consisted of 11 sequences and grouped with the NTS sequences of Allium pskemense, A. altaicum and A. × proliferum (88–92% similarity), and this clade has been designated as clade P. The second largest clade (R) consisted of five NTS sequences of A. × cornutum, which grouped with A. roylei NTS regions (89% similarity). The smallest clade (C) consisted of only three NTS sequences of A. × cornutum, which were 99% identical to the NTS sequences of A. cepa and A. vavilovii. The largest clade P, comprising 11 NTS sequences of A. × cornutum, has been Fredotović et al. BMC Plant Biology 2014, 14:24 http://www.biomedcentral.com/1471-2229/14/24 Page 5 of 14 Page 5 of 14 Table 2 List of taxa, GenBank accession numbers, and references for the previously published sequences used in this study GenBank accession number (reference) ITS1-5.8S-ITS2 NTS-5S Allium pskemense AM418380 (Gurushidze et al. 2007) [22] JF496621 (Son et al. 2012) [28] AM418382 (Gurushidze et al. 2007) JF496622 (Son et al. 2012) AJ411907 (Friesen et al. 2006) [23] Alllium roylei AJ411945 (Friesen et al. 2006) KC731587 This study AM492189 (Gurushidze et al. 2007) KC731590 This study Allium cepa FJ664287 (Hirschegger et al. 2010) [24] AB056584 (Shibata and Hizume 2002) [29] AM418367 (Gurushidze et al. 2007) AB056593 (Shibata and Hizume 2002) AM418370 (Gurushidze et al. 2007) Allium vavilovii AM418383 (Gurushidze et al. 2007) JF496618 (Son et al. 2012) JF496619 (Son et al. 2012) JF496620 (Son et al. 2012) Allium cepa var. aggregatum JF496648 (Son et al. 2012) Allium oschaninii AM418376 (Gurushidze et al. 2007) Allium praemixtum AM418379 (Gurushidze et al. 2007) Allium farctum AM492184 (Gurushidze et al. 2007) Allium asarense AM418365 (Gurushidze et al. 2007) Allium altaicum GQ412198 GQ181094 (Jang et al. unpublished) (Li et al. 2010) [25] JF496602 (Son et al. 2012) Allium fistulosum JF990845 (Guenaoui et al. 2013) [26] JF496610 (Son et al. 2012) Allium x cepiforme GU566611 (Li et al. 2010) Allium galanthum GQ181101 (Li et al. 2010) Allium x proliferum JF496645 (Son et al. 2012) Allium schoenoprasum AY427547 (Ricroch et .al. 2005) [27] AB066483 (Shibata and Hizume 2002) GQ412234 Jang et al. unpublished AB066482 (Shibata and Hizume 2002) AB066474 (Shibata and Hizume 2002) Allium maximowiczii GQ412215 Jang et al. unpublished Allium deltoidefistulosum GQ412203 Jang et al. unpublished Allium linearifolium GQ412206 Jang et al. unpublished Allium thunbergii GQ412255 Jang et al. Molecular phylogenetic analysis of ITS and NTS sequences unpublished Allium condensatum GQ412201 Jang et al. unpublished The exceptions are the sequences for the NTS-5S genes of A. roylei, which were obtained in the present study. Table 2 List of taxa, GenBank accession numbers, and references for the previously published sequences used in this study enBank accession numbers, and references for the previously published sequences used in this clearly separated from the NTS sequences of its closest relatives, A. altaicum, A. ×proliferum, and A. pskemense (Figure 1b). differed in the intensity (and likely number of repeat copies). A. pskemense possessed one subterminal locus of 35S rDNA located on one satellite chromosome pair and at most two loci of 5S rDNA on chromosome 6 (Figure 2c, d). One larger locus of 5S rDNA was located interstitially in the short arm of chromosome 6 and the other, weaker locus was situated in the pericentric region of the long arm of the same chromosome. Two homolo- gous chromosomes carrying 5S rDNA exhibited heterozy- gosity concerning the presence of a small 5S rDNA signal in the pericentromeric region of the long chromosome arm (Figure 2c,d). A. roylei possessed a single locus of 35S rDNA on a satellite-bearing chromosome pair (Figure 2e, f) and two 5S rDNA loci of similar size in the small genes and genomic in situ hybridisation The number and localisation of 5S and 35S rDNA loci were analysed in two clones of Allium × cornutum (Dubrava and Vis) and in three putative parental diploid taxa that were identified in phylogenetic analyses: Allium cepa, A. pskemense and A. roylei. Allium cepa possessed two loci of 35S rDNA in two satellite chromosome pairs and two 5S rDNA loci, both localised on the short arm of chromosome 7 (Figure 2a, b). The 35S rDNA loci Fredotović et al. BMC Plant Biology 2014, 14:24 http://www.biomedcentral.com/1471-2229/14/24 Figure 1 Phylogenetic trees resulting from a Bayesian analysis of: a) the nuclear internal transcribed spacer (ITS) of A. × cornutum and the presumptive parental species A. pskemense, A. roylei, A. cepa and other species of the section Cepa; b) the non-transcribed spacer (NTS) of the 5S rDNA sequences of A. × cornutum and the presumptive parental species A. pskemense, A. roylei, A. cepa and other species of section Cepa. The numbers above the branches depict Bayesian posterior probabilities, and the numbers below the branches indicate bootstrap support values from Maximum likelihood analysis (in the case of nodes not supported by all methods, the respective missing support values are indicated by ‘n.a.’). The bar indicates substitutions/site. Fredotović et al. BMC Plant Biology 2014, 14:24 Pag http://www.biomedcentral.com/1471-2229/14/24 Figure 1 Phylogenetic trees resulting from a Bayesian analysis of: a) the nuclear internal transcribed spacer (ITS) of A. × cornutum and the presumptive parental species A. pskemense, A. roylei, A. cepa and other species of the section Cepa; b) the non-transcribed spacer (NTS) of the 5S rDNA sequences of A. × cornutum and the presumptive parental species A. pskemense, A. roylei, A. cepa and other species of section Cepa. The numbers above the branches depict Bayesian posterior probabilities, and the numbers below the branches indicate bootstrap support values from Maximum likelihood analysis (in the case of nodes not supported by all methods, the respective missing support values are indicated by ‘n.a.’). The bar indicates substitutions/site. Page 6 of 14 Fredotović et al. BMC Plant Biology 2014, 14:24 http://www.biomedcentral.com/1471-2229/14/24 Page 7 of 14 Fredotović et al. BMC Plant Biology 2014, 14:24 Figure 2 rDNA mapping in the mitotic chromosomes of Allium × cornutum and the putative parental taxa (5S rDNA red, 35S rDNA green). (a, b) Allium cepa; (c, d) A. pskemense; (e, f) A. roylei; (g–i) A. genes and genomic in situ hybridisation × cornutum; Arrows in g, h and i indicate the smallest, barely visible 35S signal on the medium-sized metacentric chromosome originating from A. cepa. Scale bar = 10 μm. Figure 2 rDNA mapping in the mitotic chromosomes of Allium × cornutum and the putative parental taxa (5S rDNA red, 35S rDNA green). (a, b) Allium cepa; (c, d) A. pskemense; (e, f) A. roylei; (g–i) A. × cornutum; Arrows in g, h and i indicate the smallest, barely visible 35S signal on the medium-sized metacentric chromosome originating from A. cepa. Scale bar = 10 μm. The 5S rRNA genes were detected in three differently sized chromosomes in A. × cornutum (Figure 2g, h, i). The largest chromosome carrying a 5S rDNA signal re- sembled the typical A. cepa chromosome with two 5S rDNA loci localised within the long arm of chromosome 7. The medium sized chromosome carrying 5S rDNA in A. × cornutum possessed two 5S rDNA loci, which dif- fered in size and intensity, with the stronger signal posi- tioned interstitially within the short arm and the weaker signal corresponding to the pericentromeric region of the long chromosome arm. This chromosome bears resem- blance to the 5S rDNA-bearing A. pskemense chromosome. The smallest chromosome carrying 5S rDNA in triploid Al- lium possessed only one signal in the pericentromeric re- gion of the short chromosome arm (Figure 2g, h, i). This metacentric chromosome 7. One of these 5S rDNA loci was localised close to the pericentric region, and the other was localised more internally within the same arm (Figure 2e,f). In triploid A. × cornutum, two major subterminally localised signals of 35S rDNA were detected in the short arms of the two subtelocentric satellite chromosomes. A third minor 35S rDNA signal was detected on some spreads and was located in the subtelomeric region of one small sub-metacentric chromosome (white arrows in Figure 2g, h, i). The largest satellite chromosome (resembling the large NOR-bearing chromosome of A. cepa) lacked a 35S rDNA signal. Each of the three 35S rDNA signals differed in intensity and size, with the medium-sized chromosome carrying the strongest signal. Fredotović et al. BMC Plant Biology 2014, 14:24 http://www.biomedcentral.com/1471-2229/14/24 Page 8 of 14 Fredotović et al. BMC Plant Biology 2014, 14:24 Figure 3 Distribution and origin of rDNA loci in triploid A. × cornutum. (a) Chromosomes carrying 5S and 35S rDNA in A. genes and genomic in situ hybridisation × cornutum and three putative parental taxa; (b) idiogram of A. × cornutum (modified from Puizina et. al. 1999); (c) origin and localisation of the 5S and 35S rRNA genes in triploid onion. Tri-colour circles, squares and pentagons were used to label the chromosomes that carry 5S and 35S rDNA in the progenitor species and the corresponding chromosomes in the triploid hybrid A. × cornutum. Figure 3 Distribution and origin of rDNA loci in triploid A. × cornutum. (a) Chromosomes carrying 5S and 35S rDNA in A. × cornutum and three putative parental taxa; (b) idiogram of A. × cornutum (modified from Puizina et. al. 1999); (c) origin and localisation of the 5S and 35S rRNA genes in triploid onion. Tri-colour circles, squares and pentagons were used to label the chromosomes that carry 5S and 35S rDNA in the progenitor species and the corresponding chromosomes in the triploid hybrid A. × cornutum. Figure 4 Genomic in situ hybridisation (GISH); a, c) and subsequent 5S rDNA mapping (b, d) to mitotic metaphase chromosomes of Allium × cornutum. (a) GISH with genomic DNA of A. pskemense (red) and A. cepa as blocking DNA, incomplete metaphase plate; (b) 5S rDNA (green) localisation in the same chromosomal spread (c) GISH with genomic DNA of A. roylei (red) and A. cepa as blocking DNA, incomplete metaphase plate; (d) 5S rDNA (green) mapping in the same chromosomal spread. The letters C, R, and P indicate chromosomes carrying the 5S signal and belonging to the three different genomes (due to insufficient washing of the genomic probe, red subtelomeric signals (b) remained visible in majority of the chromosomes). Scale bar = 10 μm. A few of the nuclei (top left and right corners) visible in (a) were lost during reprobing and are not visible in (b). Figure 4 Genomic in situ hybridisation (GISH); a, c) and subsequent 5S rDNA mapping (b, d) to mitotic metaphase chromosomes of Allium × cornutum. (a) GISH with genomic DNA of A. pskemense (red) and A. cepa as blocking DNA, incomplete metaphase plate; (b) 5S rDNA (green) localisation in the same chromosomal spread (c) GISH with genomic DNA of A. roylei (red) and A. cepa as blocking DNA, incomplete metaphase plate; (d) 5S rDNA (green) mapping in the same chromosomal spread. genes and genomic in situ hybridisation The letters C, R, and P indicate chromosomes carrying the 5S signal and belonging to the three different genomes (due to insufficient washing of the genomic probe, red subtelomeric signals (b) remained visible in majority of the chromosomes). Scale bar = 10 μm. A few of the nuclei (top left and right corners) visible in (a) were lost during reprobing and are not visible in (b). Fredotović et al. BMC Plant Biology 2014, 14:24 http://www.biomedcentral.com/1471-2229/14/24 Page 9 of 14 Figure 5 Genomic in situ hybridisation (GISH) in Allium × cornutum; a, b) and subsequent 5S rDNA mapping (c) to mitotic metaphase chromosomes of Allium × cornutum. (a, b) GISH with genomic DNA of A. pskemense (green), A. roylei (red) and A. cepa as blocking DNA (DAPI-blue); (a) Arrows and letters (C, R, and P) indicate the putative parental origins of the three genomes (A. cepa, A. roylei and A. pskemense, respectively); (b, c) The letters C, R, and P indicate chromosomes carrying 5S (green) and 35S (red) signals that belong to the three different parental genomes. C genome The 35S rDNA-carrying chromosome could not be identified; therefore it is not indicated. One nucleus (top left corner) visible in (b) was lost during reprobing and is not visible in (c). Figure 5 Genomic in situ hybridisation (GISH) in Allium × cornutum; a, b) and subsequent 5S rDNA mapping (c) to mitotic metaphase chromosomes of Allium × cornutum. (a, b) GISH with genomic DNA of A. pskemense (green), A. roylei (red) and A. cepa as blocking DNA (DAPI-blue); (a) Arrows and letters (C, R, and P) indicate the putative parental origins of the three genomes (A. cepa, A. roylei and A. pskemense, respectively); (b, c) The letters C, R, and P indicate chromosomes carrying 5S (green) and 35S (red) signals that belong to the three different parental genomes. C genome The 35S rDNA-carrying chromosome could not be identified; therefore it is not indicated. One nucleus (top left corner) visible in (b) was lost during reprobing and is not visible in (c). chromosome might represent a truncated and/or rear- ranged chromosome 7 of A. roylei (Figure 2e, f). of A. roylei to the incomplete metaphase plate of A. × cornutum in the presence of an excess of unlabelled A. cepa total genomic DNA as blocking DNA (Figure 4c) allowed the labelling of six chromosomes of the triploid onion, which are marked by arrows. genes and genomic in situ hybridisation Among the chromo- somes labelled with A. roylei genomic DNA, one chromo- some carrying 5S rDNA is indicated (designated as R; Figure 4d). This chromosome corresponds to the smallest 5S rDNA-bearing chromosome, which is likely a truncated A. roylei-originating chromosome (Figure 3). With the aim to simultaneously discriminate between the three genomes of A. × cornutum (the putative C, R and P genomes), we performed genomic in situ hybridisation with two labelled parental DNA sequences as probes (A. pskemense genomic DNA was labelled in green; A. roylei genomic DNA was labelled in red), whereas the genomic DNA of A. cepa remained unlabelled and was used as blocking DNA The results of FISH mapping of 5S rDNA supported the inferences of the phylogenetic analyses of the 5S and 35S rDNA of triparental origin of A. × cornutum trip- loids. The three chromosomes carrying 5S rDNA genes in A. × cornutum likely originated from three different diploid Allium species (Figure 3). A combination of genomic in situ hybridisation (GISH) and FISH was attempted to identify the genomic origin of 5S rDNA-bearing chromosomes (Figures 4a–d; 5b, c). A single chromosome carrying two 5S rDNA loci (desig- nated as P) was clearly labelled with A. pskemense gen- omic DNA, in contrast to the two other chromosomes carrying the 5S rDNA signals (designated as R and C), which remained unlabelled or were only weakly labelled (Figure 4b). The hybridisation of the genomic DNA Fredotović et al. BMC Plant Biology 2014, 14:24 http://www.biomedcentral.com/1471-2229/14/24 Page 10 of 14 to the three putative parental genomes. Our previous GISH analysis [6] showed that the medium-sized NOR chromosome carrying the largest 35S rDNA locus did not hybridise either with genomic probes of A. cepa or of A. roylei, thus being assigned to the unidentified X genome. The current study showed that this locus originates from A. pskemense and was the source of the majority of the cloned ITS sequence regions (41 out of 48 ITS clones). The major 35S rDNA locus originating from the A. cepa genome has either been lost during the evolution of the triploid genome or contains only very few copies that are below the detection limit of FISH. The second (minor) A. cepa locus, which has been detected in the triploid hybrid on the medium-sized submetacentric chromosome, contains a very small copy number and might be in the process of being lost. genes and genomic in situ hybridisation The single ITS se- quence of the C-type (clone Hvar 21) that was recovered from the triploid onion genome likely represents the A. cepa minor 35S rDNA locus. (Figure 5a, b). Eight chromosomes were labelled pre- dominantly with A. pskemense genomic DNA (P-genome; green; Figure 5a); furthermore, eight chromosomes hybridised predominantly with A. roylei genomic DNA (R-genome; red), and eight chromosomes remained nearly unlabelled (C-genome, blue). The chromosomes have sub- sequently been reprobed with 35S and 5S rDNA probes. A comparison of Figure 5b and c confirms that the three different 5S-bearing chromosomes of A. × cornutum belong to three different parental genomes. (Figure 5a, b). Eight chromosomes were labelled pre- dominantly with A. pskemense genomic DNA (P-genome; green; Figure 5a); furthermore, eight chromosomes hybridised predominantly with A. roylei genomic DNA (R-genome; red), and eight chromosomes remained nearly unlabelled (C-genome, blue). The chromosomes have sub- sequently been reprobed with 35S and 5S rDNA probes. A comparison of Figure 5b and c confirms that the three different 5S-bearing chromosomes of A. × cornutum belong to three different parental genomes. NTS sequence variation and 5S rDNA mapping NTS sequence variation and 5S rDNA mapping The divergence of the NTS sequences in several well- documented allopolyploid systems proved very useful for the identification of the putative parent species: i.e., Nicotiana tabacum [48], Zingeria [53], Anemone multifida and A. baldensis [47], and Melampodium [1]. The 5S rDNA NTS sequences of A. × cornutum clustered into the three main clades (C, P, and R), which had high se- quence homology to the three putative parental species A. cepa, A. pskemense, and A. roylei, respectively. Whereas the clades C (A. cepa) and R (A. roylei) were well supported, the clade P (A. pskemense) failed to form a single well- supported clade with its closest relatives, A. pskemense, A. altaicum, and A. × proliferum. This result could have been caused by significant intra-individual variability within the NTS region [28] as well as possible high genetic variation of 5S rDNA within A. pskemense and its relatives across their wide geographical distribution. GISH allowed the detection of the three parental genomes in the hybrid, despite some level of cross- hybridisation. Using a combination of GISH and FISH, 5S and 35S rDNA-bearing chromosomes of the hybrid were shown to originate from the respective chromosomes of the putative parents. Discussion Triparental allopolyploid origin of A. × cornutum and the identification of its putative parental species Two contradictory studies have been published con- cerning the origin of the triploid onion A. × cornutum [6,11; reviewed in 12]. The suggested triparental origin of a triploid has been proposed, but only two putative parental taxa were suggested (A. cepa and A. roylei), with the third parental taxon remaining unknown (the so-called “X genome”; [6]). The present study provides phylogenetic evidence for a triparental hybrid origin of A. × cornutum, supported by the mapping of the 5S and 35S rRNA genes in the chromosomes of putative paren- tal taxa and the hybrid. Additionally, the third putative parental species, the wild Asian species A. pskemense B. Fedtsh., has been identified. These data support the complex hybrid origin of A. × cornutum and allow the rejection of previous hypotheses [11], which postulated the origin of A. × cornutum as a derivative of A. cepa. The current study clearly demonstrates that A. × cornutum contains three types of both ITS and NTS sequences, each grouping with one of three putative parental taxa, Allium pskemense, A. roylei, and A. cepa. Earlier analyses of the activity of the NOR regions in triploid onion using silver staining indicated that all three 35S rDNA loci were active with a maximum of three nucleoli detected in the interphase nuclei of all five Croatian clones of A. × cornutum [14]. In contrast, Pran, the Indian clone of triploid A. × cornutum, possessed only a single active NOR on a medium–sized satellite chromosome [14]. Such a result indicates ongoing evolu- tion of the rDNA in triploid onions over the whole species distribution range. Multiple origins of this triploid hybrid taxon are currently excluded based on the unique genome size, isozyme, RAPD and RFLP patterns of Pran, Ljutika and other analysed clones of triploid onion [6,10,11]. ITS sequence variability and 35S rDNA mapping y g The phylogenetic analysis of the ITS region of A. × cornutum revealed three major ITS types denoted as P, R, and C. These types were recovered as separate clades in combined analyses and were shown to bear high se- quence similarity to three diploid Allium species/lineages: A. pskemense, A. roylei, and A. cepa/A. vavilovii. A. cepa and A. vavilovii are closely related, with A. cepa being known only as a cultivated taxon; A. vavilovii was inferred as its closest wild relative [22]. Concordantly, three 35S rDNA loci were detected on three different chromosomes in triploid onion, in agree- ment with a previous report [52], and could be assigned The results of the NTS sequence analysis were largely congruent with the cytogenetic mapping of 5S rDNA in A. × cornutum, and 5S rDNA loci were detected in Fredotović et al. BMC Plant Biology 2014, 14:24 http://www.biomedcentral.com/1471-2229/14/24 Page 11 of 14 different positions in three chromosomes of different morphology. The two larger 5S rDNA-bearing chromo- somes greatly resembled the putative parental chromo- somes of A. cepa and A. pskemense. The 5S rDNA-bearing chromosome having 5S rDNA loci distributed in a similar manner as in A. pskemense has also been la- belled with A. pskemense genomic DNA in a GISH ex- periment. The smallest 5S rDNA-bearing chromosome of A. × cornutum has been shown to hybridise with gen- omic DNA of A. roylei ([6]; current study), thus con- firming its origin from the R genome (A. roylei). This chromosome, however, differed from the parental 5S rDNA-bearing chromosome of A. roylei and possessed only a single 5S rDNA locus in the pericentromeric re- gion of the short arm of the chromosome instead of two loci. At least two different scenarios can account for the observed truncation: i) the smallest 5S rDNA-bearing chromosome and the entire R genome could have origi- nated from a diploid ancestor closely related to A. roylei, which is characterised by smaller chromosomes and only a single 5S rDNA locus; and ii) chromosomes of A. × cor- nutum originating from the R genome have undergone re- arrangements after hybridisation and lost one of the 5S rDNA signals in the process. Based on our current under- standing of genome restructuring after allopolyploidisation and a general trend of diploidisation of both the 5S and 35S rDNA loci, the second hypothesis is more likely [43,54-57], especially because the sequences of A. Plant materials and DNA extraction Four clones of A. × cornutum (known in Croatia under the name Ljutika) were obtained from local gardens and vineyards at four well-separated localities of the Croatian seaside region (Dubrava and Kaštela) and islands (Vis, Hvar). A. pskemense B. Fedt. (CGN21442) and A. roylei Stearn (CGN20520) seeds were kindly provided by the Centre for Plant Breeding and Reproduction Research, Wageningen, The Netherlands. The commercial cultivar A. cepa cv, ‘Holland Yellow’ was used to obtain the DNA and chromosome complements of A. cepa Genomic DNA was extracted from young leaves using the CTAB method according to Saghai Maaroof et al. [62]. Conclusions The combined molecular phylogenetic and cytogenetic data obtained in this study provide evidence for a unique triparental origin of triploid onion A. × cornutum and identified all three putative diploid parental species, A. cepa, A. pskemense, and A. roylei. These results are in agreement with previously published data [6,9,14] and provide new and stronger evidence for the origin of the distinct and complex odd-ploidy allopolyploid A. × cornutum. The sequence of events leading to the origin of the triploid onion and its phylogeography cannot yet be elucidated and will be addressed using other molecular approaches. ITS sequence variability and 35S rDNA mapping × cor- nutum in clade “R” are very similar to those of A. roy- lei. The triploid genome of A. × cornutum might have been subjected to additional genomic rearrangements such as inter-chromosomal translocation, deletions, and/or inversions. possessed an identical number of 35S rDNA loci but differed in the positions of the 5S rDNA loci on chromosome 7. The localisation of 5S rDNA in A. roylei chromosome 7 resem- bles more closely that of A. cepa, whereas A. pskemense is more similar to A. fistulosum and A. altaicum. This finding supports the hypothesis of Son et al. [28] that A. pskemense is more closely related to A. fistulosum and A. altaicum. The number and position of the 5S rDNA loci, therefore, proved to be evolutionarily informative in analysing the species of Allium from section Cepa. FISH mapping of 35S and 5S rRNA genes in A. pskemense and A. roylei and inferences of the phylogenetic relationships in section Cepa Although the phylogenetic relationships among 12 species of sections of Cepa have been inferred from analyses of plastid regions and ITS [20,22], the chromosomal posi- tions of 35S and 5S rDNA have so far been determined for only three species: A. cepa, A. fistulosum and A. altaicum [58-61]. The two 5S rDNA loci in A. cepa were located on the longer arm of chromosome 7 (as confirmed by our data), whereas in A. fistulosum a single 5S rDNA locus was detected interstitially in the short arm of chromosome 7. In natural diploid homoploid hybrids of A. cepa and A. fistulosm, top onions (Allium × proliferum (Moench) Schrad. and Allium wakegi Araki (both 2n = 16), the two chromosomes carrying the 5S rDNA signals corresponded to the 5S rDNA-bearing chromosomes of the parental species [58]. In this study, 35S and 5S ribosomal genes were mapped for the first time in somatic chromosomes of diploid A. pskemense and A. roylei. These two species Additional files Additional file 1: Figure S1. Sequence variation in the nuclear internal transcribed spacer (ITS) from four different plants (clones) of A. × cornutum and its parental species, A. pskemense, A. roylei and A. cepa. Additional file 1: Figure S1. Sequence variation in the nuclear internal transcribed spacer (ITS) from four different plants (clones) of A. × cornutum and its parental species, A. pskemense, A. roylei and A. cepa. Sequence analysis The DNA sequences were assembled and prealigned using BioEdit ver. 7.0.5.3 [64]. They were then aligned in ClustalW [65] and implemented in MEGA5 [66], and the alignment was refined manually. The sequences were deposited in GenBank (Table 1). To avoid multiple submis- sions of identical sequences, we sent only one sequence of each type. To infer the phylogenetic relationships from the newly obtained ITS and NTS sequences of A. × cornutum and other closely related Allium species, the sequences were subjected to a similarity search against the non- redundant nucleotide sequence database using the NCBI (National Centre for Biotechnology Information) BLASTN network service. Sequence alignments of newly amplified regions and sequences of other related Allium species deposited in GenBank were performed using MEGA5 [66]. Polymorphic and variable sites as well as different haplotypes were generated using DnaSP Ver. 5.10 [67]. A Bayesian analysis was performed with MrBayes 3.1 [68] with 4 chains of 1,000,000 generations, trees sam- pled every 100 generations and the burn-in value set to 25% of the sampled trees. The best-fit substitution model was used as determined by the Akaike Informa- tion Criterion [69] as implemented in jModelTest 0.1.1 [70]. A maximum-likelihood analysis using starting trees obtained by neighbour-joining and TBR branch swapping with model parameters was performed using PAUP* 4.0b10 [71]. The number of bootstrap replicates was set to 1000. Phylogenetic trees were displayed in FigTree v1.3.1. p g The authors declare no competing interests. The authors declare no competing interests. Acknowledgements We sincerely thank Prof. Dr. Todd Stuessy for critically reading the manuscript and providing useful suggestions. We thank the Centre for Genetic Resources (CGN, Netherlands) for providing the specimens of A. pskemense and A. roylei used in this study. This work received funding from the Croatian Ministry of Science, Education and Sport through a grant to Jasna Puizina (no. 177-11911196-0829). The FISH method followed the procedures outlined in Weiss-Schneeweiss et al. [57,74]. Briefly, the prepara- tions were re-fixed and air dried, and chromosomal DNA was denatured in 70% (v/v) deionised formamide in 2× SSC, pH 7.0 at 70°C for 2 min, dehydrated through an ethanol series and air-dried. The hybridisation mixture PCR amplification and cloning The ITS1-5.8S-ITS2 region of 35S rDNA was amplified by PCR using the universal primers ITS1 and ITS4 and the procedures described by Bezić et al. [63]. The whole coding and non-transcribed spacer (NTS) region of the 5S rDNA gene was amplified using the primers and condi- tions from Weiss-Schneeweiss et al. [57]. The amplified products were visualised and confirmed by 1% agarose gel electrophoresis, extracted from the gel, ligated into pGEM-T Easy vectors (Promega, Madison, Wisconsin, USA) and cloned into competent JM109 E. coli cells. DNA from individual plasmids carrying inserts was iso- lated using a Plasmid Mini Kit (Qiagen, Hilden, Germany). Purified plasmid DNA was sent to Macrogen (Seoul, Korea) for sequencing of the inserts. Fredotović et al. BMC Plant Biology 2014, 14:24 http://www.biomedcentral.com/1471-2229/14/24 Page 12 of 14 containing labelled probes (100–150 ng/slide), 20–100x excess blocking DNA (for GISH) or salmon sperm DNA (for FISH), 50% formamide, 2x SSC, 10% dextran sulphate, and 0.15% sodium dodecyl sulphate (SDS) was denatured at 75°C for 10 min. The probe was applied to the slides and incubated at 37°C in a humid chamber over- night. Subsequently, the slides were washed for 5 min in 2x SSC at 39°C, for 5 min in 0.1x SSC at 39°C, and for 5 min in 2x SSC + 0.2% Tween 20 at 39°C. Biotin- and digoxygenin-labelled probes were detected using Extravidin-Cy3 (2.5 μg/mL) and anti-digoxygenin-FITC (5 μg/mL), respectively, both in 2% BSA in 2x SSC + 0.2% Tween 20 buffer at 37°C for 60 min. The slides were subsequently washed twice for 7 min in 2x SSC at 42°C and for 7 min in 2x SSC + 0.2% Tween 20 at 42°C. Finally, they were mounted in 20 μL of the antifade so- lution Vectashield containing 0.5 μg/mL DAPI (Vector Laboratories, Burlingame, CA, USA) and stored at 4°C. The slides were examined with a Zeiss Axioimager M1 epifluorescence microscope with a high-resolution micros- copy camera (Carl Zeiss AxioCam MR Rev3) using Axio Vision Rel. 4.7 software (Karl Zeiss, Vienna, Austria). For rDNA localisation, an average of 15–20 metaphase plates were analysed for each species. GISH hybridisation and detection were performed using the same protocols. Authors’ contributions h l d The experimental design was conceived by JP and HWS. The experiments were performed by ZF, IS, JK, and TY. The data were analysed by JP with assistance from HWS and IS. This paper was written by JP, HWS and ZF. All authors read and approved the final manuscript. Chromosome preparation, fluorescence in situ hybridisation (FISH) and genomic in situ hybridisation (GISH) Additional file 2: Figure S2. Sequence variation in the non- transcribed spacer (NTS) of the 5S rDNA region in three different plants (clones) of A. × cornutum. Chromosomes for FISH and GISH were prepared as described by Puizina et al. [6]. Clone pTa794 contained the complete 410-bp BamHI fragment of the 5S rRNA gene, and the spacer region of wheat [72] was used as the 5S rDNA probe. The 2.4 kb HindIII fragment of the partial 18S rRNA gene and ITS1 from Cucurbita pepo, cloned into pUC19 [73], were used as the 18S rDNA probe. The 5S rDNA probe was labelled with digoxygenin using a DIG-nick translation kit (Roche Diagnostics, Mannheim, Germany), whereas 18S rDNA was labelled with biotin using a BIO-nick translation kit (Roche Diagnostics, Mannheim, Germany). The genomic DNA (1 μg/reaction) was labelled with biotin using a BIO-nick translation kit (Roche Diagnostics, Mannheim, Germany) according to the supplier’s instructions. Additional file 1: Figure S1. 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https://openalex.org/W2159093717	https://bora.uib.no/bora-xmlui/bitstream/1956/6653/1/A9R848C.pdf	English	null	Reliability and validity of the Norwegian child and parent versions of the DISABKIDS Chronic Generic Module (DCGM-37) and Diabetes-Specific Module (DSM-10)	Health and quality of life outcomes	2,012	cc-by	8,599	* Correspondence: dag.froisland@hil.no 1Research Center for Child and Youth Competence Development, Lillehammer University College, Lillehammer, Norway Full list of author information is available at the end of the article Frøisland et al. Health and Quality of Life Outcomes 2012, 10:19 http://www.hqlo.com/content/10/1/19 Reliability and validity of the Norwegian child and parent versions of the DISABKIDS Chronic Generic Module (DCGM-37) and Diabetes-Specific Module (DSM-10) Dag Helge Frøisland1,2,12, Trond Markestad3,4, Tore Wentzel-Larsen5,6, Torild Skrivarhaug2,7,8, Knut Dahl-Jørgensen2,7,8,9 and Marit Graue10,11 RESEARCH Open Access Open Access Reliability and validity of the Norwegian child and parent versions of the DISABKIDS Chronic Generic Module (DCGM-37) and Diabetes-Specific Module (DSM-10) © 2012 Frøisland et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Correspondence: dag.froisland@hil.no 1Research Center for Child and Youth Competence Development, Lillehammer University College, Lillehammer, Norway Full list of author information is available at the end of the article © 2012 Frøisland et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: International guidelines on type 1 diabetes advocate routine screening of health-related quality of life (HRQOL). DISABKIDS questionnaires are the first instruments developed across cultures and nations to provide age-appropriate measures of HRQOL in children with chronic diseases. DISABKIDS includes a Chronic Generic Module 37 (DCGM-37) and disease-specific modules. The purpose of this study was to examine reliability and validity of the Norwegian versions of the DISABKIDS questionnaires in children and adolescents with type 1 diabetes. Methods: The DCGM-37 and the Diabetes Specific Module-10 (DDM-10) were translated into Norwegian using standard forward-backward translation. Eight to 19 year old children and adolescents with type 1 diabetes scheduled for routine follow-up at three diabetic clinics in Norway and one of their parents were invited to complete the DCGM-37 and the DDM-10. Internal consistency was determined using Cronbach’s alpha. Results were compared with those of the Child Health Questionnaire Children Form-87 (CHQ-CF87) and Child Health Questionnaire Parent Form-50 which are established generic questionnaires. DISABKIDS results were related to age, gender, duration of diabetes, mode of insulin delivery and metabolic control. Clinical data were obtained from the Norwegian Childhood Diabetes Registry. Results: Of 198 eligible child-parent dyads, 103 (52%) completed the questionnaires. Mean age was 13.6 (2.6), range 8-19 yrs, 52% were boys. Cronbach’s alpha was > 0.70 for all the DISABKIDS sub-scales except two (physical ability and social inclusion). There were moderate to high correlations (0.65-0.81) between the DISABKIDS scales and mental/emotional sub-scales of CHQ-CF87. Increasing age and higher HbA1c were significantly associated with reduced HRQOL scores. Parents tended to score their child’s HRQOL lower than the children/adolescents themselves. Conclusions: The study shows that the DISABKIDS instruments are applicable to a Norwegian childhood diabetes population. They seem to be a relevant supplement to other clinical indicators in medical practice and research. Keywords: Health-related quality of life, Type 1 diabetes, Children, Adolescents, Psychometrics, Reliability, Validity, DISABKIDS Page 2 of 11 Frøisland et al. Health and Quality of Life Outcomes 2012, 10:19 http://www.hqlo.com/content/10/1/19 Frøisland et al. Health and Quality of Life Outcomes 2012, 10:19 http://www.hqlo.com/content/10/1/19 Participants Except for families who were not able to speak or read Norwegian, all 8-19 year old children or adolescents with type 1 diabetes scheduled for follow-up at three pediatric departments in eastern Norway between Octo- ber 1st, 2009 through February 28th 2010 and one of their parents were invited by mail to participate in the study before a scheduled consultation. Whether the cohort in the present study was repre- sentative for Norwegian children and adolescents with diabetes was assessed by comparing demographic and clinical characteristics with that of the Norwegian Child- hood Diabetes Registry, which is a population based, nationwide registry covering all pediatric departments in Norway. In 2010, 95% of all children and adolescents with diabetes treated by pediatricians were included in the registry [22]. Introduction to discriminate between patients with different character- istics, i.e. age, gender, duration of disease, treatment modalities and metabolic control reflected in HbA1c. Finally, we studied whether the children and their parents assessed the child’s HRQOL differently. Type 1 diabetes is one of the most common chronic dis- eases of childhood, and the incidence in Norway of 30 new cases per 100 000 person years is one of the highest in the world [1]. Diabetes poses significant every-day challenges since optimal blood glucose control is important in order to avoid severe acute complications (i.e. hypoglycaemia and diabetes ketoacidosis) and long term consequences, such as early onset of cardiovascular disease, visual impairments, renal failure, neuropathy and premature death [2,3]. The burden of diabetes on the children and their families is well known to affect both psychological and total wellbeing [4-6], and young persons with diabetes report impaired self-perceived health-related quality of life (HRQOL) [7,8]. A good quality of life is an important treatment goal in itself [9], but is also important in order to achieve other treatment goals [10-13]. The International Society for Pediatric and Adolescent Diabetes-(ISPAD) guidelines therefore advocate assessment of quality of life as impor- tant as screening for other complications related to dia- betes [9,14]. HRQOL is a multidimensional construct including at least physical, psychological and social domains. To make international comparisons possible it is advocated that test instruments are developed in cross-cul- tural and cross-national study groups [15,16]. In line with this goal “The DISABKIDS project”, which was funded by the European Commission, was developed in seven European countries with the purpose of developing instru- ments for assessing HRQOL of children with different chronic health conditions [16,17]. The DISABKIDS instru- ments consist of questionnaires which include a generic module (DISABKIDS Chronic Generic Module (DCGM- 37)) and disease-specific modules (e.g. DISABKIDS Diabetes- Specific Module (DDM-10)) [18]. The DCGM- 37 is the only HRQOL instrument developed across cul- tures for children with chronic diseases [19]. Due to its novelty, relatively few studies using DISABKIDS have been published so far, apart from the psychometric properties reported from the European field study [17]. A literature search disclosed no recent validation studies, but Swedish and Greek groups have published results on DISABKIDS data [19-21]. Instruments DISABKIDS The DISABKIDS Chronic Generic Module (DCGM-37) is a questionnaire which measures general HRQOL and the level of distress caused by a chronic disease, and can be supplemented with condition-specific modules for asthma, arthritis, cerebral palsy, cystic fibrosis, dermati- tis, epilepsy and diabetes [18]. The instruments include one form to be filled in by children between 8 and 18 years of age, and one form for their parents. A four- week recall period is used for all items except item 11 “About symptoms” which has a one year recall in the diabetes specific module. The DCGM-37 questionnaire contains 37 items which explore six dimensions of HRQOL [16,17] (Figure 1): “Mental independence” assesses whether the child feels confident about the future and is able to live an autono- mous life without impairments caused by the condition, “Mental emotion” addresses emotional reactions, such as worries, concerns, anger and problems caused by the child’s condition, “Social exclusion” deals with the feeling of being left out and stigmatized, “Social inclusion” focuses on positive social relationships and the understanding of others, “Physical limitation” refers to somatic limitations, due to the condition and “Physical treatment” assesses the impact of taking medication, receiving injections, etc. The aims of the present study were to examine reliabil- ity and validity of the Norwegian versions of the DCGM- 37 and DDM-10 questionnaires when assessing HRQOL among children and adolescents with type 1 diabetes based on their own report and that of their parents. Internal consistency was assessed by Cronbach’s alpha coefficient. Convergent validity was assessed by compari- son with established generic instruments, in this case the Child Health Questionnaire Children Form-87 (CHQ- CF87) and Child Health Questionnaire Parent Form-50 (CHQ-PF50). We also evaluated the instruments’ ability Each item is scored on a five-point Likert scale indi- cating frequency of behaviours or feelings as 1 = never, Page 3 of 11 Frøisland et al. Health and Quality of Life Outcomes 2012, 10:19 http://www.hqlo.com/content/10/1/19 Frøisland et al. Health and Quality of Life Outcomes 2012, 10:19 http://www.hqlo.com/content/10/1/19 Frøisland et al. Health and Quality of Life Outcomes 2012, 10:19 http://www.hqlo.com/content/10/1/19 Figure 1 The structure of the DISABKIDS Chronic Generic Module-37, (DCGM-37), included rephrased, positive subscales, (in parenthesis). the DISABKIDS Chronic Generic Module-37, (DCGM-37), included rephrased, positive subscales, (in Figure 1 The structure of the DISABKIDS Chronic Generic Module-37, (DCGM-37), included rephrased, positive subscales, (in parenthesis). Instruments DISABKIDS 2 = seldom, 3 = quite often, 4 = very often, 5 = always. The scale for negatively worded items was reversed according to the manual. In computation of sum scores, missing values were substituted with the mean of non- missing items if only one item of the domain was miss- ing. If more than one item was missing the domain was not scored. The sum score of each domain is the sum of the single item scores. From the raw score a total score may be computed with a range from 0 to 100 with higher scores indicating higher self-perceived HRQOL. to the planning of treatment and the burden of carrying equipment. DDM -10 items are scored on a five-point Likert scale, and a 0-100 score is calculated for each sub- scale [23]. The DCGM-37 and DDM-10 forms were forward and backward translated from English to Norwegian according to an international scientific translations procedure [24]. The goal of this process was to keep the original meaning of the questions and simultaneously to find the most appropriate terms in the new language. The final versions were approved by the DISABKIDS research group. The diabetes specific instrument (DDM-10) consists of an “Impact” and a “Treatment” scale (Figure 2). The “Impact scale” deals with emotional reactions of blood glucose control and adhering to diets in everyday life, and the “Treatment scale” deals with emotional reactions A standard manual detailing the data collection was distributed to each of the three participating centres. In the past, presentations of HRQOL results have been criticised for being incomprehensible in relation to Page 4 of 11 Frøisland et al. Health and Quality of Life Outcomes 2012, 10:19 http://www.hqlo.com/content/10/1/19 Frøisland et al. Health and Quality of Life Outcomes 2012, 10:19 http://www.hqlo.com/content/10/1/19 Frøisland et al. Health and Quality of Life Outcomes 2012, 10:19 http://www.hqlo.com/content/10/1/19 cohorts [30-32]. A four- week recall period is used for all scales except for the “Change in Health” and “Family Cohesion” items which refer to last year, and the “General health” scale which has no recall period. Figure 2 The structure of the DISABKIDS Diabetes Module-10 (DDM-10). The questionnaires were completed at the clinic when the participants met for their follow up. As recom- mended for CHQ-87, health care personnel were avail- able to clarify questions for the age group 8-9 years, if necessary. Ethical considerations The children and adolescents and their parents gave written consent according to Norwegian requirements. The study was approved by the Regional committee on medical research ethics. Instruments DISABKIDS The child/adolescent and their parent com- pleted the questionnaire independently of each other. The completed questionnaires were scanned using Tele Form (Cardiff software, Vista, CA) and checked for scanning errors. Clinical characteristics HbA1c was analyzed at the same visit as the question- naires were filled in using Bayer DCA 2000 (Tarrytown, NY - normal reference range 3.4-6.1%). The incidence of reported ketoacidosis and hypoglycaemia was too low to allow analyses of HRQOL scores in relation to these clinical markers. Figure 2 The structure of the DISABKIDS Diabetes Module-10 (DDM-10). clinical relevance [25]. To address this critique, Osobo et al have suggested that HRQOL results will be more meaningful if negative domains were reconceptualised to positive statements [20,26]. Therefore, in the following, similar to the presentation of results from Chaplin and colleges [20]“Mental emotion” is rephrased as “Inner strength”, “Social exclusion” as “Social equality”, and “Physical limitations” as “Physical ability”. Child Health Questionnaire In additions to the DISABKIDS questionnaires, the chil- dren and adolescents were asked to complete the Child Health Questionnaire Form-87 (CHQ-CF87), and their parent the Child Health Questionnaire Parent form-50 (CHQ-PF50). The CHQ-CF87 is a generic HRQOL ques- tionnaire designed to measure physical, emotional, beha- vioural and social well being [27]. From 10 years of age children were asked to complete the CHQ-CF87 indepen- dently, while the questions could be read to younger children [28,29]. Statistical analyses l Results are presented as means with one standard devia- tion (SD) or as rates (percentages). Floor and ceiling effects are reported in numbers of patients with HRQL scores of 0 (floor) and 100 (ceiling). A percentage above 25 was characterized as high. Internal consistency refers to the degree to which the different items in a scale measure the same construct. For the DISABKIDS questionnaires reliability was assessed by tests of internal consistency of each of the subscales and the overall sum score. Cronbach’s alpha coefficients above 0.70 are generally viewed as accepta- ble when instruments are used for group level analyses [33,34]. With short scales as in DCGM-37 and DDM-10 it is often more appropriate to report mean inter item correlations. Upper and lower limits of mean inter item correlations are a matter of discussion. Some authors claim that values between 0.2 and 0.4 are optimal [35], while others argue that a mean inter item correlation consistently above 0.70, may indicate redundancy [36]. In the present article, we consider mean inter item cor- relations between 0.2 and 0.7 as satisfactory. Health is assessed over several domains i.e. general health perceptions, physical functioning, role/social- phy- sical functioning, bodily pain, role/social- emotional and behavioural functioning, parent impact-time and parent impact-emotional, self-esteem, mental health, behaviour, family activities and family cohesion. The responses are indicated on 4 to 6 point Likert scales specifying level of agreement to a certain categorical statement such as “very often” to “not at all”. The responses within each subscale are summed, and the raw scores are transformed to a score between 0 and 100, with higher scores indicat- ing better functional health and well-being. Extensive studies on the psychometric properties of the CHQ-CF87 and CHQ-PF50 suggest strong internal consistency, content validity and construct validity. Translation to Norwegian has been carried out previously, and the instruments have been used in several Norwegian patient Convergent and divergent validity of the DISABKIDS questionnaires DCGM-37 and DDM-10 were assessed with reference to the generic questionnaires CHQ- CF87 and CHQ-PF50, respectively, using Pearson correlation adjusted for age and gender. A correlation coefficient (r) Frøisland et al. Health and Quality of Life Outcomes 2012, 10:19 http://www.hqlo.com/content/10/1/19 Frøisland et al. Health and Quality of Life Outcomes 2012, 10:19 http://www.hqlo.com/content/10/1/19 Page 5 of 11 10 Cronbach’s alpha was 0.79 for both scales, and mean inter-item correlations were 0.41-0.49. Results Of 198 eligible child-parent dyads 103 (52%) completed the questionnaires. Mean age was 13.6 (2.6), range 8-19 years, and 53 (52%) were boys. Compared with the national diabetes cohort, the participants had similar gen- der distribution, mean age, mean duration of diabetes and mean body mass index (BMI), but somewhat lower mean HbA1c, lower average numbers of consultations and higher rate of insulin pump use (Table 1). Correlation coefficients between the DCGM-37 scales and the CHQ-CF87 scales “Physical function” “Role behavioral”, “Global behavior” and family related dimen- sions were low. Discriminant validity Mean scores on the children’s and adolescents’ self report forms varied between 62 and 83. Very few had floor or ceiling values (Table 2). The generic as well as the diabetes-specific module of DISABKIDS discriminated between age groups and levels of HbA1c (Table 4). Higher age and increasing HbA1c were associated with lower HRQOL scale scores. Statistical analyses l above 0.5 between measures of construct related to each other was considered as high and a coefficient between 0.3 and 0.5 as moderate convergence, while measures were not considered to be related if correlation coeffi- cients were below 0.3 [34]. For the parent’s questionnaires, Cronbach’s alpha var- ied between 0.74 and 0.89 for the DCGM-37 and was 0.83 for both scales on the DDM-10. Mean inter item correlations varied from 0.32 (“Physical ability”) to 0.55 (“Inner strengths”). DISABKIDS’ discriminant validity in relation to age, gender, duration of diabetes, mode of insulin delivery and metabolic control (i.e. levels of HbA1c) was assessed using multiple regression analysis. Convergent validity C l i ffi Paired sample t-tests were used to assess associations between scores obtained by the children and their parents. Correlation coefficients between the DCGM-37 and DDM-10 scales and the “Mental health” subscale in CHQ-CF87 were in the range 0.54-0.81 (Table 3). Cor- relation coefficients were in the range 0.65-0.81 between the DCGM-37 total score and six of the twelve sub- scales in the CHQ-CF87, and in the range 0.49-0.67 between “Role emotional” in CHQ-87 and the DCGM- 37 “Treatment” scale and the DDM-10 ("Impact” and “Treatment” scales). Significance was defined as p < 0.05. SPSS version 18.0 (SPSS IBM, NY, USA) was used for analyses. Reliability F h h Boys tended to score higher than girls, and children using insulin pump higher than those on multi-injections on total sum-scores and scores on most subscales. With respect to treatment modality, the differences in mean score between insulin pump and multi-injection users were between 3.5-4.0 on “Social equality “and “Physical treatment” subscales on DCGM-37 as well as for both subscales in the DDM-10 (results not shown). For the children’s questionnaires the internal consistency of the DCGM-37, calculated as Cronbach’s alpha, varied between 0.55 and 0.92 (Table 2.). Cronbach’s alpha was above 0.70 for all subscales except for “Physical ability” and “Social inclusion”. Mean inter item correlations were above 0.20 and below 0.50 for all subscales except for the “Physical ability” subscale which was 0.19. For the DDM- Table 1 Demographic and clinical characteristics of the children and adolescents included in the Norwegian Childhood Diabetes Registry and the study population. Norwegian Childhood Diabetes Registry (n = 2109) Study cohort (n = 103) p Number examined Number examined HbA1c (%) - mean (SD) 2048 8.65 (1.4) 101 8.04 (1.1) < 0.001 Boys - n (%) 2109 1114 (53) 102 53 (52.0) 0.92 Age(yrs)-mean (SD) 2109 12. 9 (3.8) 102 13.6 (2.6) 0.07 BMI kg/m2- mean (SD) 2081 20.4 (3.9) 100 20.8 (3.6) 0.29 Consultations last year - mean (SD) 2068 3.7 (1.7) 102 3.3 (1.3) 0.02 Diabetes duration (yrs) -mean (SD) 2109 5.2 (3.6) 102 4.6 (3.5) 0.06 Insulin pump - n (%) 2109 1073 (51) 102 74 (73) < 0.001 Table 1 Demographic and clinical characteristics of the children and adolescents included in the Norwegian Childhood Diabetes Registry and the study population. N i Childh d Di b t R i t St d h t Table 1 Demographic and clinical characteristics of the children and adolescents included in the Diabetes Registry and the study population. and clinical characteristics of the children and adolescents included in the Norwegian Childhood the study population. Frøisland et al. Health and Quality of Life Outcomes 2012, 10:19 http://www.hqlo.com/content/10/1/19 Frøisland et al. Health and Quality of Life Outcomes 2012, 10:19 http://www.hqlo.com/content/10/1/19 Page 6 of 11 Table 2 Subscale and total sum scores on the Norwegian self report version of DISABKIDS. a Pearsons’s correlation coefficient adjusted for age and gender Reliability F h h Scale n Mean score (1-100) Standard Deviation Floor/ceiling n/n “Chronbach alpha” Mean inter-item correlation Mental Mental independence 103 78 13.7 0/7 0.75 0.33 Mental Emotion (Inner strength) 103 77 15.6 0/8 0.85 0.47 Social Social Inclusion 102 80 11.5 0/3 0.60 0.22 Social exclusion (Social equality) 103 83 13 0/8 0.70 0.30 Physical Physical limitations (Physical ability) 103 77 11.4 0/5 0.55 0.19 Medication/Treatment 101 75 17.2 0/9 0.80 0.40 Total score HRQOL 100 78 16.9 0/0 0.92 0.25 Diabetes module Impact 102 70 16.9 0/6 0.79 0.41 Treatment 100 62 20.7 1/5 0.79 0.49 All subscales are scored from 0 to 100 with higher scores indicating higher self-perceived HRQOL. Table 2 Subscale and total sum scores on the Norwegian self report version of DISABKIDS. All subscales are scored from 0 to 100 with higher scores indicating higher self-perceived HRQOL. j g g p < 0.005. p < 0.01. *p < 0.001 j g g p < 0.005. p < 0.01. p < 0.001 Comparison of children’s and parents’ scores There were no differences in scores with regard to dura- tion of diabetes. Twelve (12%) of the participants had dia- betes duration of less than one year. This group scored slightly higher on all subscales as well as on the HRQOL total score, but these differences were not statistically significant. Generally, children and adolescents tended to give higher scores than their parents. This was true for all subscales in DCGM-37 and for the “Impact” scale in DDM-10. Signifi- cant mean (SD) differences were found for the DCGM-37 total score (78 ± 11.0 vs. 76 ± 11.1, p = 0.03), the subscales Table 3 Correlationsa between the DISABKIDS and the CFQ-CF87 scales in a cohort of 103 children and adolescents with type 1 diabetes CHQ-CF87 DISABKIDS DCGM-37 DDM-10 Mental Independence (Inner strength) Mental Emotion Social Excl Social Incl (Equality) Physical limitations (abilities) Treatment DCGM-37 sumscore DDM impact DDM treatment Physical function 0.21 0.15 0.21 0.21 0.06 0.20 0.22 * 0.19 0.19 Role emotional 0.30 0.39* 0.28** 0.27* 0.36 0.59* 0.81* 0.67* 0.49*** Role behavioral 0.20 0.23* 0.15* 0.11 0.32** 0.23* 0.27* 0.22* 0.19 Role physical 0.26* 0.18 0.21* 0.17 0.34*** 0.22* 0.29 0.30 0.15 Bodily pain 0.47* 0.51* 0.56* 0.49* 0.54* 0.49* 0.65* 0.38* 0.22* Behavior 0.56* 0.59* 0.45* 0.45* 0.64* 0.60* 0.70* 0.54* 0.58*** Global behavior 0.26* 0.32** 0.23* 0.315 0.32 0.19 0.34*** 0.26* 0.05 Mental health 0.69* 0.71* 0.58* 0.59* 0.71* 0.57* 0.81* 0.54* 0.55* Self-esteem 0.60* 0.58* 0.52* 0.58* 0.64* 0.50* 0.72* 0.51* 0.44* General health 0.54* 0.59* 0.54* 0.55* 0.60* 0.50* 0.70* 0.51* 0.44*** Change in Health 0.21* 0.25* 0.13 0.16 0.23* 0.07* 0.22* 0.11 0.11 Family activity 0.32* 0.33*** 0.24* 0.14 0.22* 0.34* 0.35* 0.34* 0.38* Family cohesion 0.33*** 0.26* 0.35* 0.36* 0.45* 0.31 0.43*** 0.26* 0.28** a l ff d d f d d sa between the DISABKIDS and the CFQ-CF87 scales in a cohort of 103 children and adolescents Table 3 Correlationsa between the DISABKIDS and the CFQ-CF87 scales in a cohort of 103 childre with type 1 diabetes 3 Correlationsa between the DISABKIDS and the CFQ-CF87 scales in a cohort of 103 children and 1 di b Page 7 of 11 Frøisland et al. Health and Quality of Life Outcomes 2012, 10:19 http://www.hqlo.com/content/10/1/19 Table 4 Effects of age and HbA1c on total and self reported scores in DISABKIDS. Comparison of children’s and parents’ scores Age HbA1c Subscale Unadjusted effect p Adjusted effecta* P Unadjusted effect p Adjusted effecta** p Total sum- score HRQOL -0.94 0.08 -0.94 0.04 -2.49 0.01 -2.40 0.01 Mental Mental independence -0.78 0.15 -0.87 0.13 -0.58 0.64 0.62 0.62 Mental Emotion (Inner strength) -1.58 0.01 -1.684 0.01 -2.77 0.05 -2.73 0.05 Social Social inclusion -0.75 0.11 -0.61 0.21 -2.49 0.02 -2.26 0.03 Social exclusion (Social equality) -0.57 0.29 -0.48 0.37 -2.16 0.06 -2.02 0.09 Physical Physical limitations (Physical Ability) -0.78 0.09 -0.91 0.05 -3.19 0.001 -3.39 0.001 Medication/Treatment -1.27 0.07 -0.97 0.19 -3.77 0.01 -3.37 0.03 Diabetes module Impact -1.47 0.03 -1.38 0.04 -4.43 0.002 -4.38 0.002 Treatment -3.01 0.001* -2.84 0.001 -3.44 0.06 -2.54 0.16 aAdjusted for gender, duration of diabetes and use of insulin pump vs. multi-injections using multiple linear regression analyses Effect per year of age, Effect per % increment of HbA1c. All subscales are scored from 0 to 100 with higher scores indicating higher self-perceived HRQOL. Table 4 Effects of age and HbA1c on total and self reported scores in DISABKIDS. of the DISABKIDS were compared with those obtained with a HRQOL instrument (CHQ) which is well vali- dated in Norwegian populations. Major weaknesses were the limited sample size and low participation rate. The reasons why 48% of the total population did not partici- pate were multifactorial. The main reason reported by the nurses at the participative centers was that not all families were approached during periods with large work load in the clinic. However, this happened at ran- dom and we are confident that it did not introduce a bias. “Inner strength” (77 ± 15.8 vs. 72 ± 13.1, p < 0.01) and “Social inclusion” (80 ± 11.7 vs. 74 ± 15,0 p < 0.001). In the DDM-10 “Treatment” scale parents tended to score higher than their children (66 ± 17.1 vs. 63 ± 20.4 p = 0.14). Discussion Applied to this Norwegian child and adolescent popula- tion with diabetes and their parents the internal consis- tency reliability of the DISABKIDS instruments was satisfactory for all except two scales judged by Cron- bach’s alpha. Furthermore, the scores on the DCGM-37 subscales showed moderate to high correlations with the “Mental health” subscale, but low correlations with the “Physical function” and family related subscales on the previously validated CHQ-CF87 and CHQ-PF50 ques- tionnaires. The DISABKIDS instruments discriminated between groups based on metabolic control (HbA1c) and age in that increasing HbA1c and age were associated with lower HRQOL, while no significant differences were found with respect to gender, duration of diabetes or insulin pump vs. multi-injections treatment, although there was a tendency that pump-users rated the impact of disease less and social equality higher than those on multi-injections. Parents generally scored their children’s HRQOL lower than the children and adolescents themselves. Compared to the children registered in the national dia- betes registry the study cohort had a somewhat lower mean HbA1c and higher proportion of insulin pumps users, but we still suggest that the results are representa- tive of the national cohort for the following reasons: The difference in mean HbA1c was probably too small to be of major significance, and the difference in proportion of pump users was likely due to differences in treatment tra- ditions. In Norway, pumps are, in practice, available with- out extra charge for all children, and the proportion choosing pumps is mainly a result of how familiar and confident the medical staff is with this modality. The clinics participating in the study have an active approach to encourage the use of pump, and the proportion of pump users in the study group was similar to the propor- tion among all the patients followed in the clinics. Due to the relatively small sample size we did not per- form factor analyses to assess the factor structure of the DCGM-37 or DDM-10 instruments, which is advocated when the instrument is applied to a larger population. With regard to reliability the study may be criticised for not applying test- retest reliability scores. However, this Internal consistency Most of the sub-scales showed Cronbach’s alpha coeffi- cients above 0.7, which are in agreement with the Eur- opean field study [17]. The “Physical ability” scale had a low Cronbach’s alpha compared to what was reported in the European field study where the patient populations consisted of seven different chronic conditions [17]. The “Physical ability” items in the DCGM-37 range from ques- tions about difficulties with moving and running to ques- tions on how life is ruled by the condition. Young persons with diabetes rarely experience physical complications due to the disease and, therefore, usually have no physical lim- itations. However, they experience significant practical and often emotional challenges due to repeated blood glucose measurements and administration of insulin, fear of hypo- glycaemia, ketoacidosis and long term complications on a daily basis. Therefore, a low Cronbach’s alpha was not unexpected. The Cronbach’s alpha was also low for the “Social inclusion” subscale. In line with earlier reports, we suggest that the demands of adhering to treatment may create a feeling of separation from peers [20]. Also, differ- ences in the experiences between users of pump and multi-injection treatments may create an incoherent scor- ing, explaining the lower alpha on this scale. Six of the subscales from CHQ-CF87 showed high cor- relation with DCGM -37 total score. This is as expected since these six subscales, “Role emotional”, “Bodily pain”, “Behavior”, “Mental health”, “Self esteem” and “General health” all have items that are similar to those in the DCGM-37 Questionnaire. On the other hand, the DCGM-37 does not have any sub-scales mirroring the “Change in health”, “Family activity” or “Family cohesion”. It is therefore appropriate that these scales in CHQ-CF87 showed low correlation with the subscale scores in DCGM-37. This feature may have clinical and scientific implication for children with diabetes. Other instruments than DISABKIDS may therefore be more applicable if the primary goal is to measure family related factors [37,38]. Furthermore, clinicians and researchers need to be aware that the DISABKIDS instruments seem to have their strength in measuring the mental and emotional aspects rather than detecting physical health and family related aspects of HRQOL. The different subscales of the two instruments demon- strated correlations that may seem surprisingly high or low with reference to how they are named. However, when considering the content of the respective items in the DCGM-37 and CHQ-CF87, associations were mainly as expected. Strengths and limitations The major strength of the study was that the DISAB- KIDS questionnaires were applied to a wide age-range of children and adolescents (8-19 years old). Also, a broad perspective was taken by the collection of both self-reported and parent data. Furthermore, the results Page 8 of 11 Frøisland et al. Health and Quality of Life Outcomes 2012, 10:19 http://www.hqlo.com/content/10/1/19 Frøisland et al. Health and Quality of Life Outcomes 2012, 10:19 http://www.hqlo.com/content/10/1/19 young people to connect it to “Physical ability”. Parents, however, may have interpretive abilities that cause them to answer more consistently. was not included because of concern for the patients and logistic challenges. It is suggested that the CHQ-CF87 can be read to children less than ten years of age. The medical staff, after piloting, reported that some children between 8-9 years had insufficient reading skills to respond reliably on the DISABKIDS. Use of the instruments might there- fore have a limitation in the youngest population if the questions are not read to them. Convergent validity Considering the content of the DISABKIDS question- naires, some scales were expected to correlate better than others with the CHQ-CF87 scales. The pattern of associa- tions between the subscales of the two instruments largely supported the validity of the DISABKIDS instruments. The European validation procedure used only a few of the questions from CHQ-CF87 (personal communication, John Chaplin, 2009). As far as we know a comparison with complete CHQ questionnaires has not been done earlier when examining the validity of DISABKIDS. Internal consistency For example, the names of the subscales “Physical ability” in the DCGM-37 and “Physical function” in the CHQ-CF87 give the impression that they measure similar functions. However, the “Physical function” sub- scale in CHQ-CF87 measures limitation in nine specific physical activities due to health problems, while DCGM -37’s “Physical ability” scale is constructed differently, e.g.- items like “Is your life ruled by you condition” and “Does it bother you that you have to explain to others what you can and can’t do?” measure emotional reactions to living with impairments, while the CHQ-CF87 to a larger extent addresses physical limitations. The “Physical ability” sub- scale in DCGM-37 correlated best with “Mental health” in CHQ-CF87, suggesting that the six questions measure a wider construct than physical abilities alone. In general, few items in a scale, such as six items in the DISABKIDS subscales, may make Cronbach’s alpha calcu- lations vulnerable to variations between items, and mean inter-item (MII) correlation has been suggested as an alternative analysis of consistency [35]. This method modi- fies the findings in our study, as only the “Physical ability” subscale had a MII correlation below the lower acceptable limit of 0.2 underscoring that this subscale may not be informative for young people with diabetes. Furthermore, no scales in the two questionnaires had a MII above 0.50, strongly suggesting that the items in the scales were not redundant. On the parents’ reports Cronbach’s alpha was above 0.7 on all subscales, and consistently higher than those of their children. Still, the pattern was the same as for their children in that the same subscales “Physical ability” and “Social inclusion” had the lowest Cronbach’s alpha. These findings are in accordance with the European study [24]. The heading in both the child’s and parent’s form “About your typical day” may not be explicit enough for Page 9 of 11 Frøisland et al. Health and Quality of Life Outcomes 2012, 10:19 http://www.hqlo.com/content/10/1/19 numeric differences in scores in relation to clinical importance. We believe that the statistically significant findings in our study are clinically significant as well. Generally, it has been suggested that on a scale of 0-100 a change of 5-10 points is clinically significant for an individual [34]. Internal consistency The numeric differences between those using insulin pump and multi-injections on the sub- scales “Impact” and “Treatment” on DDM-10 and on “Social equality” on the DCGM-37 may be clinically important, although they were not statistically significant in this limited study. The “Social equality” subscale con- sists of questions regarding external stigma. It has been reported that pump users have the ability to “hide” their disease better than those in need of other equipment for insulin delivery [42]. The feelings of less impact of dis- ease and smaller problems related to treatment might therefore contribute to the pump users scoring higher on this subscale. “Inner strength” (DCGM-37) correlated well with “Mental health” (CHQ-CF87) as would be expected due to the construction of single items in the two scales. The same was true for “Mental independence” in DIS- ABKIDS vs. “Mental health” and “Self esteem” subscales in CHQ-CF87. The three constructs “Mental health”, “Self esteem” and “General health” in CHQ-CF87 have 40 items covering most of the 37 item DISABKIDS questionnaire. The DCGM-37 therefore seems to be well suited for measuring the mental and emotional bur- den of a disease like diabetes. The two DDM-10 subscales “Impact” and “Treatment” and correlated well with the “Role emotional” subscale on the CHQ-CF87. The two DDM-10 subscales actually measure the emotional consequences of having a chronic illness, quite similar to what “Role emotional” measures. The “Treatment scale” in DCGM-37 and the “Role emotional” scale were similarly correlated. These findings suggest that the DISABKIDS instruments are suitable for an early detection of mental and emotional worries with possible negative influence on self-manage- ment. The fact that these instruments are available as computer programs and can be completed by the patient and automatically scored prior to consultation, make them particularly attractive in clinical practice [39]. In agreement with earlier HRQOL studies we did not exclude diabetes patients with onset of disease less than one year prior to the study [40]. Patients with diabetes less than one year had non-significantly better HRQOL scores than those with longer duration. The lack of substantial difference may partly be due to the fact that most of those with duration less than one year had duration more than 6 months. The DISABKIDS generic and diabetes specific modules showed differences between the children’s and parent’s score. Discriminant features The DISABKIDS generic instrument discriminated between patients based on age and metabolic control. It is likely that the scores on both of these variables have clini- cal significance as well [34]. Older children scored lower than the younger, a finding that is consistent with other studies on HRQOL using different instruments [31,40]. The effect of age may indicate a higher level of stress dur- ing puberty and late adolescence because of greater responsibility for own disease management and better cog- nitive ability to understand possible consequences of the disease, while parents, by taking more responsibility, tend to relieve the younger children from psychosocial burden [40]. The finding of higher HRQOL score with better meta- bolic control is also in accordance with earlier studies [41]. The fact that the DISABKIDS instruments are able to identify these differences is important, and of interest for both clinical work and further studies. Internal consistency The same findings, with parents tending to perceive their children’s HRQOL lower than the children them- selves, have been reported previously, for DISABKIDS as well as for other HRQOL instruments, and for other chronic diseases [20,43,44]. These findings may be due to changes in conceptualization of health related quality of life over the course of the disease trajectory towards better acceptance with duration of the disease [31,45]. It is also notable that the reverse was found on the DDM-10 “Impact scale”, i.e. that disease was felt to have less impact by the parents than by their children or adolescents. The reason for this finding is not obvious, but may at least partly be due to the high proportion of pump users and parents believing that treatment with pumps implies less impact of the disease. References Rubin RR, Peyrot M: Psychological issues and treatments for people with di b J l f Cl l P h l 3. The Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) Study Research Group, Nathan DM, Cleary PA, Backlund JY, Genuth SM: The effect of intensive treatment of diabetes on cardiovascular disease in patients with Type 1 diabetes. The New England Journal of Medicine 2005, 335:2643-2653. Group. European Journal of Cancer 2005, 41:280-287. 27. Mark Edward Maruish, Landgraf JM: The use of psychological testing for treatment planning and outcomes assessment 1999. g 4. Rubin RR, Peyrot M: Psychological issues and treatments for people with diabetes. Journal of Clinical Psychology 2001, 57:457-478. 28. Naar-King S, Ellis DA, Frey MA, Ondersma ML: Assessing children’s well-being, A handbook of measurers 2009. y y 5. Peyrot M, Rubin RR, Lauritzen T, Snoek FJ, Matthews DR, Skovlund SE: Psychosocial problems and barriers to improved diabetes management: results of the Cross-National Diabetes Attitudes, Wishes and Needs (DAWN) Study. Diabetic Medicine 2005, 22:1379-1385. 5. Peyrot M, Rubin RR, Lauritzen T, Snoek FJ, Matthews DR, Skovlund SE: Psychosocial problems and barriers to improved diabetes management: results of the Cross-National Diabetes Attitudes, Wishes and Needs (DAWN) Study. Diabetic Medicine 2005, 22:1379-1385. 29. Raat H, Landgraf JM, Bonsel GJ, Gemke RJBJ, Essink-Bot ML: Reliability and validity of the child health questionnaire-child form (CHQ-CF87) in a Dutch adolescent population. Quality of Life Research 2002, 11:575-581. 30. Selvaag AM, Ruperto N, Asplin L, Rygg M, Landgraf JM, Forre O, Flato B, Paediatric Rheumatology International Trials Org: The Norwegian version of the Childhood Health Assessment Questionnaire (CHAQ) and the Child Health Questionnaire (CHQ). Clinical & Experimental Rheumatology 2001, 19:Suppl-20. 6. Graue M, Wentzel-Larsen T, Hanestad BR, Sovik O: Health-related quality of life and metabolic control in adolescents with diabetes: the role of parental care, control, and involvement. Journal of Pediatric Nursing 2005, 20:373-382. 7. Varni J, Limbers C, Burwinkle T: Impaired health-related quality of life in children and adolescents with chronic conditions: a comparative analysis of 10 disease clusters and 33 disease categories/severities utilizing the PedsQLTM 4.0 Generic Core Scales. Health and Quality of Life Outcomes 2007, 5:43. 31. Graue M, Wentzel-Larsen T, Hanestad BR, Batsvik B, Sovik O: Measuring self- reported, health-related, quality of life in adolescents with type 1 diabetes using both generic and disease-specific instruments. Acta Paediatrica 2003, 92:1190-1196. 32. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. 20. Chaplin JE, Hanas R, Lind A, Tollig H, Wramner N, Lindblad B: Assessment of childhood diabetes-related quality-of-life in West Sweden. Acta Paediatrica 2009, 98:361-366. Received: 29 July 2011 Accepted: 2 February 2012 Published: 2 February 2012 Received: 29 July 2011 Accepted: 2 February 2012 Published: 2 February 2012 Received: 29 July 2011 Accepted: 2 February 2012 Published: 2 February 2012 21. Konstantinos Petsios, et al: Level of Parent-Asthmatic Child Agreement on Health-Related Quality of Life. Journal of Asthma 2011, 48:286-297. Author details 1 12. Guthrie DW, Bartsocas C, Jarosz-Chabot P, Konstantinova M: Psychosocial Issues for Children and Adolescents With Diabetes: Overview and Recommendations. Diabetes Spectr 2003, 16:7-12. 1Research Center for Child and Youth Competence Development, Lillehammer University College, Lillehammer, Norway. 2The Norwegian Childhood Diabetes Registry,Oslo University Hospital, Ullevål, Oslo, Norway. 3Department of Clinical Medicine, University of Bergen, Bergen, Norway. 4Department of Research, Innlandet Hospital Trust, Brumunddal, Norway. 5Centre for Child and Adolescent Mental Health, Eastern and Southern Norway, Oslo, Norway. 6Norwegian Centre for Violence and Traumatic Stress Studies, Oslo, Norway. 7Department of Paediatrics, Oslo University Hospital Ullevål, Oslo, Norway. 8Oslo Diabetes Research Centre, Oslo University Hospital, Oslo, Norway. 9Faculty of Medicine, University of Oslo, Oslo, Norway. 10Centre of Evidence- Based Practice, Bergen University College, Bergen, Norway. 11Department of Paediatrics, Haukeland University Hospital, Bergen, Norway. 12Pediatric Department, Innlandet Hospital Trust, Lillehammer, Norway. 13. Schiffrin A: Psychosocial issues in pediatric diabetes. [Review] [38 refs]. Current Diabetes Reports 2001, 1:33-40. 14. Cameron FJ, Northam EA, Ambler GR, Daneman D: Routine psychological screening in youth with type 1 diabetes and their parents: a notion whose time has come?[see comment]. [Review] [13335 refs]. Diabetes Care 2007, 30:2716-2724. 15. Ravens-Sieberer U, Erhart M, Wille N, Wetzel R, Nickel J, Bullinger M: Generic health-related quality-of-life assessment in children and adolescents: methodological considerations. [Review] [123 refs]. Pharmacoeconomics 2006, 24:1199-1220. 16. Petersen C, Schmidt S, Power M, Bullinger M: Development and pilot- testing of a health-related quality of life chronic generic module for children and adolescents with chronic health conditions: A European perspective. Quality of Life Research 2005, 14:1065-1077. perspective. Quality of Life Research 2005, 14:1065-1077. Conclusions The Norwegian version of the child and parent DISAB- KIDS instruments DCGM-37 and DDM-10 had accepta- ble reliability and validity in a population of children and adolescents with type 1 diabetes. DISABKIDS also discriminated between clinical characteristics that are important for disease management. We therefore sug- gest that HRQOL assessment with DISABKIDS may be of importance as a supplement to other clinical indica- tors in medical practice and research. The reason why we did not find significant differences in scores related to gender, type of treatment or duration of diabetes may be due to lack of statistical power because of limited sample size. The trends were, however, similar to what has been found as statistical significant differences in larger studies [41]. When evaluating differences in HRQOL studies, it is equally important to evaluate the statistical findings and Page 10 of 11 Frøisland et al. Health and Quality of Life Outcomes 2012, 10:19 http://www.hqlo.com/content/10/1/19 10. Delamater AM, Jacobson AM, Anderson B, Cox D, Fisher L, Lustman P, Rubin R, Wysocki T: Psychosocial therapies in diabetes - Report of the psychosocial therapies working group. Diabetes Care 2001, 24:1286-1292. Acknowledgements g The authors wish to express their gratitude to the children, teenagers and parents participating in the study, and to the diabetes nurses and doctors at The authors wish to express their gratitude to the children, teenagers and parents participating in the study, and to the diabetes nurses and doctors at the participating centers, and the employees at the Norwegian Childhood Diabetes Registry for their support. parents participating in the study, and to the diabetes nurses and doctors at the participating centers, and the employees at the Norwegian Childhood Diabetes Registry for their support 11. Grey M, Davidson M, Boland EA, Tamborlane WV: Clinical and psychosocial factors associated with achievement of treatment goals in adolescents with diabetes mellitus. Journal of Adolescent Health 2001, 28:377-385. the participating centers, and the employees at the Norwegian Childhood Diabetes Registry for their support. Authors’ contributions 17. Simeoni MC, Schmidt S, Muehlan H, Debensason D, Bullinger M, DISABKIDS Group: Field testing of a European quality of life instrument for children and adolescents with chronic conditions: the 37-item DISABKIDS Chronic Generic Module. Quality of Life Research 2007, 16:881-893. DHF designed the study, collected and analyzed the data and drafted the manuscript. TM, TW-L, MG contributed to conceptualisation and design, data analysis, interpretation of results, drafting and revising the manuscript. KD-J and TS invited DHF to research collaboration with the Norwegian Childhood Diabetes Registry, contributed to conceptualisation and design of the study, collection of data and revising the manuscript. All authors read and approved the final manuscript. 18. Bullinger M, Schmidt S, Petersen C: Assessing quality of life of children with chronic health conditions and disabilities: a European approach. International Journal of Rehabilitation Research 2002, 25:197-206. 19. Viklund G, Wikblad K: Self-perceived health and burden of diabetes in teenagers with type 1 diabetes: psychometric properties of the Swedish measure Check your health. Acta Paediatrica 2010, 99:422-426. References 22. The Norwegian Childhood Diabetes Registry: Annual report 2010 Oslo; 2011. 1. Joner G, Stene LCM, Njolstad PR, Sovik O: Marked jump in incidence of childhood onset diabetes in Norway after ten years of stable incidence: prospective nationwide study 1999-2003. Diabetologia 2005, 48:A103. 1. Joner G, Stene LCM, Njolstad PR, Sovik O: Marked jump in incidence of childhood onset diabetes in Norway after ten years of stable incidence: prospective nationwide study 1999-2003. Diabetologia 2005, 48:A103. 1. Joner G, Stene LCM, Njolstad PR, Sovik O: Marked jump in incidence of childhood onset diabetes in Norway after ten years of stable incidence: prospective nationwide study 1999-2003. Diabetologia 2005, 48:A103. 23. The DISABKIDS Group: Translation & Validation Procedure Guidelines and Documentation Form 2000. 24. Schmidt S: The DISABKIDS group. The DISABKIDS Questionnaires: Quality of life Questionnaires for children with chronic conditions. Handbook Lengerich: Pabst Science Publishers; 2006. 25. Leplège A, Sonia Hunt: The Problem of Quality of Life in Medicine. JAMA: The Journal of the American Medical Association 1997, 278:47-50. 24. Schmidt S: The DISABKIDS group. The DISABKIDS Questionnaires: Quality of life Questionnaires for children with chronic conditions. Handbook Lengerich: Pabst Science Publishers; 2006. 2. The Diabetes Control and Complications Trial Research Group: The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. The Diabetes Control and Complications Trial Research Group. N Engl J Med 1993, 329:977-986. 2. The Diabetes Control and Complications Trial Research Group: The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. The Diabetes Control and Complications Trial Research Group. N Engl J Med 1993, 329:977-986. 25. Leplège A, Sonia Hunt: The Problem of Quality of Life in Medicine. JAMA: The Journal of the American Medical Association 1997, 278:47-50. 26. Osoba D, Bezjak A, Brundage M, Zee B, Tu D, Pater J: Analysis and interpretation of health-related quality-of-life data from clinical trials: basic approach of The National Cancer Institute of Canada Clinical Trials Group. European Journal of Cancer 2005, 41:280-287. 3. The Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) Study Research Group*, Nathan DM, Cleary PA, Backlund JY, Genuth SM: The effect of intensive treatment of diabetes on cardiovascular disease in patients with Type 1 diabetes. The New England Journal of Medicine 2005, 335:2643-2653. 4. References Vederhus B, Markestad T, Eide G, Graue M, Halvorsen T: Health related quality of life after extremely preterm birth: a matched controlled cohort study. Health and Quality of Life Outcomes 2010, 8:53. 8. Cameron FJ, Northam EA: Childhood precursors of adolescent outcomes in type 1 diabetes mellitus. [Review] [108 refs]. Journal of Pediatric Endocrinology 2005, 18:223-234. 33. Crohnbach LJ: Coefficient alpha and the internal structure of tests. Psychometrika 1951, 16:297-334. 9. Delamater AM: Psychological care of children and adolescents with diabetes. Pediatric Diabetes 2007, 8:340-348. 9. Delamater AM: Psychological care of children and adolescents with diabetes. Pediatric Diabetes 2007, 8:340-348. Page 11 of 11 Page 11 of 11 Frøisland et al. Health and Quality of Life Outcomes 2012, 10:19 http://www.hqlo.com/content/10/1/19 34. Fayers PM, Machin D: Quality of Life. The assessment, analysis and interpretation of patient-reported outcomes John Wiley & Sons, Ltd; 2007, 91-116. 35. Briggs SR, Cheek JM: The Role of Factor-Analysis in the Development an E l ti f P lit S l J l f P lit 1986 54 106 148 Briggs SR, Cheek JM: The Role of Factor-Analysis in the Developme 35. Briggs SR, Cheek JM: The Role of Factor-Analysis in the Development and Evaluation of Personality-Scales. Journal of Personality 1986, 54:106-148. 36. Ponteretto JG, Ruckdeschel DE: An overview of Coefficient Aplha and a reliability matrix for estimating adequacy of internal consistency coefficients with psychological research measures. Perceptualand Motor Skills 2007, 105:997-1014. 37. Rubin RR, Young-Hyman D, Peyrot M: Parent-child responsibility and conflict in diabetes care [abstract]. Diabetes 1989, 28 A. 38. Robin AL, Koepke T, Moye A: Multidimensional assessment of parent- adolescent relations. Psychological Assessment: A Journal of Consulting and Clinical Psychology 1990, 2:451-459. y gy 39. Disabkids Computerprogram. [http://www.disabkids.de/cms/ computerprogram]. y gy 39. Disabkids Computerprogram. [http://www.disabkids.de/cms/ computerprogram]. 40. Wagner VM, Müller-Godeffroy E, von Sengbusch S, Häger S, Thyen U: Age, metabolic control and type of insulin regime influences health-related quality of life in children and adolescents with type 1 diabetes mellitus. European Journal of Pediatrics 2005, 164:491-496. 40. Wagner VM, Müller-Godeffroy E, von Sengbusch S, Häger S, Thyen U: Age, metabolic control and type of insulin regime influences health-related quality of life in children and adolescents with type 1 diabetes mellitus. European Journal of Pediatrics 2005, 164:491-496. 41. The Diabetes Control and Complications Trial Research Group: Influence of intensive diabetes treatment on quality-of-life outcomes in the diabetes control and complications trial. Diabetes Care 1996, 19:195-203. 41. The Diabetes Control and Complications Trial Research Group: Influence of intensive diabetes treatment on quality-of-life outcomes in the diabetes control and complications trial. Diabetes Care 1996, 19:195-203. 42. Brod M, Kongsoe J, Lessard S, Christensen T: Psychological insulin resistance: patient beliefs and implications for diabetes management. Quality of Life Research 2009, 18:23-32. 42. Brod M, Kongsoe J, Lessard S, Christensen T: Psychological insulin resistance: patient beliefs and implications for diabetes management. Quality of Life Research 2009, 18:23-32. y 43. Wake M, Hesketh K, Cameron F: The Child Health Questionnaire in children with diabetes: cross-sectional survey of parent and adolescent- reported functional health status. Diabetic Medicine 2000, 17:700-707. 43. doi:10.1186/1477-7525-10-19 Cite this article as: Frøisland et al.: Reliability and validity of the Norwegian child and parent versions of the DISABKIDS Chronic Generic Module (DCGM-37) and Diabetes-Specific Module (DSM-10). Health and Quality of Life Outcomes 2012 10:19. Frøisland et al. Health and Quality of Life Outcomes 2012, 10:19 http://www.hqlo.com/content/10/1/19 Wake M, Hesketh K, Cameron F: The Child Health Questionnaire in children with diabetes: cross-sectional survey of parent and adolescent- reported functional health status. Diabetic Medicine 2000, 17:700-707. p 44. Upton P, Lawford J, Eiser C: Parent-child agreement across child health- related quality of life instruments: a review of the literature. Quality of Life Research 2008, 17:895-913. 44. Upton P, Lawford J, Eiser C: Parent-child agreement across child health- related quality of life instruments: a review of the literature. Quality of Life Research 2008, 17:895-913. 45. Sprangers MAG, Schwartz CE: Integrating response shift into health- related quality of life research: a theoretical model. Social Science & Medicine 1999, 48:1507-1515. doi:10.1186/1477-7525-10-19 Cite this article as: Frøisland et al.: Reliability and validity of the Norwegian child and parent versions of the DISABKIDS Chronic Generic Module (DCGM-37) and Diabetes-Specific Module (DSM-10). Health and Quality of Life Outcomes 2012 10:19. doi:10.1186/1477-7525-10-19 Cite this article as: Frøisland et al.: Reliability and validity of the Norwegian child and parent versions of the DISABKIDS Chronic Generic Module (DCGM-37) and Diabetes-Specific Module (DSM-10). Health and Quality of Life Outcomes 2012 10:19. 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/W4224281726	https://nottingham-repository.worktribe.com/preview/8137058/1-s2.0-S0309170822000847-main.pdf	English	null	Multi-model evaluation of catchment- and global-scale hydrological model simulations of drought characteristics across eight large river catchments	Advances in water resources	2,022	cc-by	11,237	Multi-model evaluation of catchment- and global-scale hydrological model simulations of drought characteristics across eight large river catchments Jones a, Jamal Zaherpour a, Rohini Kumar b, Guoyong Leng c, Hannes Müller Schmied d,e, Jenny Kupzig f, Lutz Breuer g,h, Naota Hanasaki i, Qiuhong Tang c, Sebastian Ostberg j, Tobias Stacke k, Yadu Pokhrel l, Yoshihide Wada m, Yoshimitsu Masaki i,n Yadu Pokhrel l, Yoshihide Wada m, Yoshimitsu Masaki i,n a School of Geography, University of Nottingham, Nottingham, UK b Department of Computational Hydrosystems, Helmholtz Centre for Environmental Research – UFZ, Leipzig, Germany c Key Laboratory of Water Cycle and Related Land Surface Processes, Institute of Geographic Sciences and Natural Resources Research, Chines Beijing, China d Institute of Physical Geography, Goethe University, Frankfurt, Germany e Senckenberg Leibniz Biodiversity and Climate Research Centre (SBiK-F), Frankfurt am Main, Germany f Institute of Engineering Hydrology and Water Resources Management, Ruhr-University, Bochum, Germany g Institute for Landscape Ecology and Resources Management (ILR), Research Centre for Biosystems, Land Use and Nutrition (iFZ), Justus Lie Germany h Centre for International Development and Environmental Research (ZEU), Justus Liebig University, Giessen, Germany i National Institute for Environmental Studies, Tsukuba, Japan j Earth System Analysis, Potsdam Institute for Climate Impact Research, Potsdam, Germany k Regional Land and Atmosphere Modelling, Institute of Coastal Research, Helmholtz-Zentrum, Geesthacht, Germany l Department of Civil and Environmental Engineering, Michigan State University, Michigan, USA m International Institute for Applied Systems Analysis (IIASA), Laxenburg, Asutria n Ibaraki University, Japan a School of Geography, University of Nottingham, Nottingham, UK b Department of Computational Hydrosystems, Helmholtz Centre for Environmental Research – UFZ, Leipzig, Germany c Key Laboratory of Water Cycle and Related Land Surface Processes Institute of Geographic Sciences and Natural Res b Department of Computational Hydrosystems, Helmholtz Centre for Environmental Research – UFZ, Leipzig, Germany Key Laboratory of Water Cycle and Related Land Surface Processes, Institute of Geographic Sciences and Natural Resources Research, Chinese Aca Beijing China b Department of Computational Hydrosystems, Helmholtz Centre for Environmental Research – UFZ, Leipzig, Germany c Key Laboratory of Water Cycle and Related Land Surface Processes, Institute of Geographic Sciences and Natural Resources Resear Beijing, China c Key Laboratory of Water Cycle and Related Land Surface Processes, Institute of Geographic Sciences and Natura Beijing, China d Institute of Physical Geography, Goethe University, Frankfurt, Germany f Institute of Engineering Hydrology and Water Resources Management, Ruhr-University, Bochum, Germany g Institute for Landscape Ecology and Resources Management (ILR), Research Centre for Biosystems, Land Use and Nutrit Germany h h Centre for International Development and Environmental Research (ZEU), Justus Liebig University, Giessen, Germany i National Institute for Environmental Studies, Tsukuba, Japan f , , p j Earth System Analysis, Potsdam Institute for Climate Impact Research, Potsdam, Germany k Regional Land and Atmosphere Modelling, Institute of Coastal Research, Helmholtz-Zentrum, Geesthacht, Germany l Department of Civil and Environmental Engineering, Michigan State University, Michigan, USA m International Institute for Applied Systems Analysis (IIASA), Laxenburg, Asutria n Ibaraki University, Japan Contents lists available at ScienceDirect Contents lists available at ScienceDirect A R T I C L E I N F O Keywords: Global hydrological models Catchment hydrological models Hydrological droughts Model evaluation Model validation ISIMIP Although global- and catchment-scale hydrological models are often shown to accurately simulate long-term runoff time-series, far less is known about their suitability for capturing hydrological extremes, such as droughts. Here we evaluated simulations of hydrological droughts from nine catchment scale hydrological models (CHMs) and eight global scale hydrological models (GHMs) for eight large catchments: Upper Amazon, Lena, Upper Mississippi, Upper Niger, Rhine, Tagus, Upper Yangtze and Upper Yellow. The simulations were conducted within the framework of phase 2a of the Inter-Sectoral Impact Model Intercomparison Project (ISI MIP2a). We evaluated the ability of the CHMs, GHMs and their respective ensemble means (Ens-CHM and Ens- GHM) to simulate observed hydrological droughts of at least one month duration, over 31 years (1971–2001). Hydrological drought events were identified from runoff-deficits and the Standardised Runoff Index (SRI). In all catchments, the CHMs performed relatively better than the GHMs, for simulating monthly runoff-deficits. The number of drought events identified under different drought categories (i.e. SRI values of -1 to -1.49, -1.5 to -1.99, and ≤-2) varied significantly between models. All the models, as well as the two ensemble means, have limited abilities to accurately simulate drought events in all eight catchments, in terms of their occurrence and magnitude. Overall, there are opportunities to improve both CHMs and GHMs for better characterisation of hydrological droughts. Advances in Water Resources 165 (2022) 104212 Advances in Water Resources 165 (2022) 104212 Advances in Water Resources 165 (2022) 104212 * Corresponding author. E-mail address: amit1792kumar@gmail.com (A. Kumar). Multi-model evaluation of catchment- and global-scale hydrological model simulations of drought characteristics across eight large river catchments Amit Kumar a,*, Simon N. Gosling a, Matthew F. Johnson a, Matthew D. 1. Introduction resulting in a failure to fulfil the water demands of different natural systems and socioeconomic sectors (WMO, 1986). From 1991 to 2005, 950 million people were affected by droughts worldwide and economic A drought is an event where water availability is lower than normal, A. Kumar et al. Advances in Water Resources 165 (2022) 104212 decision-making, it is critical to understand their strengths and limita tions when specifically focusing on drought assessment and prediction. damage of 100 billion US dollars was reported UN and UNISDR, UNDP, I., (2009). Droughts are usually the consequence of a prolonged period of below normal precipitation that also affects many other environ mental, climate and socio-economic variables (Lloyd-Hughes, 2014; Van Loon, 2015). Drought can be difficult to identify in space and time, which makes it one of the most complex natural hazard (Wilhite, 1993; Wilhite et al., 2000). Researchers, managers and policy makers quantify drought events using drought indices based on climate data (reviewed in Heim, 2002; Keyantash and Dracup, 2002; Mishra and Singh, 2010). Whilst precipitation is a key input to calculate these indices, other climate and environmental variables that affect water storage and availability, are also significant. Whilst some studies have assessed the ability of multiple CHMs (Huang et al., 2017) and GHMs (Zaherpour et al., 2018) to simulate historical low flows, there has to date been no study that compares the performance of CHMs and GHMs with a focus on drought characteris tics. Thus the main novelty of this study is that it is the first cross-scale model evaluation of drought event frequency, intensity/severity and duration, together referred to as ‘droughts’ hereafter. This was achieved through the computation of observed and simulated runoff-deficits and the Standardised Runoff Index (SRI). The main objective of this study was to systematically evaluate the performance of several global scale and catchment scale hydrological models to simulate droughts. We also discuss the opportunities for improving the simulation of drought events by both GHMs and CHMs. i Droughts are complex natural disasters as their onset and magnitude are related to the interaction between many hydrological and climato logical processes. Droughts can be classified into different types namely meteorological, hydrological, agricultural and socioeconomic droughts. 2.1. Study catchments Eight large (> 65,000 km2) catchments were selected to cover diverse climate zones and hydrological systems around the globe. These were, the Upper Amazon, Lena, Upper Mississippi, Upper Niger, Rhine, Tagus, Upper Yangtze and Upper Yellow (Fig. 1). The same eight catchments used in previous GHM-CHM comparisons (Gosling et al., 2017; Hattermann et al., 2017). For the analysis, only the upper part of the Amazon, Mississippi, Niger, Yangtze and Yellow were modelled due to their complicated geomorphological structure and human alterations further downstream (Krysanova and Hattermann, 2017). Catchment boundaries were defined according to Drainage Direction Maps at 30′ (DDM30; D¨oll and Lehner, 2002) for the GHMs and CHMs, and ac cording to the Global Runoff Data Centre (GRDC; http://grdc.bafg.de) for the observed data. Studies on hydrological droughts at global or continental scales increasingly use Land Surface Models (LSMs), Global Hydrological Models (GHMs) and Catchment Scale Hydrological Models (CHMs) to quantify and predict drought events (Gosling, Zaherpour, et al., 2017; Hattermann et al., 2017). GHMs, LSMs and CHMs have been widely used to model flood hazards and risk (Arnell and Gosling 2016), climate change mitigation (Irvine et al., 2017), forecasting at shorter time scales (Emerton et al., 2016) and food security (Elliott et al., 2014). The use of these models to study droughts is also relatively common (Van Huijge voort, et al., 2013; Prudhomme et al., 2014) but there is relatively less information on the performance of the models for simulating drought. Given the societal significance of drought prediction under climate change scenarios (Pokhrel et al., 2021) using these tools, and the in fluence of results on climate change adaptation and mitigation 1. Introduction Meteorological droughts represent below normal precipitation and are mainly presented by precipitation driven indices such as the Stand ardised Precipitation Index (SPI; McKee et al., 1993), Regional Drought Area Index (RDAI; Fleig et al., 2011) and Effective drought index (EDI; Byun and Wilhite, 1999). In contrast, hydrological droughts define ef fects on freshwater storage, which are represented by indices that use stream flows, reservoir levels, groundwater levels or other similar var iables. Hydrological droughts are often closely related to meteorological droughts and can also be exacerbated by environmental changes, anthropogenic activities, and mismanagement of water resources (Tal laksen et al., 2004). 2.2. Models and input data We used CHM and GHM simulations from ISIMIP2a (Gosling et al., 2017) to identify historical drought events from their respective monthly runoff simulations, and evaluated how these droughts compared to the observed record. The simulations are the most up-to-date opportunity that exists to robustly and consistently compare Fig. 1. Location of the eight study catchments labelled as (1) Upper Amazon, (2) Lena, (3) Upper Mississippi, (4) Upper Niger, (5) Rhine, (6) Tagus, (7) Upper Yangtze, and (8) Upper Yellow. atchments labelled as (1) Upper Amazon, (2) Lena, (3) Upper Mississippi, (4) Upper Niger, (5) Rhine, (6) Tagus, (7) Upper Fig. 1. Location of the eight study catchments labelled as (1) Upper Amazon, (2) Lena, (3) Upper Mississippi, (4) Upper Niger, (5) Rhine, (6) Tagus, (7) Upper Yangtze, and (8) Upper Yellow. 2 Advances in Water Resources 165 (2022) 104212 A. Kumar et al. performance of the calibration evaluated in a separate validation period using the WATCH ERA-40 climate forcing data (Huang et al., 2017). the performance of several CHMs and GHMs. the performance of several CHMs and GHMs. Simulated runoff data from nine CHMs and eight GHMs along with corresponding observed runoff data from the GRDC, for 1971–2001, were used in this study. Observed monthly runoff data was acquired from the most downstream gauge in the GRDC catalogue for each catchment. All GHMs and CHMs were run with daily input climate data from WATCH ERA-40 (Weedon et al., 2011) for the period 1971–2001, which we aggregated to monthly temporal resolution. Output from the models is openly available from the Earth System Grid Federation (ESGF; https://esg.pik-potsdam.de/search/isimip) for the GHMs (Gosling et al., 2017) and the CHMs (Krysanova et al., 2017). All the GHMs provided outputs for all catchments; however, the number of CHMs with simulated runoff varied by catchment (Table 1). Following the method described by Haddeland et al. (2011), monthly observed and simulated runoff data was converted to catchment-mean monthly runoff by using the area upstream of the gauge according to the DDM30 river network. Thus, an area correction factor was applied to the GRDC runoff data to account for the fact that the river network, which is at 0.5◦ spatial resolution, may not perfectly overlap with the GRDC river catchment boundaries (Table 1). In addition to the individual model results, we calculated the cor responding ensemble mean (using monthly runoff values) for the GHMs (denoted Ens-GHM) and CHMs (Ens-CHM) for every catchment, and included them in the analysis (SM 1). All the calculations of runoff- deficits and SRI for Ens-GHM and Ens-CHM are based on ensemble monthly runoff values. Table 1 h Table 1 Catchments, gauging station, upstream area of gauging station, and the GHMs and CHMs that comprise ensemble (marked with x). Catchment Upper Amazon Lena Upper Mississippi Upper Niger Rhine Tagus Upper Yangtze Upper Yellow GRDC number 3,623,100 2,903,430 4,119,800 1,134,100 6,435,060 6,113,050 — — Gauging station Sao Paulo de Olivenca Stolb Alton Koulikoro Lobith Almourol Cuntan Tangnaihai Upstream drainage area (km2) - GRDC 990,781 2460,000 444,185 120,000 160,800 67,490 804,859 121,000 Upstream drainage area (km2) – DDM30 994,469 2456,513 448,575 121,058 162,092 71,007 851,303 117,543 Difference between GRDC and DDM30 areas (%) −0.4 0.1 −1.0 −0.9 −0.8 −5.2 −5.8 2.9 GHM ensemble (Ens-GHM) CLM (Oleson et al., 2010) x x x x x x x x DBH (Tang et al., 2007) x x x x x x x x H08 (Hanasaki et al., 2008) x x x x x x x x MATSIRO (Pokhrel et al., 2015) x x x x x x x x MPI-HM (Hagemann and Dümenil, 1998) x x x x x x x x PCR-GLOBWB (Wada et al., 2014) x x x x x x x x WaterGAP2 (Muller Schmied et al., 2016) x x x x x x x x LPJmL (Bondeau et al., 2007) x x x x x x x x Number of GHM simulations 8 8 8 8 8 8 8 8 CHM ensemble (Ens-CHM) ECOMAG (Motovilov et al., 1999) x HBV (Bergstrom and Forsman, 1973) x x x x x x x HYMOD (Boyle, 2001) x x x x x HYPE (Lindstr¨om et al., 2010) x x x mHM (Samaniego et al., 2010) x x x x x SWAT (Arnold et al., 1993) x x x x x SWIM (Krysanova et al., 1998) x x x x x x x VIC (Liang et al., 1994) x x x x x x x x WaterGAP3 (Verzano 2009) x x x x x x x Number of CHM simulations 7 5 7 7 8 4 4 6 Table 1 Catchments, gauging station, upstream area of gauging station, and the GHMs and CHMs that comprise ensemble (marked with x). For the purposes of this study, all the models were treated as independent even though some of the models employ similar model parameterisations for some hydrological processes. No hydrological model was excluded or weighted based on their ability to simulate runoff. 3.1. Runoff-deficit We calculated runoff-deficit values based on a variable threshold and compared the simulated runoff-deficit with observed values. The threshold was defined as the 80th exceedance percentile runoff for each month separately, a value between the 70th and 95th percentile, commonly used for perennial rivers (Hisdal et al., 2001; Andreadis et al., 2005; Fleig et al., 2006; Tallaksen et al., 2009; van Loon, 2015). A positive runoff deficit indicates dry conditions i.e. when the runoff falls below the threshold value. Both CHMs and GHMs simulate the full hydrological cycle with predominantly daily precipitation and temperature as input data. All the GHMs simulated hydrological processes at a spatial resolution of 0.5◦x 0.5◦across the global land surface. In contrast, CHMs operated using various approaches; three CHMs run on a grid (mHM, VIC, WaterGAP3), four by splitting the catchment into sub-catchments and smaller hy drological response units (HBV, HYPE, SWAT, SWIM) and one by considering the whole catchment as a single entity (HYMOD). The GHMs were not calibrated to catchment specific conditions, except WaterGAP2 (which was calibrated against long-term average annual streamflow for 1319 gauges worldwide) while the CHMs were calibrated and the 3.2. Standardised runoff index (SRI) Several standardised indices have been developed for identifying hydrological droughts, such as the Palmer Hydrological Drought Index (PDHI; Jacobi et al., 2013), Water Supply Index (WSI; Garen, 1993) and 3.3. Drought characteristics and performance evaluation R2 and NSE values for the SRI series are displayed in Table 3. Both R2 and NSE vary widely across all catchments for both CHMs and GHMs. The R2 values of the ensemble series varied between 0.18 (Ens-GHM, Upper Niger) and 0.94 (Ens-CHM, Rhine), while the NSE ranged from −0.14 (Ens-GHM, Upper Niger) to 0.95 (Ens-CHM, Rhine). For only three catchments, Ens-CHM had R2 and NSE values >0.70 and Ens-GHM for only two. SRI drought events (drought events based on SRI values) were defined as temporally continuous SRI values ˂−1 lasting until the SRI is above −1 again. Runoff-deficit drought events (drought events based on runoff-deficit values) were defined as temporally continuous positive runoff-deficit values lasting until it is zero or less. i The catchments where most of the models (both CHMs and GHMs) performed well were the Upper Mississippi and Rhine, both with R2 and NSE values for Ens-CHM and Ens-GHM >0.85. For both these catch ments, individual models always achieved R2 >0.50 and NSE values >0.40. No individual CHM or GHM achieved R2 and NSE values >0.7 for the other six catchments, with the exception of HBV for the Tagus catchment (R2 = 0.75 and NSE = 0.74). The catchment where all the models performed the worst was the Upper Niger, where no individual GHM or CHM (except SWIM) achieved an R2 value >0.35 and NSE values were always close to, or below zero. i We calculated three drought characteristics for both runoff-deficit and SRI drought events from the observed and simulated data: drought intensity (or severity in case of runoff-deficit drought events), drought duration and frequency of drought events. Run theory (Yevjevich and Ica Yevjevich, 1967) was used for the extraction of drought char acteristics from the SRI and runoff deficit time series. The Run theory method is based on a threshold level approach which considers the statistical properties of runs (here, length of negative or positive de viations in SRI or runoff-deficit values respectively) above or below a given threshold level. The drought duration (in months) was taken as the period in months for which the SRI remained less than a threshold value of −1 or runoff-deficit was greater than a threshold value of 0. 4.1. Comparison of observed and simulated runoff deficits 4.1. Comparison of observed and simulated runoff deficits 4.1. Comparison of observed and simulated runoff deficits Table 2 shows the R2 and NSE values for all the CHMs and GHMs, as well as the Ens-CHM and Ens-GHM. An R2 value >0.7 was interpreted as satisfactory model performance, as was an NSE >0.70, following Moriasi et al. (2007), (2015). For all eight catchments, the CHMs were better than GHMs for the estimation of monthly runoff-deficits because the R2 values for Ens-CHM were higher than those for Ens-GHM. Both ensem bles performed well in four catchments according to R2 because the Ens-CHM and Ens-GHM R2 values were greater than 0.7 (Moriasi et al., 2007, 2015): the Upper Mississippi, Rhine, Tagus and Upper Yellow. NSE values for Ens-CHM were greater than 0.7 for five catchments, and closer to 1 for the Rhine and Tagus catchments, while for Ens-GHM only two catchments (Upper Mississippi and Tagus) had NSE values greater than 0.7. All individual CHMs that simulated runoff for the Rhine, Tagus and Upper Yangtze catchments had R2 and NSE values >0.70 except WaterGAP3 for the Tagus catchment achieving 0.77 (R2) and 0.60 (NSE). Both the R2 and NSE values were <0.70 for all other catchments, the only exception being mHM for the Upper Mississippi and Upper Yellow catchments. The “SPEI” package in R (Beguería and Vicente-Serrano, 2017) was used for the calculations of SRI. “SPEI” i.e. Standardized Precipitation-Evapotranspiration Index package, facilitates computation of SPI and other variants of SPI (SRI in our study) by providing defined functions that can be used directly in the R working environment. Any positive SRI values indicate runoff values greater than the mean monthly runoff and vice versa. SRI values less than −1 were considered to indi cate drought conditions. These drought conditions were categorised based on SRI values into three drought categories, moderate, severe and extreme droughts. SRI values from −1 to −1.49 indicate moderate drought, from −1.5 to −1.99 severe drought, and all the SRI values less than −2 indicate extreme drought (McKee et al., 1993a; Lloyd-Hughes and Saunders, 2002; Mishra et al., 2007; Bloomfield and Marchant, 2013; Wasko et al., 2021). None of the individual CHMs or GHMs had consistently satisfactory R2 and NSE values across all catchments. The Rhine and Tagus catch ments had individual GHMs with R2 values >0.70 for over half the models but with unsatisfactory NSE values. Table 1 h Catchment Upper Amazon Lena Upper Mississippi Upper Niger Rhine Tagus Upper Yangtze Upper Yellow GRDC number 3,623,100 2,903,430 4,119,800 1,134,100 6,435,060 6,113,050 — — Gauging station Sao Paulo de Olivenca Stolb Alton Koulikoro Lobith Almourol Cuntan Tangnaihai Upstream drainage area (km2) - GRDC 990,781 2460,000 444,185 120,000 160,800 67,490 804,859 121,000 Upstream drainage area (km2) – DDM30 994,469 2456,513 448,575 121,058 162,092 71,007 851,303 117,543 Difference between GRDC and DDM30 areas (%) −0.4 0.1 −1.0 −0.9 −0.8 −5.2 −5.8 2.9 GHM ensemble (Ens-GHM) CLM (Oleson et al., 2010) x x x x x x x x DBH (Tang et al., 2007) x x x x x x x x H08 (Hanasaki et al., 2008) x x x x x x x x MATSIRO (Pokhrel et al., 2015) x x x x x x x x MPI-HM (Hagemann and Dümenil, 1998) x x x x x x x x PCR-GLOBWB (Wada et al., 2014) x x x x x x x x WaterGAP2 (Muller Schmied et al., 2016) x x x x x x x x LPJmL (Bondeau et al., 2007) x x x x x x x x Number of GHM simulations 8 8 8 8 8 8 8 8 CHM ensemble (Ens-CHM) ECOMAG (Motovilov et al., 1999) x HBV (Bergstrom and Forsman, 1973) x x x x x x x HYMOD (Boyle, 2001) x x x x x HYPE (Lindstr¨om et al., 2010) x x x mHM (Samaniego et al., 2010) x x x x x SWAT (Arnold et al., 1993) x x x x x SWIM (Krysanova et al., 1998) x x x x x x x VIC (Liang et al., 1994) x x x x x x x x WaterGAP3 (Verzano 2009) x x x x x x x Number of CHM simulations 7 5 7 7 8 4 4 6 area of gauging station, and the GHMs and CHMs that comprise ensemble (marked with x). ging station, upstream area of gauging station, and the GHMs and CHMs that comprise ensemble (marked with x). 3 Advances in Water Resources 165 (2022) 104212 A. Kumar et al. 3.3. Drought characteristics and performance evaluation The minimum SRI value in the drought period was used as the drought in tensity for SRI drought events, while the collective sum of all positive runoff-deficits over the drought period was taken as drought severity for runoff-deficit drought events. i 4.2. Comparison of observed and simulated SRI 4.1. Comparison of observed and simulated runoff deficits In addition, the Upper Amazon, Lena and Upper Niger catchments had no individual GHMs with R2 or NSE values >0.70. Individual GHMs showed good perfor mance in the Tagus catchment, where both indicators were >0.70. 4. Results Standardised Streamflow/Runoff Index (SSI/SRI; Vicente-Serrano et al., 2012). In comparison with PHDI and WSI, SSI is more commonly accepted because it is simple to calculate, can be used on various time scales and requires fewer inputs. SSI is extensively used in many studies (Shukla and Wood, 2008; Vicente-Serrano et al., 2012; Wu et al., 2018; Liu et al., 2019). Calculation of SSI/SRI is similar to that of Standardised Precipitation Index (SPI) proposed by McKee et al. (1993b) but considering runoff instead of precipitation. The SRI values are deter mined based on long-term runoff records (preferably >30 years) by aggregating the monthly runoff over an accumulation period (1, 3, 6, 12, or 24 months). The new series formed after accumulation (1 month for this study) is then fitted to a probability distribution that subsequently is transformed to a normal distribution such that the mean SRI is zero. We tested three different probability distributions, Gamma, Normal and Weibull, to see which had the best fit to observed runoff. The Gamma distribution was the best fit for all eight catchments (SM 2) and was used for all the SRI calculations. Table 2 Upper Amazon Lena Upper Mississippi Upper Niger Rhine Tagus Upper Yangtze Upper Yellow GHMs CLM 0.36 (−0.13) 0.05 (−0.04) 0.58 (0.09) 0.49 (−1.33) 0.47 (−1.13) 0.59 (0.53) 0.19 (0.08) 0.42 (0.40) DBH 0.38 (0.32) 0.07 (−0.16) 0.69 (0.03) 0.41 (−2.37) 0.73 (0.01) 0.82 (−0.08) 0.54 (0.5) 0.64 (0.62) H08 0.37 (0.20) 0.01 (−0.17) 0.60 (−0.01) 0.40 (−3.67) 0.64 (−0.47) 0.85 (−1.26) 0.44 (0.38) 0.45 (0.39) MATSIRO 0.30 (0.24) 0.31 (0.26) 0.69 (0.49) 0.63 (−0.87) 0.55 (0.51) 0.85 (0.79) 0.58 (0.56) 0.58 (0.35) MPI-HM 0.36 (0.32) 0.10 (−0.05) 0.74 (0.50) 0.56 (−0.08) 0.67 (0.36) 0.76 (0.55) 0.39 (0.37) 0.50 (0.42) PCR-GLOBWB 0.38 (0.34) 0.41 (0.36) 0.74 (0.67) 0.31 (0.15) 0.81 (0.67) 0.88 (0.87) 0.77 (0.70) 0.70 (0.70) WaterGAP2 0.46 (0.39) 0.39 (0.35) 0.75 (0.70) 0.44 (0.42) 0.86 (0.86) 0.86 (0.85) 0.62 (0.61) 0.65 (0.56) LPJmL 0.37 (0.23) 0.23 (−1.73) 0.45 (−0.33) 0.41 (−4.05) 0.84 (0.12) 0.86 (−0.89) 0.66 (0.66) 0.54 (0.53) CHMs ECOMAG x 0.67 (0.58) x x x x x x HBV 0.52 (0.51) x 0.65 (0.63) 0.55 (0.41) 0.86 (0.86) 0.94 (0.88) 0.80 (0.79) 0.69 (0.67) HYMOD 0.43 (0.39) x 0.54 (0.48) 0.56 (0.46) 0.86 (0.84) x x 0.61 (0.59) HYPE x 0.72 (0.62) x x 0.91 (0.88) 0.94 (0.92) x x mHM 0.50 (0.48) x 0.85 (0.83) 0.54 (0.51) 0.90 (0.90) x x 0.76 (0.73) SWAT 0.49 (0.43) x 0.77 (0.68) 0.49 (0.47) 0.94 (0.92) x 0.79 (0.78) x SWIM 0.55 (0.51) 0.58 (0.47) 0.70 (0.65) 0.67 (0.59) 0.89 (0.89) x 0.79 (0.72) 0.71 (0.68) VIC 0.49 (0.45) 0.53 (0.52) 0.65 (0.56) 0.59 (0.49) 0.91 (0.90) 0.91 (0.87) 0.83 (0.82) 0.59 (0.46) WaterGAP3 0.17 (−0.35) 0.37 (0.31) 0.67 (0.65) 0.35 (0.22) 0.85 (0.85) 0.77 (0.60) x 0.59 (0.56) Ens-GHM 0.49 (0.47) 0.37 (0.30) 0.82 (0.80) 0.53 (−0.35) 0.82 (0.65) 0.9 (0.71) 0.63 (0.62) 0.72 (0.62) Ens-CHM 0.50 (0.48) 0.73 (0.73) 0.83 (0.82) 0.61 (0.59) 0.96 (0.95) 0.96 (0.95) 0.85 (0.85) 0.73 (0.66) differing by 6 or less runoff deficit drought events and only by 2 or less and observed drought intensity or severity There are marked differences Upper Amazon Lena Upper Mississippi Upper Niger Rhine Tagus Upper Yangtze Upper Yellow GHMs CLM 0.36 (−0.13) 0.05 (−0.04) 0.58 (0.09) 0.49 (−1.33) 0.47 (−1.13) 0.59 (0.53) 0.19 (0.08) 0.42 (0.40) DBH 0.38 (0.32) 0.07 (−0.16) 0.69 (0.03) 0.41 (−2.37) 0.73 (0.01) 0.82 (−0.08) 0.54 (0.5) 0.64 (0.62) H08 0.37 (0.20) 0.01 (−0.17) 0.60 (−0.01) 0.40 (−3.67) 0.64 (−0.47) 0.85 (−1.26) 0.44 (0.38) 0.45 (0.39) MATSIRO 0.30 (0.24) 0.31 (0.26) 0.69 (0.49) 0.63 (−0.87) 0.55 (0.51) 0.85 (0.79) 0.58 (0.56) 0.58 (0.35) MPI-HM 0.36 (0.32) 0.10 (−0.05) 0.74 (0.50) 0.56 (−0.08) 0.67 (0.36) 0.76 (0.55) 0.39 (0.37) 0.50 (0.42) PCR-GLOBWB 0.38 (0.34) 0.41 (0.36) 0.74 (0.67) 0.31 (0.15) 0.81 (0.67) 0.88 (0.87) 0.77 (0.70) 0.70 (0.70) WaterGAP2 0.46 (0.39) 0.39 (0.35) 0.75 (0.70) 0.44 (0.42) 0.86 (0.86) 0.86 (0.85) 0.62 (0.61) 0.65 (0.56) LPJmL 0.37 (0.23) 0.23 (−1.73) 0.45 (−0.33) 0.41 (−4.05) 0.84 (0.12) 0.86 (−0.89) 0.66 (0.66) 0.54 (0.53) CHMs ECOMAG x 0.67 (0.58) x x x x x x HBV 0.52 (0.51) x 0.65 (0.63) 0.55 (0.41) 0.86 (0.86) 0.94 (0.88) 0.80 (0.79) 0.69 (0.67) HYMOD 0.43 (0.39) x 0.54 (0.48) 0.56 (0.46) 0.86 (0.84) x x 0.61 (0.59) HYPE x 0.72 (0.62) x x 0.91 (0.88) 0.94 (0.92) x x mHM 0.50 (0.48) x 0.85 (0.83) 0.54 (0.51) 0.90 (0.90) x x 0.76 (0.73) SWAT 0.49 (0.43) x 0.77 (0.68) 0.49 (0.47) 0.94 (0.92) x 0.79 (0.78) x SWIM 0.55 (0.51) 0.58 (0.47) 0.70 (0.65) 0.67 (0.59) 0.89 (0.89) x 0.79 (0.72) 0.71 (0.68) VIC 0.49 (0.45) 0.53 (0.52) 0.65 (0.56) 0.59 (0.49) 0.91 (0.90) 0.91 (0.87) 0.83 (0.82) 0.59 (0.46) WaterGAP3 0.17 (−0.35) 0.37 (0.31) 0.67 (0.65) 0.35 (0.22) 0.85 (0.85) 0.77 (0.60) x 0.59 (0.56) Ens-GHM 0.49 (0.47) 0.37 (0.30) 0.82 (0.80) 0.53 (−0.35) 0.82 (0.65) 0.9 (0.71) 0.63 (0.62) 0.72 (0.62) Ens-CHM 0.50 (0.48) 0.73 (0.73) 0.83 (0.82) 0.61 (0.59) 0.96 (0.95) 0.96 (0.95) 0.85 (0.85) 0.73 (0.66) differing by 6 or less runoff-deficit drought events and only by 2 or less SRI drought events. Table 2 For half of the catchments, the number of SRI drought events estimated by the Ens-GHM was closer to the observed data than the Ens-CHM simulations, while for observed runoff-deficit drought events estimation by the Ens-CHM was better in the majority of catchments. and observed drought intensity or severity. There are marked differences across catchments in whether the models over- or under-estimated the intensity (or severity) of observed droughts, and in the ability to simulate the very occurrence of an observed drought itself. We observed cases where ensemble models simulated a drought that never occurred in the observed record, and cases where a model fails to simulate a drought that occurred in the observed record, in most catchments for extreme SRI and severe runoff-deficit drought events. Despite the comparable estimates of the total number of SRI and runoff-deficit drought events across most catchments, both Ens-CHM and Ens-GHM struggled in estimating the frequency of droughts within SRI drought categories. Both, Ens-CHM and Ens-GHM underestimated the number of extreme SRI droughts (≤−2) in the Upper Amazon, Upper Niger and Tagus catchments. While in the Upper Yellow catchment, Ens- CHM and Ens-GHM both overestimated the number of extreme SRI droughts, showing 3 and 4 extreme events respectively when no such SRI drought events were identified in the observed data. i Out of the 27 observed extreme SRI drought events across all catch ments, Ens-CHM and Ens-GHM failed to identify 1 and 3 drought events, respectively. In total 14 extreme SRI drought events were identified by the models which were not observed (shaded grey under ‘Observed’ in Fig. 2), and 5 of these events were identified by both Ens-CHM and Ens- GHM. For the Upper Amazon, individual CHMs and GHMs performed similarly, with most models under-estimating drought intensity for 6 of the observed extreme SRI drought events. For one event in the Upper Amazon, an observed SRI drought event went undetected by 10 out of 17 models. All the individual models did not accurately simulate hydro logical conditions in the Upper Yellow catchment, with 4 extreme drought events simulated that were not observed in reality. Model performance is more nuanced in the other catchments, with some observed droughts within a catchment under-estimated and other events over-estimated (Upper Niger, Rhine, and Upper Yangtze). Table 2 Table 2 R2 and NSE (in parentheses) values for simulated (individual GHMs and CHMs including both ensembles) versus observed monthly runoff-deficit across all eight catchments. Cells with bold text denotes where both R2 and NSE values are above 0.7. Cells marked with x denote that the particular model was not run for the specific catchment. Table 2 R2 and NSE (in parentheses) values for simulated (individual GHMs and CHMs including both ensembles) versus observed monthly runoff-deficit across all eight catchments. Cells with bold text denotes where both R2 and NSE values are above 0.7. Cells marked with x denote that the particular model was not run for the specific catchment. Table 2 R2 and NSE (in parentheses) values for simulated (individual GHMs and CHMs including both ensembles) versus observed monthly runoff-deficit across all eight catchments. Cells with bold text denotes where both R2 and NSE values are above 0.7. Cells marked with x denote that the particular model was not run for the specific h differing by 6 or less runoff-deficit drought events and only by 2 or less SRI drought events. For half of the catchments, the number of SRI drought events estimated by the Ens-GHM was closer to the observed data than the Ens-CHM simulations, while for observed runoff-deficit drought events estimation by the Ens-CHM was better in the majority of catchments. Despite the comparable estimates of the total number of SRI and runoff-deficit drought events across most catchments, both Ens-CHM and Ens-GHM struggled in estimating the frequency of droughts within SRI drought categories. Both, Ens-CHM and Ens-GHM underestimated the number of extreme SRI droughts (≤−2) in the Upper Amazon, Upper Niger and Tagus catchments. While in the Upper Yellow catchment, Ens- CHM and Ens-GHM both overestimated the number of extreme SRI droughts, showing 3 and 4 extreme events respectively when no such SRI drought events were identified in the observed data. 4 4 E l ti f d ht i t it and observed drought intensity or severity. There are marked differences across catchments in whether the models over- or under-estimated the intensity (or severity) of observed droughts, and in the ability to simulate the very occurrence of an observed drought itself. Table 2 We observed cases where ensemble models simulated a drought that never occurred in the observed record, and cases where a model fails to simulate a drought that occurred in the observed record, in most catchments for extreme SRI and severe runoff-deficit drought events. Out of the 27 observed extreme SRI drought events across all catch ments, Ens-CHM and Ens-GHM failed to identify 1 and 3 drought events, respectively. In total 14 extreme SRI drought events were identified by the models which were not observed (shaded grey under ‘Observed’ in Fig. 2), and 5 of these events were identified by both Ens-CHM and Ens- GHM. For the Upper Amazon, individual CHMs and GHMs performed similarly, with most models under-estimating drought intensity for 6 of the observed extreme SRI drought events. For one event in the Upper Amazon, an observed SRI drought event went undetected by 10 out of 17 catchment. 4.3. Evaluation of the frequency of drought events Table 4 presents the number of runoff deficit and SRI drought events, identified from the observed and simulated runoff (Ens-CHM and Ens- GHM) for all eight catchments. The total number of individual drought events identified by Ens-GHM and Ens-CHM were comparable in most catchments. For all eight catchments, 136 individual SRI drought events were identified from the observed data, and the ensemble models successfully simulated 133 and 123 drought events for Ens-GHM and Ens-CHM, respectively. Similarly, Ens-GHM and Ens-CHM successfully simulated 233 and 220 individual drought events respec tively from 244 individual runoff-deficit drought events, identified from observed records. i We used the coefficient of determination i.e. the square of Pearson product moment correlation coefficient (R2), and the Nash-Sutcliffe Ef ficiency coefficient (NSE; Nash and Sutcliffe, 1970) to evaluate the goodness of fit between observed and simulated runoff-deficits and SRI values, for each CHM, GHM, and the Ens-CHM and Ens-GHM. R2 indi cated the strength of relationship between the simulated and observed runoff, and NSE indicated the models’ efficiency (range -ꝏ–1). NSE values approaching 1 indicate a perfect match of simulated and observed runoff values while negative values show that the observed mean is a better predictor than the model. Ens-CHM and Ens-GHM performed similarly in their estimation of the number of drought events for most catchments, with five catchments 4 Advances in Water Resources 165 (2022) 104212 A. Kumar et al. 5. Discussion For all the observed runoff-deficit and SRI drought events (inde pendently under each drought category i.e. moderate, severe and extreme for SRI droughts events), we calculated the absolute error in drought duration for each model. The mean of all the absolute errors (under each drought category for SRI droughts events) for a model yielded the mean absolute error (MAE) for that model. Figs. 3 and 4 shows the MAE values for SRI (under the three drought categories) and runoff-deficit drought events respectively from the Ens-CHM and Ens-GHM, for every catch ment along with their respective mean observed drought duration. SM 4 (supplementary materials) presents MAE values for the individual models averaged across all catchments for both SRI and runoff-deficit drought events. For all the observed runoff-deficit and SRI drought events (inde pendently under each drought category i.e. moderate, severe and extreme for SRI droughts events), we calculated the absolute error in drought duration for each model. The mean of all the absolute errors (under each drought category for SRI droughts events) for a model yielded the mean absolute error (MAE) for that model. Figs. 3 and 4 shows the MAE values The aim of this study was to assess the performance of CHMs and GHMs in simulating observed drought events using runoff-deficit and SRI as indicators of hydrological drought. No two drought events are the same and, as such, drought events cannot be judged based on a single characteristic. Here we used three characteristics; the intensity or severity, duration and frequency of drought events. We used R2 and NSE to judge the ability of the models and their ensemble means to replicate observed monthly runoff-deficits and SRI. for SRI (under the three drought categories) and runoff-deficit drought events respectively from the Ens-CHM and Ens-GHM, for every catch ment along with their respective mean observed drought duration. SM 4 (supplementary materials) presents MAE values for the individual models averaged across all catchments for both SRI and runoff-deficit drought events. Table 3 2 Cells with bold text denotes where both R2 and NSE values are >0.70 and marked with x denotes that the particular model was not run for the specific catchment. Table 3 R2 and NSE (in parentheses) values for simulated (individual GHMs and CHMs including both ensembles) versus observed SRI across all eight catchments. Cells with bold text denotes where both R2 and NSE values are >0.70 and marked with x denotes that the particular model was not run for the specific catchment. severe runoff-deficit drought events, 19 additional runoff-deficit drought events were identified by the models which were not observed and 9 of these additional events were identified as severe runoff-deficit drought events by both Ens-GHM and Ens-CHM. Performance of CHMs and GHMs was analogous in estimating drought severity across all catch ments, however there were fewer cases in CHMs (than GHMs) where observed severe runoff-deficit drought events were identified only by one individual model. Two runoff-deficit drought events went undetected by all individual models in the Upper Yellow catchment. for Ens-GHM (Fig. 3). Both ensembles display MAE’s smaller than all individual models (SM 4), for extreme SRI droughts across all three drought categories. The models simulate drought duration for extreme SRI droughts better than for lower intensity SRI droughts (Fig. 3). However for SRI drought events, overall, both the CHMs and GHMs struggle to accurately model drought duration, with MAE consistently >1 month (SM 4). For runoff-deficit droughts, the MAE was similar between Ens-CHM and Ens-GHM, except for the Lena with a difference of 1 month between the two ensembles (Fig. 4). MAEs were generally lower for runoff-deficit droughts than for SRI droughts. 4.4. Evaluation of drought intensity Fig. 2 and SM 3 (supplementary materials) show extreme SRI and severe runoff deficit drought events respectively, and the performance of individual models in representing these events with regards to drought intensity or severity along with drought duration. Substantial variation was seen in the ability of individual CHMs and GHMs to simulate drought intensity or severity. The over- or under-estimation of drought intensity in Fig. 2 (drought severity in SM 3) shown as colour coded cells from shades of green to brown is based on the difference in estimated Ens-CHM and Ens-GHM each failed to identify 12 drought events across all eight catchments from 70 observed severe runoff-deficit droughts (SM 3, supplementary material). In addition to 70 observed 5 Advances in Water Resources 165 (2022) 104212 A. Kumar et al. Table 3 2 ii Table 3 R2 and NSE (in parentheses) values for simulated (individual GHMs and CHMs including both ensembles) versus observed SRI across all eight catchments. Cells with bold text denotes where both R2 and NSE values are >0.70 and marked with x denotes that the particular model was not run for the specific catchment. Upper Amazon Lena Upper Mississippi Upper Niger Rhine Tagus Upper Yangtze Upper Yellow GHMs CLM 0.42 (0.30) 0.17 (−0.29) 0.64 (0.6) 0.15 (−0.20) 0.50 (0.43) 0.38 (0.24) 0.37 (0.22) 0.4 (0.27) DBH 0.44 (0.34) 0.12 (−0.43) 0.67 (0.64) 0.11 (−0.32) 0.76 (0.75) 0.32 (0.15) 0.45 (0.35) 0.27 (0.05) H08 0.44 (0.34) 0.14 (−0.35) 0.66 (0.63) 0.11 (−0.32) 0.63 (0.59) 0.31 (0.13) 0.34 (0.18) 0.23 (−0.03) MATSIRO 0.32 (0.14) 0.30 (0.01) 0.71 (0.7) 0.24 (−0.01) 0.55 (0.49) 0.42 (0.31) 0.44 (0.33) 0.34 (0.18) MPI-HM 0.39 (0.26) 0.19 (−0.24) 0.75 (0.74) 0.27 (0.05) 0.71 (0.7) 0.65 (0.62) 0.39 (0.25) 0.43 (0.32) PCR-GLOBWB 0.48 (0.39) 0.33 (0.06) 0.75 (0.74) 0.01 (−0.75) 0.80 (0.79) 0.56 (0.50) 0.52 (0.45) 0.45 (0.35) WaterGAP2 0.53 (0.47) 0.28 (−0.02) 0.78 (0.77) 0.24 (−0.01) 0.87 (0.88) 0.65 (0.62) 0.54 (0.48) 0.5 (0.43) LPJmL 0.50 (0.43) 0.12 (−0.44) 0.53 (0.46) 0.12 (−0.28) 0.80 (0.79) 0.21 (−0.07) 0.47 (0.37) 0.29 (0.09) CHMs ECOMAG x 0.45 (0.28) x x x x x x HBV 0.55 (0.49) x 0.68 (0.65) 0.25 (0.01) 0.88 (0.88) 0.75 (0.74) 0.59 (0.54) 0.25 (0.01) HYMOD 0.50 (0.42) x 0.59 (0.54) 0.28 (0.07) 0.78 (0.77) x x 0.41 (0.29) HYPE x 0.44 (0.27) x x 0.89 (0.89) 0.64 (0.61) x x mHM 0.56 (0.50) x 0.87 (0.87) 0.27 (0.05) 0.88 (0.89) x x 0.54 (0.47) SWAT 0.54 (0.47) x 0.72 (0.70) 0.31 (0.13) 0.93 (0.93) x 0.56 (0.5) x SWIM 0.62 (0.58) x 0.73 (0.72) 0.49 (0.4) 0.88 (0.88) x 0.51 (0.43) 0.51 (0.44) VIC 0.52 (0.45) 0.30 (0.01) 0.64 (0.60) 0.27 (0.05) 0.91 (0.91) 0.34 (0.18) 0.55 (0.49) 0.32 (0.13) WaterGAP3 0.23 (−0.02) 0.29 (0) 0.77 (0.76) 0.16 (−0.17) 0.86 (0.86) 0.57 (0.51) x 0.49 (0.41) Ens-GHM 0.56 (0.50) 0.35 (0.10) 0.86 (0.86) 0.18 (−0.14) 0.85 (0.85) 0.47 (0.38) 0.57 (0.52) 0.56 (0.46) Ens-CHM 0.56 (0.50) 0.46 (0.30) 0.88 (0.88) 0.33 (0.15) 0.94 (0.95) 0.73 (0.71) 0.65 (0.62) 0.53 (0.46) Table 3 R2 and NSE (in parentheses) values for simulated (individual GHMs and CHMs including both ensembles) versus observed SRI across all eight catchments. Table 4 Table 4 Number of runoff-deficit and SRI drought events identified from observed and simulated runoff (Ens-GHM and Ens-CHM) for all eight catchments, SRI drought events are classified into moderate, severe and extreme drought events based on SRI values. SRI drought events Runoff-deficit drought events Catchments(↓) Drought category (→) Moderate(−1 to −1.49) Severe(−1.5 to −1.99) Extreme(−2 or Below) Total individualdrought events(−1 or Below) Total individualdrought events(with positive drought deficit) Upper Amazon Ens-GHM 3 5 4 12 24 Ens-CHM 6 5 4 15 29 Obs 6 4 6 16 27 Lena Ens-GHM 7 4 3 14 19 Ens-CHM 7 5 3 15 25 Obs 11 4 3 18 36 Upper Mississippi Ens-GHM 8 4 2 14 28 Ens-CHM 6 3 3 12 23 Obs 9 3 2 14 27 Upper Niger Ens-GHM 10 7 2 19 40 Ens-CHM 9 4 2 15 31 Obs 11 6 5 22 30 Rhine Ens-GHM 7 7 4 18 32 Ens-CHM 8 6 3 17 30 Obs 10 6 3 19 31 Tagus Ens-GHM 11 6 0 17 38 Ens-CHM 2 5 1 8 16 Obs 5 3 2 10 27 Upper Yangtze Ens-GHM 12 5 5 22 26 Ens-CHM 12 5 7 24 38 Obs 10 5 6 21 34 Upper Yellow Ens-GHM 6 7 4 17 26 Ens-CHM 9 5 3 17 28 Obs 7 9 0 16 32 Total individual drought events across all catchments Ens-GHM 64 45 24 133 233 Ens-CHM 59 38 26 123 220 Obs 69 40 27 136 244 Table 4 Number of runoff-deficit and SRI drought events identified from observed and simulated runoff (Ens-GHM and Ens-CHM) for all eight catchments, SRI drought events are classified into moderate, severe and extreme drought events based on SRI values. different formulations to compute potential evapotranspiration (Telteu et al., 2021), which contributes to differences in simulated runoff be tween the GHMs (Beck et al., 2017). For monthly runoff-deficits, the Ens-GHM outperformed individual GHMs because the individual models over- or under-estimated low-flow conditions. The computation of an ensemble mean for the GHMs essentially balances out the over- and under-estimation of runoff-deficits by individual models. However the use of a large number of models does not necessarily ensure better performance of the ensemble mean. Table 4 For the Upper Amazon, Upper Mississippi and Rhine where the number of models used for the ensemble calculation was higher, the performance of the Ens-CHM was better at estimating drought frequency but showed higher MAE for drought duration with exception of the Rhine. drought events was higher than that of SRI drought events (Table 4), which resulted in almost consistent mean observed drought duration and very less variation was seen in the MAE between Ens-CHM and Ens-GHM across all catchments. There was a marked difference in performance of CHMs and GHMs when estimating drought intensity and duration with the SRI. This is owing to the data used for identifying simulated drought events, which here is simulated monthly runoff data. The accuracy of the SRI computation is directly proportional to the quality of data used for its calculation (Hayes et al., 1999). Huang et al. (2017) reported that CHMs accurately reproduced monthly runoff, seasonal dynamics, moderate or high-flows but simulations of low-flows were problematic in most catchments. Zaherpour et al. (2018) found that the majority of GHMs overestimated low-flows considerably more than they overestimated high-flows and that GHMs overestimated minimum flow return periods. The majority of the GHMs showed a tendency for overestimating monthly runoff with a wider magnitude range (Veldkamp et al., 2018). Previous studies highlight that this wider spread around ensembles in every catchment is due to the structure of GHMs (Haddeland et al., 2011; Gudmundsson et al., 2012). Physical processes such as transmission losses, having less presence in the GHMs is one main reason for some of the differences between simulated and observed runoff (Gosling and Arnell, 2011). In addition, evapotranspiration simulation has been re ported to vary widely among the GHMs (Wartenburger et al., 2018). 5.3. Implications for model calibration The performance of WaterGAP2 for runoff-deficit estimation was similar to the other GHMs. Despite WaterGAP2 being the only GHM to be calibrated with long-term annual river discharge, it did not perform noticeably better than other GHMs, and independent of calibration, the identification of hydrological extremes was not satisfactory across all models. This suggests that it is important to calibrate models with high temporal resolution data that allows extreme events to be accounted for in the calibration process. The use of multiple criteria during model calibration, specifically for low- or no-flow could be helpful (Krysanova et al., 2018). 5.1. Comparison of CHM and GHM performance Different thresholds defining runoff-deficit droughts for each catch ment meant there were not standard drought classes, which limited the distinctive analysis of drought occurrences. Frequency of runoff-deficit For SRI droughts, the largest total MAE (i.e. the MAE summed across the three drought categories) was seen for Upper Niger for both en sembles, followed by Upper Mississippi for Ens-CHM and Upper Amazon 6 Advances in Water Resources 165 (2022) 104212 A. Kumar et al. 5.4. Representation of processes and quality of observed data Although the R2 and NSE values for the SRI series are less than satisfactory for many individual GHMs and CHMs across catchments, the ensemble models were better at estimating drought frequency for most of the catchments. Moreover, both the ensembles display, overall, a better performance compared to the individual models that make up each respective ensemble, and showed comparable outputs (runoff-deficit and SRI values) despite the GHMs having a wider spread across the ensemble. Although the GHMs used the same climate forcing, they used Our results indicate relatively better performances of GHMs for runoff-deficits and SRI patterns in the Upper Mississippi and Rhine compared to other catchments. Furthermore, none of the GHMs missed any observed SRI drought events, while only few GHMs failed to simu late observed runoff-deficit drought events. However, tendency of GHMs (across all catchments) towards simulating drought events that were not 7 Advances in Water Resources 165 (2022) 104212 A. Kumar et al. Advances in Water Resources 165 (202 Observed and simulated extreme SRI drought events (SRI ≤−2). Displayed is each observed drought’s start and end date, with the numbers indic d and simulated drought duration in months. Drought events are colour coded based on the difference in simulated and observed drought intensity ( arked from shades of green to brown (under- and over-estimated respectively). Blue cells are observed droughts not present in simulated records. Grey ed droughts (either by Ens-CHM or Ens-GHM or both) not present in the observed record. Yellow cells denote that the particular model was not ru catchment. r et al. ed, can be attributed to a dry bias introduced by the choice of l t i ti f l ti f i di id l d l F likely to have an impact on drought timing (initiation and dur d ht t ) Observed and simulated extreme SRI drought events (SRI ≤−2). Displayed is each observed drought’s start and end date, with the numbers indic d and simulated drought duration in months. Drought events are colour coded based on the difference in simulated and observed drought intensity ( rked from shades of green to brown (under- and over-estimated respectively). Blue cells are observed droughts not present in simulated records. Grey ed droughts (either by Ens-CHM or Ens-GHM or both) not present in the observed record. Yellow cells denote that the particular model was not ru catchment. Fig. 2. 5.4. Representation of processes and quality of observed data Observed and simulated extreme SRI drought events (SRI ≤−2). Displayed is each observed drought’s start and end date, with the numbers indicating the observed and simulated drought duration in months. Drought events are colour coded based on the difference in simulated and observed drought intensity (from 2 to −2), marked from shades of green to brown (under- and over-estimated respectively). Blue cells are observed droughts not present in simulated records. Grey cells are simulated droughts (either by Ens-CHM or Ens-GHM or both) not present in the observed record. Yellow cells denote that the particular model was not run for the specific catchment. likely to have an impact on drought timing (initiation and duration of drought events). l observed, can be attributed to a dry bias introduced by the choice of potential evapotranspiration formulation for individual models. For example, PCR-GLOBWB consistently appeared near the dry end of Ens- GHM, perhaps because it includes a temperature based evaporation formulation (Hamon) that has been shown to induce a large bias when applied outside its calibration range (Milly and Dunne, 2017). In gen eral, for GHMs it is difficult to estimate a drought event at the right time because multiple errors propagate from the inputs (meteorological pa rameters) and some GHMs struggle to capture the magnitude and timing of processes like abstraction losses and snowmelt accurately, which is For CHMs, large biases have been reported in simulating low-flow conditions across majority of the catchments we studied, especially the Upper Yangtze (Huang et al., 2017). Inaccuracies of low-flow ob servations may be a factor affecting the estimation of observed drought conditions, as might river ice in some catchments, while the inability of individual CHMs to replicate low-flow or no-flow may be due to the choice of objective functions for calibration of the CHMs (Huang et al., 2017). Similarities in performance of both sets of individual models in 8 8 A. Kumar et al. Advances in Water Resources 165 (2022) 104212 Fig. 3. MAE for SRI drought duration under each drought category for Ens-GHM and Ens-CHM, for all eight catchments, along with respective mean observed drought duration. Fig. 3. MAE for SRI drought duration under each drought category for Ens-GHM and Ens-CHM, for all eight catchments, along with respective mean observed drought duration. Fig. 4. MAE for runoff-deficit drought duration for Ens-GHM and Ens-CHM, for all eight catchments, along with respective mean observed drought duration. Fig. CRediT authorship contribution statement D¨oll, P., Lehner, B., 2002. Validation of a new global 30-min drainage direction map. J. Hydrol. 258 (1–4) https://doi.org/10.1016/S0022-1694(01)00565-0. Elliott, J., et al., 2014. Constraints and potentials of future irrigation water availability on agricultural production under climate change. Proc. Natl. Acad. Sci. U. S. A. https://doi.org/10.1073/pnas.1222474110 [Preprint]. l Amit Kumar: Investigation, Data curation, Writing – original draft. Simon N. Gosling: Methodology, Data curation, Writing – original draft, Writing – review & editing. Matthew F. Johnson: Methodology, Data curation, Writing – original draft, Writing – review & editing. Matthew D. Jones: Methodology, Writing – original draft, Writing – review & editing. Jamal Zaherpour: Data curation, Writing – original draft, Writing – review & editing. Rohini Kumar: Writing – original draft, Writing – review & editing. Guoyong Leng: Writing – original draft, Writing – review & editing. Hannes Müller Schmied: Writing – original draft, Writing – review & editing. Jenny Kupzig: Writing – original draft, Writing – review & editing. Lutz Breuer: Writing – original draft, Writing – review & editing. Naota Hanasaki: Writing – original draft, Writing – review & editing. Qiuhong Tang: Writing – original draft, Writing – review & editing. Sebastian Ostberg: Writing – original draft, Writing – review & editing. Tobias Stacke: Writing – original draft, Writing – review & editing. Yadu Pokhrel: Writing – original draft, Writing – review & editing. Yoshihide Wada: Writing – original draft, Writing – review & editing. Yoshimitsu Masaki: Writing – original draft, Writing – review & editing. Emerton, R.E., et al., 2016. Continental and global scale flood forecasting systems. WIREs Water. https://doi.org/10.1002/wat2.1137 [Preprint]. l Fleig, A.K., et al., 2006. 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Representation of processes and quality of observed data 4. MAE for runoff-deficit drought duration for Ens-GHM and Ens-CHM, for all eight catchments, along with respective mean observed drought duration. drought duration for Ens-GHM and Ens-CHM, for all eight catchments, along with respective mean observed drought duratio Fig. 4. MAE for runoff-deficit drought duration for Ens-GHM and Ens-CHM, for all eight catchments, along with respectiv unreliable due to inaccuracies in observed precipitation records, caused by fog/mist (Strauch et al., 2017). WaterGAP3 among all CHMs comparatively showed the weakest performance, which may be attrib uted to fewer parameters used for model calibration compared to other unreliable due to inaccuracies in observed precipitation records, caused by fog/mist (Strauch et al., 2017). WaterGAP3 among all CHMs comparatively showed the weakest performance, which may be attrib uted to fewer parameters used for model calibration compared to other unreliable due to inaccuracies in observed precipitation records, caused by fog/mist (Strauch et al., 2017). WaterGAP3 among all CHMs comparatively showed the weakest performance, which may be attrib uted to fewer parameters used for model calibration compared to other the Upper Amazon, Lena, Tagus, and Upper Yangtze catchments for drought events in and SM 3 (supplementary materials) can likely be attributed towards the quality of the meteorological data used for sim ulations. Some studies have reported WATCH ERA-40 data to be the Upper Amazon, Lena, Tagus, and Upper Yangtze catchments for drought events in and SM 3 (supplementary materials) can likely be attributed towards the quality of the meteorological data used for sim ulations. Some studies have reported WATCH ERA-40 data to be the Upper Amazon, Lena, Tagus, and Upper Yangtze catchments for drought events in and SM 3 (supplementary materials) can likely be attributed towards the quality of the meteorological data used for sim ulations. Some studies have reported WATCH ERA-40 data to be 9 9 CHMs. A. Kumar et al. A. Kumar et al. Advances in Water Resources 165 (2022) 104212 CHMs. CRediT authorship contribution statement Simulating current global river runoff with a global hydrological model: model revisions, validation, and sensitivity analysis. Hydrol. Process. https://doi.org/10.1002/hyp.7727 [Preprint]. Gudmundsson, L., et al., 2012. Comparing large-scale hydrological model simulations to observed runoff percentiles in Europe. J. Hydrometeorol. https://doi.org/10.1175/ JHM-D-11-083.1 [Preprint]. original draft, Writing – review & editing. Yoshimitsu Masaki: Writing – original draft, Writing – review & editing. Haddeland, I., et al., 2011. 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Clim. https://doi.org/10.1175/1520-0442(1999)012<2747: OQODSA>2.0.CO;2 [Preprint]. 6. Conclusion Our study focused upon the effectiveness of catchment- and global- scale hydrological models to estimate drought conditions at the catch ment scale, for 8 large catchments. We found comparably lower per formance by most GHMs in simulating monthly runoff-deficits, while CHMs and GHMs were similar in estimating SRI. Both sets of models show limited ability to simulate the finer, more granular and detailed characteristics (intensity or severity, duration and frequency) of observed droughts but the ensembles performed better compared to the individual models that make up each respective ensemble. Whilst the Ens-CHM and Ens-GHM simulated drought frequency well for runoff-deficit and SRI drought events, marked differences were observed in ability to simulate the occurrence of observed and simulated drought events (not simu lating several observed drought events and simulating drought events out of observed records). For both the ensembles, the error is, overall, smallest for duration of extreme SRI droughts across all three drought categories. However, it can also be concluded that both CHMs and GHMs struggled in accurately modelling drought duration for moderate and severe SRI drought categories. We believe that there is still room for improvement in runoff simulations to facilitate drought identification and accurate estimation of drought characteristics. Funding Hattermann, F.F., et al., 2017. Cross-scale intercomparison of climate change impacts simulated by regional and global hydrological models in eleven large river basins. Clim. Chang. 141 (3), 561–576. https://doi.org/10.1007/s10584-016-1829-4. The research was funded by the University of Nottingham Vice Chancellor’s Scholarship for Research Excellence (International). Hayes, M.J., et al., 1999. Monitoring the 1996 drought using the standardized precipitation index. Bull. Am. Meteorol. Soc. https://doi.org/10.1175/1520-0477 (1999)080<0429:MTDUTS>2.0.CO;2 [Preprint]. Availability of data and material Declaration of Competing Interest Hagemann, S., Dümenil, L., 1998. A parametrization of the lateral waterflow for the global scale. Clim. Dyn. https://doi.org/10.1007/s003820050205 [Preprint]. The authors declare no conflict of interest. Hanasaki, N., et al., 2008. An integrated model for the assessment of global water resources - part 1: model description and input meteorological forcing. Hydrol. Earth Syst. Sci. https://doi.org/10.5194/hess-12-1007-2008 [Preprint]. Availability of data and material Heim, R.R., 2002. A review of twentieth-century drought indices used in the United States. Bull. Am. Meteorol. Soc. 83 (8), 1149–1166. https://doi.org/10.1175/1520- 0477-83.8.1149. l Output from the models used for this study are openly available from the Earth System Grid Federation (ESGF; https://esg.pik-potsdam. de/search/isimip) for the GHMs (Gosling et al., 2017) and the CHMs (Krysanova et al., 2017). Hisdal, H., et al., 2001. Have streamflow droughts in Europe become more severe or frequent? Int. J. Climatol. 21 (3) https://doi.org/10.1002/joc.619. Huang, S., et al., 2017. Evaluation of an ensemble of regional hydrological models in 12 large-scale river basins worldwide. Clim. Chang. https://doi.org/10.1007/s10584- 016-1841-8 [Preprint]. Van Huijgevoort, M.H.J., et al., 2013. Global multimodel analysis of drought in runofffor the second half of the twentieth century. J. Hydrometeorol. https://doi.org/ 10.1175/JHM-D-12-0186.1 [Preprint]. Code availability Code availability Not applicable. 10 A. 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Dyn. https://doi.org/10.5194/esd-5-15-2014 [Preprint]. Milly, P.C.D., Dunne, K.A., 2017. A hydrologic drying bias in water-resource impact analyses of anthropogenic climate change. J. Am. Water Resour. Assoc. https://doi. org/10.1111/1752-1688.12538 [Preprint]. Wartenburger, R., et al., 2018. Evapotranspiration simulations in ISIMIP2a-Evaluation of spatio-temporal characteristics with a comprehensive ensemble of independent datasets. Environ. Res. Lett. 13 (7) https://doi.org/10.1088/1748-9326/aac4bb. Mishra, A.K., Desai, V.R., Singh, V.P., 2007. Drought forecasting using a hybrid stochastic and neural network model. J. Hydrol. Eng. 12 (6) https://doi.org/ 10.1061/(asce)1084-0699(2007)12:6(626. Wasko, C., et al., 2021. Understanding trends in hydrologic extremes across Australia. J. Hydrol. 593. https://doi.org/10.1016/j.jhydrol.2020.125877. Code availability Mishra, A.K., Singh, V.P., 2010. A review of drought concepts. J. 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https://openalex.org/W2891903109	https://rbmfc.org.br/rbmfc/article/download/1856/943	es		La Medicina Familiar y Comunitaria como fuente de cuidados de Salud Mental	Revista Brasileira de Medicina de Família e Comunidade	2,018	cc-by	7,965	www.rbmfc.org.br La Medicina Familiar y Comunitaria como fuente de cuidados de Salud Mental Family and Community Medicine as source of Mental Health Care ENSAYOS Macarena Moral Lópeza Diana Yuruhán Mohrbachb Carmen Ruiz Puyanac Maria Inez Padula Andersond Medicina Familiar e Comunitária como fonte de cuidados em Saúde Mental Paula Andrea Carmona Mejíae Grupo de Trabajo: Amanda Astudillo (Colombia), Alfonso Avila (Colombia), Garibaldi Baldovino (Colombia); Virginia Cardozo (Uruguay), Ximena Cruz (Bolivia), Carmen Daza (Colombia), Sonia De La Portilla (Colombia), José Ignacio Díaz (Colombia), Ysabel Díaz (Rep. Dominicana); Giuliano Dimarzio (Brasil), Elizabeth Escobar (Colombia), Margarita García (Colombia), Luz Girón (Colombia), Carlos Guevara (Colombia), Melissa Gutiérrez (Colombia), Jakeline Jolkh (Colombia), Fátima González (Paraguay), Carlos Guevara (Colombia), Giuliano Dimarzio (Brasil), Mauricio Molina (Colombia), Marta A. Mejía (Venezuela), Alvaro Pérez (Colombia), Marcela Pérez (Chile), Olga Polo (Perú), Lina Quintero (Colombia), Katherine Rocha (Colombia), Diana Rodríguez (Colombia), Julieth Salazar (Colombia), Martha Sánchez (Bolivia), Mónica Sánchez (Colombia), Gladys Sandoval (Paraguay), Heyder Satizabal (Colombia), Melba Vásquez (Costa Rica), José Manuel Vivas (Colombia), Vilma Velásquez (Colombia). María Luisa Vera Gonzálezh Sandra Fortesf José Rubén Quirozg a Facultad de Medicina, Universidad de Chile; Confederación Iberoamericana de Medicina Familiar (CIMF/WONCA); Sociedad Chilena de Medicina Familiar (SOCHIMEF). Chile. moralmacarena@hotmail.com (Autora correspondiente) b Universidad Católica “Nuestra Señora de la Asunción” (UC); Universidad del Norte (UN); Sociedad Paraguaya de Medicina Familiar (SPMF). Paraguay. dyuruhan@hotmail.es Universidad Militar Nueva Granada (UMNG); Universidad El Bosque (UEB); Hospital Militar Central; Sociedad Colombiana Medicina Familiar (SOCMEF). Colombia. cruizpuyana@gmail.com c Resumen En la Séptima Cumbre Iberoamericana de Medicina Familiar, Cali – Colombia 2018, el grupo de trabajo Salud Mental (SM) reflexionó sobre como la Medicina Familiar (MF) puede actuar para apoyar a las personas que enfrentan situaciones de estrés frente a la vida diaria, conflictos (armados/no armados), emergencias y desastres naturales. Estudio descriptivo de corte transversal basado en una encuesta de 42 preguntas a 99 profesionales sanitarios iberoamericanos provenientes de 15 países, 98 médicos y 1 psicólogo, 8% residentes de MF, 85% especialistas en MF, 4% médicos generales, 2% psiquiatras y 1% internista. El 47% de los médicos percibe como buena la capacidad de los médicos de familia en el abordaje de la SM. En cuanto a los problemas de SM observados, 30% indica Trastorno de Ansiedad, 27% depresión, 17% insomnio, 10% alcoholismo, 7% adicción a drogas ilícitas, 5% trastornos alimentarios y 4% trastorno de estrés postraumático. En este contexto se realizaron las recomendaciones para la Carta de Cali que consideran la formación en SM necesaria para los Médicos Familiares, con estrategias de autocuidado costo efectivas, mediante el fortalecimiento del trabajo comunitario. El cuerpo docente de las residencias de MF debe hacerse cargo de acciones tendientes al autocuidado de los alumnos, propendiendo a facilitar el proceso de aprendizaje y la preparación para ejercer la profesión en un medio tan complejo como son los centros del primer nivel de atención o en cualquier contexto en el que se trabaje con la estrategia de Atención Primaria de Salud. Palabras clave: Medicina Familiar y Comunitaria; Salud Mental; Emergencias; Desastres d Faculdade de Ciencias Médicas, Universidade do Estado do Rio de Janeiro (UERJ); Confederación Iberoamericana de Medicina Familiar (CIMF). Rio de Janeiro, RJ, Brasil. inezpadula@gmail.com e Universidad de La Coruña (UDC); Sociedad Chilena de Medicina Familiar (SOCHIMEF). España. pa.carmona@hotmail.com f Faculdade de Ciencias Médicas, Universidade do Estado do Rio de Janeiro (UERJ); Guía de Intervención del Mental Health Gap, Organización Panamericana de Salud (OPS/OMS). Brasil. sandrafortes@gmail.com g Instituto de la Familia Asociación Civil (IFAC). México. joserubenquiroz@gmail.com h Caja Nacional de Salud (CNS). Bolivia maluverag@gmail.com Financiación: ninguna declarada. Aprobación ética: no aplicable. Conflicto de intereses: ninguna declarada. Cómo citar: Moral M, Yuruhán D, Ruiz C, Anderson MIP, Carmona PA, Fortes S, et al. La Medicina Familiar y Comunitaria como fuente de cuidados de Salud Mental. Rev Bras Med Fam Comunidade. 2018;13(Suppl 1):54-68. http://dx.doi.org/10.5712/rbmfc13(40)1856 54 Rev Bras Med Fam Comunidade. Rio de Janeiro, 2018 Oct; 13(Suppl 1):54-68 Procedencia y revisión por pares: revisado por pares. Recibido el: 25/07/2018. Aceptado el: 27/08/2018. La Medicina Familiar y Comunitaria como fuente de cuidados de Salud Mental Abstract During the Seventh Iberoamerican Summit of Family Medicine, Cali Colombia 2018, the Mental Health (MH) working group reflected on how Family Medicine (FM) can act to support people facing stressful situations in daily life as well as in conflicts (armed/unarmed), emergencies and natural disasters. Descriptive cross-sectional study, based on a survey of 42 questions to 99 Iberoamerican health professionals from 15 countries; 98 physicians and 1 psychologist. 8% residents of family medicine, 85% family physicians (FP), 4% general doctors, 2% psychiatrists and 1% internists. 47% of physicians perceive as good the ability of FP in the approach to MH. Concerning the MH problems observed, 30% where anxiety disorder, 27% depression, 17% insomnia, 10% alcoholism, 7% illicit drug abuse, 5% eating disorders and 4% post-traumatic stress disorder. Wich such results, recommendations for the Cali Declaration consider the necessary MH training for Family physicians, with cost-effective self-care strategies through strengthening community work. The teachers of FP must take actions tending to the self-care of their students, to facilitate the learning process and the preparation to practice the profession in such a complex environment as the Primary Care (PC) centers or in any place based on PC strategy. Keywords: Family Practice; Mental Health; Emergencies; Disasters Resumo Na Sétima Cúpula Ibero-Americana de Medicina Familiar, Cali – Colombia 2018, o grupo de trabalho Saúde Mental (SM) refletiu sobre como a Medicina de Família (MF) pode atuar em para apoiar a saúde integral das pessoas que enfrentam situações de estresse na vida diaria, como conflitos armados/desarmados, emergências e desastres naturais. Estudo descritivo transversal com base em um levantamento de 42 perguntas a 99 profissionais de saúde ibero-americanos provenientes de 15 países; 98 médicos e 1 psicólogo. 8% de residentes de MF, 85% de especialistas em MF, 4% de clínicos gerais, 2% de psiquiatras e 1% de internistas. 47% dos médicos percebem como boa a capacidade dos médicos de família na abordagem da SM. Em relação aos problemas SM, 30% indicam Transtorno de Ansiedade, 27% de depressão, 17% de insônia, 10% de alcoolismo, 7% de dependência de drogas ilícitas, 5% de transtornos alimentares e 4% de transtorno de estresse pós-traumático. Neste contexto, foram feitas recomendações para a Carta de Cali que consideram o treinamento de SM necessário para médicos de família, com estratégias de autocuidado custo-efetivas através do fortalecimento do trabalho comunitário. O corpo docente das residências do MF deve se encarregar de ações que promovam o autocuidado dos alunos, visando facilitar o processo de aprendizagem e o preparo para a prática da profissão em um ambiente tão complexo como os centros de Atenção Primária à Saúde (APS) ou em qualquer contexto em que se trabalhe com a estratégia APS. Palavras-chave: Medicina de Família e Comunidade; Saúde Mental; Emergências; Desastres Introducción La Séptima Cumbre Iberoamericana de Medicina Familiar, Cali Colombia 2018, a través del grupo que tuvo como lema “La Medicina Familiar y Comunitaria como fuente de cuidados de Salud Mental”, nos dio la oportunidad de reflexionar sobre el cómo la medicina familiar puede y debe actuar en relación a la problemática de Salud Mental (SM), buscando aportes concretos conforme al lema central de la cumbre: “Medicina Familiar y Políticas Públicas en territorios de equidad y paz”. En el resumen ejecutivo sobre Prevención del Suicidio de la Organización Mundial de la Salud (OMS) de 2014, se plantea que más de 800.000 personas se suicidan al año y que por cada persona que se suicida habría al menos otras 20 personas que han intentado suicidarse. Es además la segunda causa de muerte entre las personas de 15 a 29 años.1 Habría entonces cada año 16 millones de personas en riesgo vital por intento suicida por patologías de Salud Mental en el mundo. Por otro lado, el principal factor de riesgo frente al suicidio es un intento suicida previo, por lo que esas 16 millones de personas tienen potencialmente un riesgo de muerte mayor que el común de la población. Las causas son múltiples y relacionadas con crisis que determinan una disminución de la capacidad para afrontar las tensiones de la vida tanto aquellas de la cotidianeidad (problemas financieros, rupturas de relaciones, dolores y enfermedades crónicas, etc.), como las de situaciones más complejas: las emergencias y los desastres1 tanto naturales (inundaciones, terremotos, tsunamis, etc.) como antrópicos2 (conflictos políticos y/o armados, incendios, etc.). Rev Bras Med Fam Comunidade. Rio de Janeiro, 2018 Oct; 13(Suppl 1):54-68 55 Moral M, Yuruhán D, Ruiz C, Anderson MIP, Carmona PA, Fortes S, et al. Incluso se ha acuñado el término tsunami de la salud mental3, en el contexto del desarrollo de los niños y adolescentes que son víctimas de violencia física o sexual grave, que inician carreras delictuales alrededor de los 13 años dependiendo de los países y que aumentan progresivamente el consumo de drogas y alcohol. Una amenaza latente para el futuro de nuestras naciones. Por la forma de vida y las situaciones creadas o no por el ser humano, se está produciendo un contexto en el que un número importante de personas es potencialmente incapaz de afrontar las dificultades de la vida diaria y determina una mayor incapacidad para enfrentar las dificultades inherentes a emergencias y desastres, en particular las antrópicas, que son aquellas en las que el ser humano es la causa directa; y en especial, las determinadas por conflictos políticos, armados o no armados. Si bien toda la información anterior pudiera parecer apocalíptica, el informe de la OMS de 2013: “Volver a construir mejor”4 plantea como paradoja que las emergencias son una oportunidad para volver a construir mejor el sistema de atención en SM, sin importar lo débil que haya sido el sistema antes de la emergencia ni la gravedad de la misma. Se comenta que las emergencias y los desastres, naturales o antrópicos pueden dar lugar a situaciones donde la SM requiere especial consideración debido a tres problemas comunes: • aumento de las tasas de problemas de SM, • la debilidad de la infraestructura de SM y • las dificultades que se generan en la coordinación de los organismos que proveen servicios en el área de la SM. En estas situaciones, la prevalencia de la depresión y de los trastornos por estrés postraumático aumenta sustancialmente. Se produce también un aumento de la vulnerabilidad y las necesidades de las personas que ya tenían trastornos mentales graves como esquizofrenia, trastorno bipolar, ansiedad y dependencia a alcohol y drogas. Como resultado de las emergencias y desastres puede debilitarse la infraestructura de atención en SM. Hay mayor necesidad de servicios y los mismos trabajadores de la salud pueden ser víctimas de la emergencia, en muchos casos necesitan cuidar de sus propias familias o amigos antes de cumplir con sus deberes profesionales. Esto puede llevar consigo una escasez de trabajadores de salud calificados. En emergencias mayores con inicio agudo pueden crearse situaciones caóticas por la aparición de múltiples agencias de apoyo. Frente a este gran impulso inicial de organizaciones de todo tipo, gubernamentales y no gubernamentales debe ponerse especial hincapié en la coordinación de la oferta de servicios. El rol fundamental de la ayuda humanitaria es fortalecer las estructuras públicas, logrando así coordinar las acciones y la sostenibilidad a largo plazo de la estructura funcional de los servicios de salud.5 La importancia de la sostenibilidad a largo plazo radica en que no sólo se logra un mejor estado de salud general, sino que también mayor educación, mayor productividad y mejores relaciones interpersonales, y por ende una mejor calidad de vida. Con este actuar no sólo se beneficia la SM de las personas, sino que también el funcionamiento general del país afectado y la resiliencia de la sociedad que es capaz de recuperarse de una situación de emergencia. Las acciones en el marco de la recuperación temprana sientan las bases para una mentalidad a largo plazo. La atención en SM debe centrarse en los servicios que son accesibles a la comunidad. En el mencionado informe de la OMS se plantea que la descentralización de los sistemas de SM hacia sistemas 56 Rev Bras Med Fam Comunidade. Rio de Janeiro, 2018 Oct; 13(Suppl 1):54-68 La Medicina Familiar y Comunitaria como fuente de cuidados de Salud Mental de cuidados basados en la comunidad es una estrategia fundamental. En especial para hacer frente a situaciones de emergencias y desastres. Es necesario para la recuperación temprana, que las directrices de los organismos a cargo generen estándares basados en el consenso entre los servicios de salud y la comunidad. La comunidad debe constituirse en actor estratégico. Es importante destacar que la mayor inversión debe realizarse en las personas, comunidad y trabajadores de la salud, más que en infraestructura. Se debe fortalecer y expandir la atención en SM basada en la comunidad, debe redefinirse el actuar de los centros hospitalarios psiquiátricos de larga estadía, considerando incluso disminuir su tamaño. Ningún nivel de servicio, de primario a secundario y terciario, puede cumplir todas las necesidades de salud mental, es necesario buscar la combinación adecuada entre todos ellos. El autocuidado, el cuidado informal de la comunidad y la atención primaria en SM son la base del modelo piramidal de cuidados. Es a este nivel donde debieran coordinarse la mayor parte de ellos. Para aquellos que requieren servicios más intensivos en algún momento de su vida, el nivel secundario debiera proveer de atención ambulatoria e internación de corta estadía. Entendiendo al nivel secundario como centros comunitarios de salud mental y hospitales generales. Sólo una fracción muy pequeña de las personas con problemas severos de SM necesita hospitalizaciones de larga estadía, considerando un nivel terciario, pero sin que por ello se considere estructurar hospitales psiquiátricos de estadía permanente. La meta es siempre lograr atención y control ambulatorio. En todos los niveles, el modelo enfatiza que las personas con problemas de SM deben participar en la autogestión de sus condiciones.5 En este contexto general un informe del 2017 de la Organización para la Cooperación y el Desarrollo Económico (OECD) sobre la SM5 plantea que se debe promover e invertir en la introducción de programas que promuevan la buena salud mental y la prevención de enfermedad mental. El establecer acciones que prevengan la depresión y la ansiedad conlleva beneficios económicos para las familias, mientras que ciertas intervenciones en el lugar de trabajo pueden reducir en más de un tercio el costo de la baja productividad. Se observa, sin embargo, un compromiso gubernamental desigual entre la promoción de la salud mental y otras patologías en los sistemas de salud, con tendencia a inclinarse hacia las últimas, como son por ejemplo el fuerte foco en la promoción de patologías cardiovasculares. El documento de la OECD realiza varias recomendaciones para los gobiernos de los países, entre ellas: • Implementar programas integrados de salud mental, herramientas de aplicación y política laboral; • Priorizar el bienestar mental de los ciudadanos de todas las edades. Para algunos países esto puede demandar actividades a lo largo de la vida del individuo y para otros implicará promover esfuerzos enfocados a grupos particulares como ancianos o desempleados. Se puede realizar mediante un enfoque escalonado de la acción haciendo uso de nuevas oportunidades, como son por ejemplo los sistemas informáticos en línea. • Desarrollar y respaldar una estrategia permanentemente actualizada y exhaustiva para promocionar la SM a lo largo de la vida. Basada en la mejor evidencia de la efectividad de las intervenciones con foco en el contexto local. • Monitorizar y evaluar en forma permanente la efectividad de lo implementado, de modo tal que permita realizar cálculos estimativos del retorno económico de lo invertido en actividades de promoción y prevención. Esto puede mejorar la comprensión del costo/efectividad de las Rev Bras Med Fam Comunidade. Rio de Janeiro, 2018 Oct; 13(Suppl 1):54-68 57 Moral M, Yuruhán D, Ruiz C, Anderson MIP, Carmona PA, Fortes S, et al. inversiones orientadas a mejorar la SM de las personas, favoreciendo que otros actores, externos al sector de la provisión de atención de salud, visualicen mejor el retorno económico de ésta inversión, impulsándolos a invertir y participar en la realización de actividades de promoción y prevención. • Establecer redes de colaboración intersectorial, reconociendo su importancia más allá de los sistemas de cuidado de la salud y de la SM, involucrando en la promoción y prevención en SM a otros sectores interesados incluyendo asistencia social, sistemas educativos y lugares de trabajo. En el informe de la Organización Panamericana de la Salud (OPS 2013) sobre los sistemas de SM en América Latina y El Caribe6 se refleja la importancia de abarcar los trastornos mentales y neurológicos desde el ámbito de la Atención Primaria en Salud (APS), ya que representan casi la cuarta parte de la carga total de enfermedad en América Latina y el Caribe. “Un sistema de salud mental eficiente es vital tanto para poder ofrecer una respuesta apropiada y reducir esa carga que se traduce en morbilidad, mortalidad y discapacidad, como para cerrar la alta brecha de personas enfermas que no están recibiendo ningún tipo de tratamiento”.7 Se desarrolla la evaluación de los sistemas de atención en SM desde la relevancia de su reestructuración. En el informe se reconoce que en América del Sur, América Central, México y el Caribe latino, la formación en pregrado de medicina y enfermería, además de las capacitaciones en el ambiente laboral en APS, relacionadas con la dedicación y carga horaria con respecto a la salud mental, son bajas en general, siendo la capacitación en servicio levemente mejor en América del Sur. La formación y capacitación en servicios de APS son al menos insatisfactorias, no permiten dar respuesta adecuada a la problemática de SM. Son insuficientes para mejorar la capacidad de resolver frente a la demanda de atención por problemas psicosociales y de SM. La integración de la SM en los servicios de APS por lo general es limitada. Esto restringe considerablemente la capacidad de la APS para cumplir con las funciones en relación a la SM y el nivel de resolutividad que se le confía en el contexto de un modelo comunitario de salud mental. Según éste informe la disponibilidad de protocolos de evaluación y tratamiento es muy dispar, de casi inexistente a no necesariamente disponibles o conocidos según la región. Se suma además la escasa interacción y limitada integración, a veces por falta de información, de los profesionales de APS con profesionales especializados en SM y con agentes del sistema alternativo de atención (medicina complementaria integrativa). Respecto del plan terapéutico a instaurar a las personas que consultan en APS por problemáticas de SM, como plantea el documento, es evidente que el acceso a los psicofármacos es una condición necesaria para que pueda cumplir la función de atenderlas apropiadamente. En ese contexto se informa que los medicamentos sí están disponibles, aunque, en muchos casos, de manera parcial. En cuanto a los recursos humanos los datos muestran la escasez de los mismos y la desigual distribución en los países, con una marcada variabilidad entre las subregiones. Es importante hacer mención de los factores que limitan el acceso a la salud mental son entre otros: • la distribución entre el sistema privado y público de los profesionales capacitados en SM, muchas veces en desmedro del sistema público, donde se concentra a la mayor parte de la población, 58 Rev Bras Med Fam Comunidade. Rio de Janeiro, 2018 Oct; 13(Suppl 1):54-68 La Medicina Familiar y Comunitaria como fuente de cuidados de Salud Mental • el desequilibrio en la distribución del personal capacitado en SM con tendencia hacia mayor cantidad en los hospitales psiquiátricos, con excepción de América del Sur en la que se está iniciando una tendencia a la existencia de mayor cantidad de personal capacitado en centros de atención ambulatoria intermedia, pero sin que exista aún una tendencia comparable en la APS, • una distribución desigual de los psicofármacos disponibles, y • una distribución desigual geográfica del personal capacitado, en que la mayoría se concentra en las ciudades y en especial en las capitales de los países de la región. Otro aspecto fundamental en el desarrollo de estrategias para la intervención en patologías de SM es el rol de la sociedad civil – comunidad, asociaciones de usuarios y familiares. Si bien el documento de la OMS sobre la región plantea que por el momento es muy limitado e incluso inexistente (no participa ni en la discusión ni en la toma de decisiones respecto de la prestación de servicios de salud mental), otros documentos revisados en éste análisis destacan la importancia de la participación de las comunidades, en especial en situaciones de emergencia y desastres. Quizás entonces falta desarrollar dispositivos que permitan la participación de los usuarios en contextos habituales, ya que en situaciones de emergencias y desastres, la comunidad adquiere un rol espontáneo y vital. Frente al rol espontáneo y vital de la sociedad civil es que surge la necesidad de generar estrategias fáciles de implementar para el manejo de problemas y enfermedades de SM, tanto en el diario vivir como en situaciones de emergencias y desastres. Es así como se desarrollan estrategias como el “Programa de Acción para superar las Brechas en SM: mhGAP” (por sus siglas en inglés: mental health Gap Action Program). Este programa fue lanzado en el 2008 por la OMS como una forma de enfrentar el gran desafío de desarrollar estrategias de bajo costo, accesibles a la población, en especial de países de ingresos bajos a medios y en particular en situaciones de emergencias humanitarias. Es así que surgen diferentes guías y programas de fácil implementación y accesibles a personas sin entrenamiento formal o con recomendaciones muy específicas del entrenamiento requerido. Existen por tanto guías clínicas para profesionales de la salud que permiten un apoyo rápido y eficaz en situaciones humanitarias. Una de ellas, es la Guía de Intervención Humanitaria mhGAP (GIH – mhGAP).8 Se trata de una guía clínica básica sobre los trastornos mentales, neurológicos y por uso de sustancias dirigida a los trabajadores del área de la salud: médicos generales, enfermeras, parteras y auxiliares clínicos, así como médicos con especialidades ajenas a la psiquiatría o la neurología que trabajan en servicios no especializados, en particular para los países de ingresos bajos y medianos. Contiene consejos para los directores de servicios clínicos respecto de principios generales de atención aplicables a las emergencias humanitarias, relevando la importancia del apoyo multisectorial. Plantea los siguientes principios generales de la atención a personas con trastornos mentales, neurológicos y por uso de sustancias en emergencias humanitarias: 1. Principio de la comunicación: Se pondera la comunicación directa, concisa, respetuosa confidencial, con escucha activa, incluyente para el paciente, y, si es necesario, con intérpretes capacitados. 2. Principio de evaluación: Se da importancia a la plena identificación del trastorno mental, neurológico o uso de sustancias y a la interpretación que le da el paciente a su problema de salud. Debe haber un interrogatorio que incluya antecedentes familiares, antecedentes de la persona, Rev Bras Med Fam Comunidade. Rio de Janeiro, 2018 Oct; 13(Suppl 1):54-68 59 Moral M, Yuruhán D, Ruiz C, Anderson MIP, Carmona PA, Fortes S, et al. estrategias utilizadas para la resolución del problema y el apoyo social con el que cuenta. Se aconseja hacer preguntas sobre el suicidio de manera sensible. 3. Principio para el manejo: La capacitación y el entendimiento del manejo que tendrá el paciente por parte de los cuidadores. 4. Principio de la reducción del estrés y del fortalecimiento del apoyo social: Fundamental es la reducción del estrés que puedan presentar el paciente o sus cuidadores. Se recomienda el uso de la Guía del IASC sobre salud mental y apoyo psicosocial en emergencias humanitarias y catástrofes, además de ejercicios de relajamiento con técnicas de respiración. 5. Principio de protección de los derechos humanos: Proteger los derechos de las personas con afecciones mentales o neurológicas e integrarlos a la comunidad. 6. Principio de la atención al bienestar general: Ayudar a las personas afectadas al acceso sin peligro a los servicios que necesiten para sobrevivir, llevar una vida digna y velar por la salud física general. Orienta sobres los síntomas y signos del estrés agudo y postraumático, el duelo, la depresión mayor, la psicosis, la epilepsia, la adicción al alcohol y otros trastornos emocionales; mencionando los puntos importantes para la evaluación del cuadro clínico; y especifica un plan de manejo básico que incluye las intervenciones farmacológicas y las psicosociales. Existen además programas de consejería breve orientada a problemas, de fácil implementación por profesionales de la salud no especializados en SM. Es el caso del Programa de Manejo de Problemas “Plus” – Ayuda, para adultos afectados por la angustia en comunidades expuestas a la adversidad, dentro del programa mhGAP ya descrito; programa conocido con las siglas PM+.9 La estrategia PM+ requiere que los profesionales tengan habilidades de ayuda básica, con foco en la comunicación y en el construir una relación con las personas que van a atender. Los Médicos de Familia, especialmente aquellos formados en el abordaje centrado en el paciente y en las habilidades comunicacionales, están especialmente preparados para ello. Por otra parte, el PM +, ayuda a mejorar las habilidades para tratar pacientes que han tenido que enfrentar situaciones vitales complejas. Las intervenciones o estrategias conductuales consideradas son: 1. MANEJO DEL ESTRÉS: se utiliza la estrategia de respiración lenta, la más apropiada en la mayoría de las situaciones que producen ansiedad y estrés. Se puede combinar con métodos de relajación localizada cuando la situación es percibida como más compleja. Esta intervención conductual es introducida desde el inicio en el PM+ y debiera ser practicada al término de cada sesión. 2. MANEJO DE LOS PROBLEMAS: en situaciones en las que las personas se enfrentan a problemas prácticos (cesantía, conflictos familiares, etc.). El profesional y el usuario trabajarán en conjunto para considerar soluciones posibles al problema que más preocupa a la persona. Pueden plantear en conjunto soluciones para la resolución del problema y generar una estrategia para llevar a cabo las soluciones. 3. PONERSE EN MARCHA, MANTENERSE ACTIVO: el objetivo es recuperar y mantener el nivel de actividad, lo que tiene un impacto inmediato en el humor, ya que las personas con depresión con frecuencia dejan de hacer sus actividades usuales. 60 Rev Bras Med Fam Comunidade. Rio de Janeiro, 2018 Oct; 13(Suppl 1):54-68 La Medicina Familiar y Comunitaria como fuente de cuidados de Salud Mental 4. FORTALECIMIENTO DEL APOYO SOCIAL: los individuos con problemas emocionales pueden aislarse de sus redes de apoyo tanto las personales como las organizacionales. Si las personas tienen una buena red de apoyo social y la usan con regularidad, puede que todo lo que se necesite es alentarlas a continuar haciéndolo. En el caso de las personas que no las tienen, puede que tome más tiempo analizar el cómo pueden mejorar sus redes de apoyo social y se les debe ayudar a que desarrollen un plan para que reciban más apoyo social. 5. MANTENERSE BIEN Y MIRAR AL FUTURO: de la misma manera que lo hacen las personas que se recuperan de heridas o enfermedades físicas, las personas con problemas o enfermedades mentales sufren “alzas” y “bajas” de sus emociones durante la recuperación. Es importante al finalizar las sesiones aclarar que el practicar las estrategias después de terminada la consejería es fundamental para mantenerse bien. En la eventualidad de que reaparezca una situación problemática que cause angustia, es probable que la persona sea capaz de responder utilizando estas estrategias. Objetivo General Analizar el rol del médico familiar en la Salud Mental en Iberoamérica con el fin de apoyar la salud integral de las personas que padecen trastornos a consecuencia de enfrentar situaciones de conflictos (armados o no armados), de emergencias y desastres naturales, así como los presentes en la cotidianeidad de la vida. Objetivos específicos 1. Identificar el rol del médico familiar en la detección y el abordaje de los trastornos prevalentes de Salud Mental, así como la capacidad de apoyar la detección de trastornos en la población afectada por conflictos armados y no armados y situaciones de emergencia y desastres naturales, en Iberoamérica. 2. Conocer el rol del Médico Familiar en la detección precoz de trastornos de salud mental en todas las situaciones de la vida y en especial el estrés post traumático en población del post-conflicto y emergencias y desastres. 3. Analizar la disponibilidad de Médicos Familiares y de recursos humanos en general en los países de Iberoamérica para el abordaje de la problemática de salud mental en la población a cargo. 4. Reconocer la capacidad de participación de Médicos de Familia en el desarrollo de estrategias para el abordaje de problemáticas que se generan en conflictos armados y no armados, emergencias y desastres naturales. 5. Identificar las fortalezas del modelo de atención integral, centrada en la persona, su familia y la comunidad, como una forma permanente de detección precoz y manejo de problemáticas de salud mental en la práctica clínica habitual de los Médicos de Familia. 6. Identificar las destrezas con las que cuenta el Médico de Familia para la prevención de problemas de salud mental, de acuerdo al ciclo vital individual y familiar. Rev Bras Med Fam Comunidade. Rio de Janeiro, 2018 Oct; 13(Suppl 1):54-68 61 Moral M, Yuruhán D, Ruiz C, Anderson MIP, Carmona PA, Fortes S, et al. Método Estudio descriptivo de corte transversal, que recoge la opinión de diferentes profesionales de la salud en Iberoamérica sobre las capacidades propias y ajenas para abordar problemas de salud mental. Para la recolección de la información, se diseñó una encuesta orientada por los objetivos del trabajo, que constó de 42 preguntas, 12 de identificación general (país, ciudad de residencia, edad, género, profesión, etc.), 21 preguntas cerradas y 9 abiertas, dirigidas a conocer la opinión, actitudes o experiencias, en relación a salud mental. Para la validación del instrumento se realizó una prueba piloto que permitió la realización de correcciones por los autores principales del trabajo. El link de la encuesta fue enviado vía correo electrónico a miembros de la Confederación Iberoamericana de Medicina Familiar, y se les solicitó que la promocionaran entre sus contactos. Se estableció como criterio de inclusión que todos los encuestados fueran profesionales de la salud graduados o en formación de postgrado, excluyéndose los estudiantes en formación de pregrado. Se obtuvieron 100 respuestas a la encuesta, de las cuales 99 cumplieron los criterios de inclusión y 1 no los cumplió por tratarse de 1 estudiante de pregrado en medicina. Las 99 restantes fueron respondidas por Médicos con o sin especialidad y 1 Psicólogo. Al proceder al análisis de las respuestas de las 99 encuestas en 8 de ellas se suprimieron 2 preguntas al detectarse errores en la selección de las alternativas planteadas o en la interpretación de las preguntas abiertas. En esas preguntas el análisis se basó en 91 encuestas. El análisis final se basó en 99 encuestas, excepto en los ítems 16 y 17 donde se obtuvieron 91 respuestas. Revisión Bibliográfica Se realizó una revisión bibliográfica del tema de Salud Mental (SM) en situaciones de emergencias y desastres, naturales y antrópicas, y en contextos de la vida cotidiana, con búsqueda activa orientada a artículos que entreguen información global de SM (Regiones, Subregiones) y oriente hacia el manejo de problemas de SM. El criterio de selección de la bibliografía fue fundamentalmente por la búsqueda de los últimos 10 años de informes de organizaciones de la salud y aportes de los mismos autores en relación a sus países de origen. La revisión bibliográfica se realizó mediante la distribución de diferentes textos en grupos de interés. Se distribuyeron18 documentos entre los 12 interesados en participar, solicitando que realizaran un resumen de los documentos que les habían sido asignados. Se obtuvieron 9 resúmenes de los documentos distribuidos de 7 participantes. Presentación en Plenario de la Cumbre Se presentó en plenario en la Cumbre la temática de Salud Mental en Iberoamérica y las necesidades de abordaje, como una forma de introducir el tema para los el trabajo de grupo. 62 Rev Bras Med Fam Comunidade. Rio de Janeiro, 2018 Oct; 13(Suppl 1):54-68 La Medicina Familiar y Comunitaria como fuente de cuidados de Salud Mental Trabajo de grupo Durante el evento de la Cumbre se procedió al trabajo de grupo para establecer recomendaciones sobre la problemática de Salud Mental para la Carta de Cali. Inicialmente se hizo una presentación sobre la temática de Salud Mental. Luego los participantes se distribuyeron en grupos pequeños (5 a 6 personas como máximo) con preguntas claves respecto de los objetivos del presente trabajo. Específicamente se les indicó proponer tres recomendaciones para la Carta de Cali. Al finalizar se realizó un catastro de todas las recomendaciones, agrupándolas según calidad y acción en el área de la Salud Mental, para finalizar con tres recomendaciones construidas en base a las recomendaciones. Resultados de la Encuesta De las 99 encuestas admitidas en el estudio, se desprende que los encuestados provenían de 15 (75%) países que conforman la Confederación Iberoamericana de Medicina Familiar (CIMF), registrándose una abstención en el llenado del instrumento en 5 (25%) países de la región (Cuba, El Salvador, Nicaragua, Portugal y República Dominicana). La participación por país fue desigual (Gráfica 1), determinando un sesgo que se deberá considerar en futuros estudios. Gráfica 1. Representación (%) de los países en las respuestas a la encuesta (n=83). En la distribución por sexo se encuentra un 58,6% mujeres y 41,4% hombres. Según la profesión, el 99% declaró ser médico y 1% psicólogo. El 93% de los médicos señaló que estaban vinculados a la Medicina Familiar (MF), con un 8% de residentes, 85% especialista en MF cumpliendo residencia completa. 4% de médicos generales, 2% psiquiatra y 1% de medicina interna. De los médicos de familia, la mayoría (83%) trabaja como médico de atención primaria de la salud (entendida en el contexto de esta investigación como primer nivel de Rev Bras Med Fam Comunidade. Rio de Janeiro, 2018 Oct; 13(Suppl 1):54-68 63 Moral M, Yuruhán D, Ruiz C, Anderson MIP, Carmona PA, Fortes S, et al. atención), mientras que un 8% contestó que eran tutores de residentes de MF. Es necesario destacar, que la pregunta estuvo orientada a la labor principal que desempeñaban los médicos familiares, razón por la cual, no se puede inferir, si los que ejercen en atención primaria de la salud, también cumplen labores de tutoría de residentes (Gráfica 2). Grafica 2. Distribución (%) de los Medicos Familiares que han contestado la encuesta segundo el labor (n=83). De los MF con formación de postgrado universitaria, descartando aquellos países en los que sólo respondió 1 persona, destaca que la mayoría de los MF que respondieron de Argentina (64%), Bolivia (71%) y Brasil (83%), opinaron que la capacidad de los MF para abordar problemas de SM es Regular. Por su parte, los MF de México respondieron 45,5% Regular y 45,5% Buena, con un 9% Mala. Los MF de países como Chile (100%), Colombia (86%), Costa Rica (100%), Ecuador (80%), Panamá (88%), Paraguay (67%), Uruguay (75%) y Venezuela (100%) declaran que la capacidad de los MF de su país es Buena y Muy buena. (Gráfica 3). Gráfica 3. Comparación (%) entre la percepción de los Médicos Familiares respecto su propia capacidad en manejo de Salud Mental y la capacidad de los otros MF y (n=83). Fuente: Encuesta del Grupo de Trabajo 64 Rev Bras Med Fam Comunidade. Rio de Janeiro, 2018 Oct; 13(Suppl 1):54-68 La Medicina Familiar y Comunitaria como fuente de cuidados de Salud Mental Al hablar de la propia capacidad para abordar problemáticas de Salud Mental, se observa una mejor percepción de sí mismos. Así se dan casos como por ejemplo, los MF de Argentina perciben que un 27% de sus colegas de MF tienen una buena y muy buena capacidad, y que ellos mismos tienen un 64% entre buena y muy buena capacidad. Así mismo, en Brasil, varía de 17% a 100% la autopercepción de calidad, mientras que en México fluctúa de un 45% a un 82% y Uruguay varía de un 75% a un 100%. Este cambio podría hacer pensar que son más estrictos respecto de los demás médicos de familia que de sí mismos, o que hay un sesgo por mayor interés respecto de la problemática de SM al responder la encuesta y que se saben con mayores capacidades que sus colegas. Una comparación porcentual entre la percepción de los MF acerca de su propia capacidad y la de sus colegas, puede ser vista en la Grafica 3. Por otro lado, el único psicólogo que responde la encuesta es de Argentina y declara que la capacidad y el interés de los MF en la problemática de SM es regular, lo que coincide con la percepción de los médicos de Argentina, que en su mayoría (64,3%) consideran que es regular o mala. Sólo un 35,7% la considera como buena o muy buena. El interés sigue una tendencia similar, los encuestados declaran que el interés de los MF de su país es en su mayoría regular (78,6%) El número de médicos generales (MG) sin especialidad que respondió la encuesta fue muy pequeño (2 de Chile, 1 de Panamá y 1 de Colombia).Sin embargo, se analiza ya que pudiera orientar en una tendencia sobre la percepción que tienen de los MF. Los MG de Colombia, Panamá y uno de Chile, indicaron que la capacidad de los MF era buena en la problemática de SM, el otro de Chile, la consideró regular, y al mismo tiempo también señaló como regular su propia capacidad. Los demás calificaron como muy buena su propia capacidad sin ser MF, en contraposición con su percepción de que la capacidad de los MF es buena. Destaca que dan como justificación de ésta diferencia su propio interés en el tema de SM y, por lo tanto, su interés en capacitarse. Por otra parte, uno de ellos plantea que la capacidad de los MF se debe al tipo de atención integral que proveen. Respecto del interés en el tema de SM, los MG lo informan como bueno y muy bueno en un 100%, pero que no siempre lo pueden desarrollar por las limitaciones de tiempo de las consultas. Los otros especialistas encuestados, 2 psiquiatras y 1 médico internista (MI), consideraron que la capacidad de los MF para detectar problemas de SM es buena. Respecto del interés de los MF en la detección y abordaje de los problemas de salud mental dicen que es bueno y muy bueno. El MI destaca que es por el conocimiento que tienen los MF de cómo los trastornos de SM inciden en la salud física. En relación a la pregunta sobre el abordaje de las personas que presentan problemas de SM en el nivel primario de atención, los tres eligen la respuesta: “Pienso que los médicos familiares son profesionales de gran relevancia porque los conceptos y herramientas de esta especialidad pueden traer nuevas miradas y posibilidades de intervención en los trastornos mentales”. Sin embargo, cuando se amplía la revisión a todos los médicos, la dispersión de las respuestas y el responder a varias posibilidades a la vez, incluso contrapuestas, hace difícil observar un patrón en el resto de los médicos. En cuanto a la frecuencia de los problemas de SM observados, de las respuestas válidas de los médicos, el 30% indica al Trastorno de Ansiedad como el más frecuente, le sigue la depresión con 27%, insomnio 17%, alcoholismo 10%, adicción a drogas ilícitas 7%, trastornos alimentarios 5% y trastorno de estrés postraumático (TEPT) 4%. Rev Bras Med Fam Comunidade. Rio de Janeiro, 2018 Oct; 13(Suppl 1):54-68 65 Moral M, Yuruhán D, Ruiz C, Anderson MIP, Carmona PA, Fortes S, et al. Gráfico 4. Frecuencia de los problemas de salud mental según la percepción de encuestados de Iberoamérica. Marzo 2018 (n 99). En relación a la pregunta sobre ¿qué otras patologías podría incluir como frecuentes?, en los encuestados de varios países, aparece en un 12% la Esquizofrenia. Esto podría reflejar que los MF también están jugando un rol en el abordaje de los trastornos crónicos de SM, al igual que lo hacen por ejemplo en las enfermedades crónicas cardiovasculares. Incluso algunos lo justifican en el contexto de la cronicidad, incluyendo también como control de seguimiento al Trastorno Bipolar, al Trastorno por Déficit de Atención y otros. Uno de los MF nos hace recordar que el Tabaquismo como cualquier adicción es un problema de SM. El no haberla incluido en el listado refleja la normalización de su uso, incluso entre nosotros los médicos. En el mismo contexto un 5,5% nos hacen recordar que la violencia en las relaciones en general y el maltrato familiar no son infrecuentes. En cuanto al rol del MF en los conflictos armados, un 31% plantea que tiene competencias específicas y un especial vínculo con la población, un 21% dice que tiene competencias adecuadas para formar parte del equipo, un 17% que existe la necesidad de mayor formación y un 2% plantea que no tienen ningún rol. En cuanto al rol del MF en los desastres naturales hay una distribución muy similar a la de los conflictos armados. Un 41 % dice que el MF tiene competencias específicas y vínculo con la población, un 30% que tiene competencias adecuadas para formar parte del equipo, un 24% que es necesaria mayor capacitación, un 4% que no es su rol y 1% no lo sabe. En cuanto a las Fortalezas que tiene el MF para actuar en dichas situaciones, se expresan: integralidad, longitudinalidad y enfoque familiar, en el contexto de la continuidad de la atención, la cercanía con la comunidad - física y afectiva - y la formación adecuada, aunque se plantea que siempre se necesita más. En cuanto a las Barreras, hay un claro desconocimiento de la participación en políticas públicas. Un 60% no sabe o cree que los MF no participamos en definiciones de políticas públicas respecto de la Salud Mental de las personas. Un 65% no sabe o no cree que participemos en el desarrollo de estrategias de capacitación y protocolos de abordaje de problemas de Salud Mental. 66 Rev Bras Med Fam Comunidade. Rio de Janeiro, 2018 Oct; 13(Suppl 1):54-68 La Medicina Familiar y Comunitaria como fuente de cuidados de Salud Mental Resultados del Trabajo de Grupo Durante el proceso de trabajo de grupo se generaron varias inquietudes respecto a la salud mental de los equipos de salud. Así mismo, surgió una marcada preocupación por la SM de los residentes de Medicina Familiar, resaltando que durante su formación, los tutores de MF deberían proporcionar las herramientas necesarias para que los futuros Médicos Familiares desarrollen las capacidades de autocuidado, y que además, les permita implementar y fortalecer la atención de SM en los centros en los que trabajen. Por otra parte, el grupo manifestó que se debe garantizar la salud mental de los prestadores de servicios, entre ellos los MF, garantizando un espacio de descarga emocional y salarios dignos. También se consideró prioritario que los MF participen en la creación de protocolos de intervención en equipo, que especifiquen los alcances y el límite de cada intervención. Para ello se debe fortalecer la capacitación sobre SM, para trabajar en la prevención en SM y realizar un diagnóstico precoz cuando es necesario. Estructurando además capacitaciones en intervenciones comunitarias en SM, para desarrollar la estrategia de rehabilitación basada en la comunidad y fortalecimiento de las redes de apoyo. Se destacó además, la importancia de desarrollar competencias específicas de acuerdo al perfil epidemiológico de cada país. Orientando el quehacer del MF a generar el más alto impacto en la salud de la población. Conclusión Dada la alta prevalencia e incidencia de la problemática de SM y el influjo que tienen en el desarrollo de las personas, las familias y las comunidades, los MF debemos incorporar en nuestro diario quehacer, en la mirada integral, preguntas orientadas a conocer situaciones del área emocional. En cada atención, sea cual sea el motivo de ella, debemos preguntar respecto del ámbito de salud mental, no sólo “¿Cómo está usted, cómo le va?”. Debemos preguntar específicamente cómo está su ánimo, ha tenido algún problema emocional en el último tiempo. Debemos recordar siempre que las personas no consultan por problemas del ámbito mental. No sólo por el estigma que aún persiste en el mundo respecto de éste tipo de problema, sino por la misma condición del problema de salud mental. Está entonces el desafío, que es al mismo tiempo una oportunidad, de visibilizara la Medicina Familiar como una especialidad que puede y debe abordar la problemática de Salud Mental por su formación y la cercanía con la población a cargo en sus territorios. En este contexto se realizan las siguientes recomendaciones para ser incluidas en la Carta de Cali: En este contexto se realizan las siguientes recomendaciones para ser incluidas en la Carta de Cali: 1. Incorporar y/o fortalecer, según sea el caso, la formación en salud mental y comunitaria necesaria para los espacios de atención en los que se desenvuelven los médicos de familia, sin considerar las situaciones vitales como algo patológico, con el desarrollo de estrategias de autocuidado de las personas, sustentables tanto en el tiempo como en la capacidad financiera de los países de Iberoamérica; con el fin de desarrollar la capacidad de enfrentar situaciones estresantes de la vida diaria, que permitan desarrollar fortalezas para situaciones de emergencias y desastres. 2. Fortalecer y empoderar el trabajo comunitario con el fin de que sea la propia comunidad empoderada quien establezca redes de apoyo en problemáticas de salud mental y esté preparada Rev Bras Med Fam Comunidade. Rio de Janeiro, 2018 Oct; 13(Suppl 1):54-68 67 Moral M, Yuruhán D, Ruiz C, Anderson MIP, Carmona PA, Fortes S, et al. en conjunto para enfrentar situaciones de la vida diaria y le permita desarrollar acciones inmediatas en situaciones de emergencias y desastres naturales o no. 3. Priorizar estrategias de autocuidado costo efectivas y orientadas a herramientas de desarrollo personal y de las familias, incluidos los equipos de salud, y los profesores, tutores de Medicina Familiar. De modo tal que se establezcan relaciones virtuosas orientadas a un trato cordial y constructivo. El cuerpo docente de las residencias de MF debe hacerse cargo de acciones tendientes al autocuidado de los alumnos, propendiendo a facilitar el proceso de aprendizaje y la preparación para ejercer la profesión en un medio tan complejo como son los centros de Atención Primaria de Salud o en cualquier contexto en el que se trabaje con la estrategia de APS. Referencias 1. Resumen Ejecutivo Prevención del Suicidio OMS-OPS Nota descriptiva Enero 2018. Disponible en: http://www.who.int/mediacentre/ factsheets/fs398/es/ 2. Manual para la protección y cuidado de la Salud Mental en situaciones de Emergencias y Desastres: JICA (Agencia de Cooperación Internacional del Japón) – Ministerio de Salud Chile, 2015: Disponible en: http://web.minsal.cl/wp-content/uploads/2015/09/Manualpara-la-protecci%C3%B3n-y-cuidado-de-la-Salud-Mental-en-situaciones-de-Emergencias-y-Desastres.pdf 3. Mario Waissbluth, El Tsunami de la Patología Mental En Chile, Reflexiones de Valor Público Nº 1, Centro de Sistemas Públicos/Ingeniería Industrial – Universidad de Chile Octubre 2017. Disponible en: http://www.sistemaspublicos.cl/wp-content/uploads/2017/10/Reflexionesde-Valor-P%C3%BAblico-N%C2%B01.-El-Tsunami-de-la-Patolog%C3%ADa-Mental-en-Chile.pdf 4. Organización Mundial de la Salud (OMS). Volver a construir mejor: atención de salud mental sostenible después de una emergencia. Información general. OMS: Ginebra, 2013. Disponible en: http://apps.who.int/iris/bitstream/handle/10665/85619/WHO_MSD_MER_13.1_ spa.pdf;jsessionid=0B3B48F7C9B8CFB6037AAF7C114EFB96?sequence=1 5. McDaid, D., E. Hewlett and A. Park, “Understanding effective approaches to promoting mental health and preventing mental illness”, OECD Health Working Papers, No. 97, OECD Publishing, Paris, Oct 2017. Disponible en: http://dx.doi.org/10.1787/bc364fb2-en 6. WHO-AIMS: Informe sobre los Sistemas de Salud de Salud Mental en América Latina y El Caribe, OPS 2013. Disponible en:http://www. paho.org/per/images/stories/FtPage/2013/WHO-AIMS.pdf 7. WHO-AIMS (Assessment Instrument for Mental Health Systems): Informe sobre los Sistemas de Salud de Salud Mental en América Latina y El Caribe, OPS 2013; Disponible en: http://www.paho.org/per/images/stories/FtPage/2013/WHO-AIMS.pdf 8. Organización Panamericana de la Salud. Guía de intervención humanitaria mhGAP (GIH-mhGAP). El manejo clínico de los trastornos mentales neurológicos y por uso de sustancias en las emergencias humanitarias. Washington, DC : OPS, 2016. Disponible: http://iris. paho.org/xmlui/bitstream/handle/123456789/28418/9789275319017_spa.pdf 9. Organización Mundial de la Salud, Enfrentando Problemas Plus (EP+): Ayuda psicológica individual para adultos afectados por la angustia en comunidades expuestas a adversidad. (Versión genérica de prueba de mercado 1.0). Ginebra, OMS, 2016. http://apps.who.int/iris/ bitstream/handle/10665/259696/WHO-MSD-MER-16.2-spa.pdf?sequence=1 68 Rev Bras Med Fam Comunidade. Rio de Janeiro, 2018 Oct; 13(Suppl 1):54-68
https://openalex.org/W3118608929	https://kclpure.kcl.ac.uk/portal/files/141628134/Russell_et_al_Accepted_Elife.pdf	English	null	Pituitary stem cells produce paracrine WNT signals to control the expansion of their descendant progenitor cells	eLife	2,021	cc-by	21,469	Citation for published version (APA): Russell, J. P., Lim, X., Santambrogio, A., Yianni, V., Kemkem, Y., Wang, B., Fish, M., Haston, S., Grabek, A., Hallang, S., Lodge, E. J., Patist, A. L., Schedl, A., Mollard, P., Nusse, R., & Andoniadou, C. L. (2021). Pituitary stem cells produce paracrine WNT signals to control the expansion of their descendant progenitor cells. eLife, 10, 1-23. Article e59142. https://doi.org/10.7554/eLife.59142 Citing this paper Pl h C t g t s pape Please note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this may differ from the final Published version. If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you are again advised to check the publisher's website for any subsequent corrections. Citation for published version (APA): Russell, J. P., Lim, X., Santambrogio, A., Yianni, V., Kemkem, Y., Wang, B., Fish, M., Haston, S., Grabek, A., Hallang, S., Lodge, E. J., Patist, A. L., Schedl, A., Mollard, P., Nusse, R., & Andoniadou, C. L. (2021). Pituitary stem cells produce paracrine WNT signals to control the expansion of their descendant progenitor cells. eLife, 10, 1-23. Article e59142. https://doi.org/10.7554/eLife.59142 24 Hills Road, P 01223 855346 Cambridge W elifesciences.org CB2 1JP T @elife_sciences 24 Hills Road, P 01223 855346 Cambridge W elifesciences.org CB2 1JP T @elife_sciences 24 Hills Road, P 01223 855346 Cambridge W elifesciences.org CB2 1JP T @elife_sciences FOR PEER REVIEW - CONFIDENTIAL Funding: g UKRI \| Medical Research Council (MRC): Cynthia Lilian Andoniadou, MR/L016729/1; UKRI \| Medical Research Council (MRC): Cynthia Lilian Andoniadou, MR/T012153/1; Deutsche Forschungsgemeinschaft (DFG): Cynthia Lilian Andoniadou, 314061271 - TRR 205; Howard Hughes Medical Institute (HHMI): Roel Nusse; Agence Nationale de la Recherche (ANR): Patrice Mollard, ANR-18-CE14-0017; Fondation pour la Recherche Médicale (FRM): Patrice Mollard, DEQ20150331732; Lister Institute of Preventive Medicine: Cynthia Lilian Andoniadou The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Author contributions: John Russell: Conceptualization; Formal analysis; Investigation; Methodology; Writing - original draft; Writing - review and editing Xinhong Lim: Resources; Writing - review and editing Alice Santambrogio: Formal analysis; Investigation Val Yianni: Software; Formal analysis; Investigation Yasmine Kemkem: Resources; Investigation Bruce Wang: Resources Matthew Fish: Resources; Investigation Scott Haston: Investigation Anaëlle Grabek: Resources Shirleen Hallang: Investigation Emily Lodge: Investigation Amanda Patist: Investigation Andreas Schedl: Resources; Supervision Patrice Mollard: Resources; Supervision; Funding acquisition; Methodology; Writing - review and editing Roel Nusse: Resources; Supervision; Funding acquisition; Methodology; Writing - review and editing Cynthia Andoniadou: Conceptualization; Supervision; Funding acquisition; Investigation; Methodology; Writing - original draft; Writing - review and editing Data Availability: y Sequencing data can be accessed through the following link: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA421806 Datasets Generated: Pituitary stem cells produce paracrine WNT signals to control the expansion of their descendant progenitor cells: Andoniadou CL et al, 2020, https://www.ncbi.nlm.nih.gov/bioproject/PRJNA421806, NCBI Bioproject, PRJNA421806 Reporting Standards: N/A Pituitary stem cells produce paracrine WNT signals to control the expansion of their descendant progenitor cells Tracking no: 20-05-2020-RA-eLife-59142R1 Impact statement: Stem cells of the pituitary gland contribute to organ growth cell non-autonomously by promoting proliferation of committed progenitors through WNT ligand secretion. 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Download date: 24. Oct. 2024 eLife Sciences Publications, Ltd is a limited liability non-profit nonstock corporation incorporated in the State of Delaware, USA with company number 5030732, and is registered in the UK with company number FC030576 and branch number BR015634 at the address 24 Hills Road, Cambridge, CB2 1JP. Ethics: Ethics: Human Subjects: No Animal Subjects: Yes Ethics Statement: This study was performed under compliance of the Animals (Scientific Procedures) Act 1986, Home Office License (P5F0A1579) and KCL Biological Safety approval for project 'Function and Regulation of Pituitary Stem Cells in Mammals' Pituitary stem cells produce paracrine WNT signals to control the 1 expansion of their descendant progenitor cells 2 John Parkin Russell1, Xinhong Lim2,3, Alice Santambrogio1,4, Val Yianni1, Yasmine 3 Kemkem5, Bruce Wang6,7, Matthew Fish6, Scott Haston8, Anaëlle Grabek9, Shirleen 4 Hallang1, Emily Jane Lodge1, Amanda Louise Patist1, Andreas Schedl9, Patrice 5 Mollard5, Roeland Nusse6, Cynthia Lilian Andoniadou1,4,* 6 7 1 Centre for Craniofacial and Regenerative Biology, King’s College London, Floor 27 Tower 8 Wing, Guy’s Campus, London, SE1 9RT, United Kingdom 9 2 Skin Research Institute of Singapore, Agency for Science, Technology and Research, 10 Singapore 308232 11 3 Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 12 308232 13 4 Department of Medicine III, University Hospital Carl Gustav Carus, Technische Universität 14 Dresden, 01307 Dresden, Germany 15 5 Institute of Functional Genomics (IGF), University of Montpellier, CNRS, INSERM, F- 16 34094 Montpellier, France 17 6 Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 18 94305-5329; Department of Developmental Biology, Stanford University School of 19 Medicine, Stanford, CA 94305-5329, USA 20 7 Department of Medicine and Liver Center, University of California San Francisco, San 21 Francisco, CA 94143-1346, USA 22 8 Developmental Biology and Cancer, Birth Defects Research Centre, UCL GOS Institute of 23 Child Health, London, WC1N 1EH, United Kingdom 24 9 Université Côte d'Azur, Inserm, CNRS, iBV, Nice 06108, France. 25 26 * Corresponding author 27 E-mail: cynthia.andoniadou@kcl.ac.uk. Phone: +44 20 7188 7389. Fax: +44 20 7188 1674 28 29 30 Pituitary stem cells produce paracrine WNT signals to control the 1 expansion of their descendant progenitor cells 2 John Parkin Russell1, Xinhong Lim2,3, Alice Santambrogio1,4, Val Yianni1, Yasmine 3 Kemkem5, Bruce Wang6,7, Matthew Fish6, Scott Haston8, Anaëlle Grabek9, Shirleen 4 Hallang1, Emily Jane Lodge1, Amanda Louise Patist1, Andreas Schedl9, Patrice 5 Mollard5, Roeland Nusse6, Cynthia Lilian Andoniadou1,4,* 6 4 Department of Medicine III, University Hospital Carl Gustav Carus, Technische Universität 14 Dresden, 01307 Dresden, Germany 15 5 Institute of Functional Genomics (IGF), University of Montpellier, CNRS, INSERM, F- 16 34094 Montpellier, France 17 6 Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 18 94305-5329; Department of Developmental Biology, Stanford University School of 19 Medicine, Stanford, CA 94305-5329, USA 20 7 Department of Medicine and Liver Center, University of California San Francisco, San 21 Francisco, CA 94143-1346, USA 22 8 Developmental Biology and Cancer, Birth Defects Research Centre, UCL GOS Institute of 23 Child Health, London, WC1N 1EH, United Kingdom 24 9 Université Côte d'Azur, Inserm, CNRS, iBV, Nice 06108, France. 25 E-mail: cynthia.andoniadou@kcl.ac.uk. Phone: +44 20 7188 7389. Fax: +44 20 7188 1674 28 29 E-mail: cynthia.andoniadou@kcl.ac.uk. Phone: +44 20 7188 7389. Fax: +44 20 7188 1674 28 1 32 32 ABSTRACT 33 34 In response to physiological demand, the pituitary gland generates new hormone- 35 secreting cells from committed progenitor cells throughout life. It remains unclear to 36 what extent pituitary stem cells (PSCs), which uniquely express SOX2, contribute to 37 pituitary growth and renewal. Moreover, neither the signals that drive proliferation 38 nor their sources have been elucidated. We have used genetic approaches in the 39 mouse, showing that the WNT pathway is essential for proliferation of all lineages in 40 the gland. We reveal that SOX2+ stem cells are a key source of WNT ligands. By 41 blocking secretion of WNTs from SOX2+ PSCs in vivo, we demonstrate that 42 proliferation of neighbouring committed progenitor cells declines, demonstrating that 43 progenitor multiplication depends on the paracrine WNT secretion from SOX2+ 44 PSCs. Our results indicate that stem cells can hold additional roles in tissue expansion 45 and homeostasis, acting as paracrine signalling centres to coordinate the proliferation 46 of neighbouring cells. 47 48 ABSTRACT 33 SOX2, paracrine signal, WNT, pituitary gland, stem cell, feedforward regulation 52 2 INTRODUCTION 53 INTRODUCTION 53 How stem cells interact with their surrounding tissue has been a topic of 54 investigation since the concept of the stem cell niche was first proposed (Schofield, 55 1978). Secreted from supporting cells, factors such as WNTs, FGFs, SHH, EGF and 56 cytokines, regulate the activity of stem cells (Nabhan et al., 2018; Palma et al., 2005; 57 Tan and Barker, 2014). Furthermore, communication is known to take place in a bi- 58 directional manner (Doupe et al., 2018; Tata and Rajagopal, 2016). 59 The anterior pituitary (AP) is a major primary endocrine organ that controls 60 key physiological functions including growth, metabolism, reproduction and the stress 61 responses and exhibits tremendous capability to remodel its constituent hormone 62 populations throughout life, in response to physiological demand. It contains a 63 population of Sox2 expressing stem cells that self-renew and give rise to lineage- 64 committed progenitors and functional endocrine cells (Andoniadou et al., 2013; 65 Rizzoti et al., 2013). During embryonic development, SOX2+ undifferentiated 66 precursor cells of Rathke’s pouch, the pituitary anlage (Arnold et al., 2011; Castinetti 67 et al., 2011; Fauquier et al., 2008; Pevny and Rao, 2003), generate all committed 68 endocrine progenitor lineages, defined by the absence of SOX2 and expression of 69 either POU1F1 (PIT1), TBX19 (TPIT) or NR5A1 (SF1) (Bilodeau et al., 2009; Davis 70 et al., 2011). These committed progenitors are proliferative and give rise to the 71 hormone-secreting cells. Demand for hormone secretion rises after birth, resulting in 72 dramatic organ growth and expansion of all populations by the second postnatal week 73 (Carbajo-Perez and Watanabe, 1990; Taniguchi et al., 2002). SOX2+ pituitary stem 74 cells (PSCs) are most active during this period, but the bulk of proliferation and organ 75 expansion during postnatal stages derives from SOX2- committed progenitors. The 76 How stem cells interact with their surrounding tissue has been a topic of 54 investigation since the concept of the stem cell niche was first proposed (Schofield, 55 1978). Secreted from supporting cells, factors such as WNTs, FGFs, SHH, EGF and 56 cytokines, regulate the activity of stem cells (Nabhan et al., 2018; Palma et al., 2005; 57 Tan and Barker, 2014). Furthermore, communication is known to take place in a bi- 58 directional manner (Doupe et al., 2018; Tata and Rajagopal, 2016). 59 directional manner (Doupe et al., 2018; Tata and Rajagopal, 2016). INTRODUCTION 53 The signals driving proliferation of committed progenitor cells are not 82 known, and neither is it known if SOX2+ PSCs can influence this process beyond 83 their minor contribution of new cells. 84 The self-renewal and proliferation of numerous stem cell populations relies 85 upon WNT signals (Basham et al., 2019; Lim et al., 2013; Takase and Nusse, 2016; 86 Wang et al., 2015; Yan et al., 2017). WNTs are necessary for the initial expansion of 87 Rathke’s pouch as well as for PIT1 lineage specification (Osmundsen et al., 2017; 88 Potok et al., 2008). In the postnatal pituitary, the expression of WNT pathway 89 components is upregulated during periods of expansion and remodelling. Gene 90 expression comparisons between neonatal and adult pituitaries or in GH-cell ablation 91 experiments (Gremeaux et al., 2012; Willems et al., 2016), show that the WNT 92 pathway is upregulated during growth and regeneration. 93 Our previous work revealed that during disease, the paradigm of supporting 94 cells signalling to the stem cells may be reversed; mutant stem cells expressing a 95 degradation-resistant -catenin in the pituitary, promote cell non-autonomous 96 development of tumours through their paracrine actions (Andoniadou et al., 2013; 97 Gonzalez-Meljem et al., 2017). Similarly, degradation-resistant -catenin expression 98 in hair follicle stem cells led to cell non-autonomous WNT activation in neighbouring 99 cells promoting new growth (Deschene et al., 2014). In the context of normal 100 homeostasis, stem cells have been shown to influence daughter cell fate in the 101 mammalian airway epithelium and the Drosophila gut via ‘forward regulation’ 102 The self-renewal and proliferation of numerous stem cell populations relies 85 upon WNT signals (Basham et al., 2019; Lim et al., 2013; Takase and Nusse, 2016; 86 Wang et al., 2015; Yan et al., 2017). WNTs are necessary for the initial expansion of 87 Rathke’s pouch as well as for PIT1 lineage specification (Osmundsen et al., 2017; 88 Potok et al., 2008). In the postnatal pituitary, the expression of WNT pathway 89 components is upregulated during periods of expansion and remodelling. Gene 90 expression comparisons between neonatal and adult pituitaries or in GH-cell ablation 91 experiments (Gremeaux et al., 2012; Willems et al., 2016), show that the WNT 92 pathway is upregulated during growth and regeneration. INTRODUCTION 53 59 The anterior pituitary (AP) is a major primary endocrine organ that controls 60 key physiological functions including growth, metabolism, reproduction and the stress 61 responses and exhibits tremendous capability to remodel its constituent hormone 62 populations throughout life, in response to physiological demand. It contains a 63 population of Sox2 expressing stem cells that self-renew and give rise to lineage- 64 committed progenitors and functional endocrine cells (Andoniadou et al., 2013; 65 Rizzoti et al., 2013). During embryonic development, SOX2+ undifferentiated 66 precursor cells of Rathke’s pouch, the pituitary anlage (Arnold et al., 2011; Castinetti 67 et al., 2011; Fauquier et al., 2008; Pevny and Rao, 2003), generate all committed 68 endocrine progenitor lineages, defined by the absence of SOX2 and expression of 69 either POU1F1 (PIT1), TBX19 (TPIT) or NR5A1 (SF1) (Bilodeau et al., 2009; Davis 70 et al., 2011). These committed progenitors are proliferative and give rise to the 71 hormone-secreting cells. Demand for hormone secretion rises after birth, resulting in 72 dramatic organ growth and expansion of all populations by the second postnatal week 73 (Carbajo-Perez and Watanabe, 1990; Taniguchi et al., 2002). SOX2+ pituitary stem 74 cells (PSCs) are most active during this period, but the bulk of proliferation and organ 75 expansion during postnatal stages derives from SOX2- committed progenitors. The 76 activity of SOX2+ PSCs gradually decreases and during adulthood is minimally 77 3 3 activated even following physiological challenge (Andoniadou et al., 2013; Gaston- 78 Massuet et al., 2011; Gremeaux et al., 2012; Zhu et al., 2015). By adulthood, 79 activated even following physiological challenge (Andoniadou et al., 2013; Gaston- 78 Massuet et al., 2011; Gremeaux et al., 2012; Zhu et al., 2015). By adulthood, 79 progenitors carry out most of the homeostatic functions, yet SOX2+ PSCs persist 80 throughout life in both mice and humans (Gonzalez-Meljem et al., 2017; Xekouki et 81 al., 2018). The signals driving proliferation of committed progenitor cells are not 82 known, and neither is it known if SOX2+ PSCs can influence this process beyond 83 their minor contribution of new cells. 84 progenitors carry out most of the homeostatic functions, yet SOX2+ PSCs persist 80 throughout life in both mice and humans (Gonzalez-Meljem et al., 2017; Xekouki et 81 al., 2018). INTRODUCTION 53 93 The self-renewal and proliferation of numerous stem cell populations relies 85 upon WNT signals (Basham et al., 2019; Lim et al., 2013; Takase and Nusse, 2016; 86 Wang et al., 2015; Yan et al., 2017). WNTs are necessary for the initial expansion of 87 Rathke’s pouch as well as for PIT1 lineage specification (Osmundsen et al., 2017; 88 Potok et al., 2008). In the postnatal pituitary, the expression of WNT pathway 89 components is upregulated during periods of expansion and remodelling. Gene 90 expression comparisons between neonatal and adult pituitaries or in GH-cell ablation 91 experiments (Gremeaux et al., 2012; Willems et al., 2016), show that the WNT 92 pathway is upregulated during growth and regeneration. 93 Our previous work revealed that during disease, the paradigm of supporting 94 cells signalling to the stem cells may be reversed; mutant stem cells expressing a 95 degradation-resistant -catenin in the pituitary, promote cell non-autonomous 96 development of tumours through their paracrine actions (Andoniadou et al., 2013; 97 Gonzalez-Meljem et al., 2017). Similarly, degradation-resistant -catenin expression 98 in hair follicle stem cells led to cell non-autonomous WNT activation in neighbouring 99 cells promoting new growth (Deschene et al., 2014). In the context of normal 100 homeostasis, stem cells have been shown to influence daughter cell fate in the 101 mammalian airway epithelium and the Drosophila gut via ‘forward regulation’ 102 4 4 models, where the fate of a daughter cell is directed by a stem cell via juxtacrine 103 Notch signalling (Ohlstein and Spradling, 2007; Pardo-Saganta et al., 2015). It 104 remains unknown if paracrine stem cell action can also promote local proliferation in 105 normal tissues. 106 Here, we used genetic approaches to determine if paracrine stem cell action 107 takes place in the anterior pituitary and to discern the function of WNTs in pituitary 108 growth. Our results demonstrate that postnatal pituitary expansion, largely driven by 109 committed progenitor cells, depends on WNT activation. Importantly, we show that 110 SOX2+ PSCs are the key regulators of this process, acting through secretion of WNT 111 ligands acting in a paracrine manner on neighbouring progenitors. Identification of 112 this forward-regulatory model elucidates a previously unidentified function for stem 113 cells during tissue expansion. 114 115 116 RESULTS 117 WNT-responsive cells in the pituitary include progenitors driving major 118 postnatal expansion. INTRODUCTION 53 We carried out double immunofluorescence staining using antibodies 129 against uncommitted (SOX2), lineage committed (PIT1, TPIT, SF1), and hormone- 130 expressing endocrine cells (GH, PRL, TSH, ACTH or FSH/LH) together with 131 antibodies against GFP labelling the WNT-activated cells. We detected WNT- 132 responsive cells among all the different cell types of the anterior pituitary including 133 SOX2+ PSCs, the three committed populations and all hormone-secreting cells 134 (Figure 1A, Figure 1 – figure supplement 1A). 135 Axin2CreERT2/+;ROSA26mTmG/+ mice and pituitaries were analysed 2 days post- 128 induction. We carried out double immunofluorescence staining using antibodies 129 against uncommitted (SOX2), lineage committed (PIT1, TPIT, SF1), and hormone- 130 expressing endocrine cells (GH, PRL, TSH, ACTH or FSH/LH) together with 131 antibodies against GFP labelling the WNT-activated cells. We detected WNT- 132 responsive cells among all the different cell types of the anterior pituitary including 133 SOX2+ PSCs, the three committed populations and all hormone-secreting cells 134 (Figure 1A, Figure 1 – figure supplement 1A). 135 To confirm if the three committed lineages as well as uncommitted SOX2+ 136 PSCs all expand in response to WNT, we further lineage-traced Axin2-expressing 137 cells for 14 days after tamoxifen administration at P14. Double labelling revealed an 138 increase in all four populations between 2 and 14 days (Figure 1A, B). This increase 139 reached significance for the PIT1 (13.7% at 2 days to 30.3% at 14 days, P=0.000004) 140 and TPIT (3.78% to 11.03%, P=0.008) populations, but not SF1 (0.5% to 4%, n.s.). 141 As this time course ends at P28 at the commencement of puberty, we repeated the 142 analysis for SF1 cells to P42, which spans puberty and the expansion of gonadotrophs 143 (Figure 1 – figure supplement 1B). This reveals a significant expansion in WNT- 144 responsive SF1+ cells as a proportion of the total SF1+ population (P=0.0048, n=3). 145 Lineage tracing of the PIT1-derivates (GH+ somatotrophs, PRL+ lactotrophs, TSH+ 146 thyrotrophs) reveals that there is a preferential expansion of somatotrophs and 147 thyrotrophs (Figure 1 – figure supplement 1C). Only a minority of SOX2+ PSCs were 148 WNT-responsive at 2 days (0.57%) and this population expanded to 2% at 14 days 149 (n.s.), suggesting that these are self-renewing. INTRODUCTION 53 119 To clarify which cells respond to WNT signals in the postnatal anterior pituitary, we 120 first characterised the anterior pituitary cell types activating the WNT pathway at P14, 121 a peak time for organ expansion and a time point when a subpopulation of SOX2+ 122 stem cells are proliferative. The Axin2-CreERT2 mouse line (van Amerongen et al., 123 2012) has been shown to efficiently label cells with activated WNT signalling in the 124 liver, lung, breast, skin, testes and endometrium among other tissues (Lim et al., 2013; 125 Moiseenko et al., 2017; Syed et al., 2020; van Amerongen et al., 2012; Wang et al., 126 2015). Axin2 positive cells were labelled by GFP following tamoxifen induction in 127 models, where the fate of a daughter cell is directed by a stem cell via juxtacrine 103 Notch signalling (Ohlstein and Spradling, 2007; Pardo-Saganta et al., 2015). It 104 remains unknown if paracrine stem cell action can also promote local proliferation in 105 normal tissues. 106 Here, we used genetic approaches to determine if paracrine stem cell action 107 takes place in the anterior pituitary and to discern the function of WNTs in pituitary 108 growth. Our results demonstrate that postnatal pituitary expansion, largely driven by 109 committed progenitor cells, depends on WNT activation. Importantly, we show that 110 SOX2+ PSCs are the key regulators of this process, acting through secretion of WNT 111 ligands acting in a paracrine manner on neighbouring progenitors. Identification of 112 this forward-regulatory model elucidates a previously unidentified function for stem 113 cells during tissue expansion. 114 Here, we used genetic approaches to determine if paracrine stem cell action 107 takes place in the anterior pituitary and to discern the function of WNTs in pituitary 108 growth. Our results demonstrate that postnatal pituitary expansion, largely driven by 109 committed progenitor cells, depends on WNT activation. Importantly, we show that 110 SOX2+ PSCs are the key regulators of this process, acting through secretion of WNT 111 ligands acting in a paracrine manner on neighbouring progenitors. Identification of 112 this forward-regulatory model elucidates a previously unidentified function for stem 113 cells during tissue expansion. 114 5 5 Axin2CreERT2/+;ROSA26mTmG/+ mice and pituitaries were analysed 2 days post- 128 induction. INTRODUCTION 53 GFP+ cells were traced for a period of 8 150 weeks post-induction, which revealed that WNT-responsive descendants continued to 151 expand at the same rate as the rest of the pituitary (n=4-8 mice per time point at P16, 152 To confirm if the three committed lineages as well as uncommitted SOX2+ 136 PSCs all expand in response to WNT, we further lineage-traced Axin2-expressing 137 cells for 14 days after tamoxifen administration at P14. Double labelling revealed an 138 increase in all four populations between 2 and 14 days (Figure 1A, B). This increase 139 reached significance for the PIT1 (13.7% at 2 days to 30.3% at 14 days, P=0.000004) 140 and TPIT (3.78% to 11.03%, P=0.008) populations, but not SF1 (0.5% to 4%, n.s.). 141 As this time course ends at P28 at the commencement of puberty, we repeated the 142 analysis for SF1 cells to P42, which spans puberty and the expansion of gonadotrophs 143 (Figure 1 – figure supplement 1B). This reveals a significant expansion in WNT- 144 responsive SF1+ cells as a proportion of the total SF1+ population (P=0.0048, n=3). 145 Lineage tracing of the PIT1-derivates (GH+ somatotrophs, PRL+ lactotrophs, TSH+ 146 thyrotrophs) reveals that there is a preferential expansion of somatotrophs and 147 thyrotrophs (Figure 1 – figure supplement 1C). Only a minority of SOX2+ PSCs were 148 WNT-responsive at 2 days (0.57%) and this population expanded to 2% at 14 days 149 (n.s.), suggesting that these are self-renewing. GFP+ cells were traced for a period of 8 150 weeks post-induction, which revealed that WNT-responsive descendants continued to 151 expand at the same rate as the rest of the pituitary (n=4-8 mice per time point at P16, 152 6 6 P21, P28, P42, P70) (Figure 1C, D). The time period between 2 and 7 days saw the 153 greatest increase in GFP+ cells, during which, the labelled population nearly tripled in 154 size (Figure 1D). The persistence of labelled cells was evident in longer-term traces 155 using the ROSA26lacZ/+ reporter (Axin2CreERT2/+;ROSA26lacZ/+), up to a year following 156 induction at P14 (Figure 1E, n=4). Clonal analysis using the Confetti reporter, 157 demonstrated that individual Axin2-expressing cells (Axin2CreERT2/+;ROSA26Confetti/+) 158 gave a greater contribution after four weeks compared to lineage-tracing from Sox2- 159 expressing cells (Sox2CreERT2/+;ROSA26Confetti/+), in support of predominant expansion 160 from WNT-responsive lineage-committed progenitors (Figure 1 – figure supplement 161 1D). INTRODUCTION 53 162 To establish if signalling mediated by -catenin is necessary for organ 163 expansion we carried out deletion of Ctnnb1 in the Axin2+ population from P14 164 during normal growth (Axin2CreERT2/+;Ctnnb1lox(ex2-6)/lox(ex2-6) hereby 165 Axin2CreERT2/+;Ctnnb1LOF/LOF). Due to morbidity, likely due to Axin2 expression in 166 other organs, we were limited to analysis up to 5 days post-induction. Deletion of 167 Ctnnb1 resulted in a significant reduction in the number of dividing cells, marked by 168 pH-H3 (40% reduction, Figure 1 – figure supplement 2A, P=0.0313, n=3), confirming 169 that activation of the WNT pathway is necessary for expansion of the pituitary 170 populations. This deletion did not result in significant differences in overall numbers 171 among the three lineages, as determined by the numbers of PIT1+, SF1+ or ACTH+ 172 cells among the targeted population (Figure 1 – figure supplement 2B, n=4 controls, 2 173 mutants). The number of SOX2+ stem cells and cells undergoing cell death also 174 remained unaffected during the 5 day period (Figure 1 – figure supplement 2C and 175 D). Taken together, these results confirm that postnatal AP expansion depends on 176 7 7 WNT-responsive progenitors across all lineages, in addition to SOX2+ PSCs (Figure 177 1F). 178 179 WNT/-catenin signalling is required for long-term anterior pituitary expansion 180 from SOX2+ pituitary stem cells. 181 We further explored the role of WNT pathway activation in postnatal SOX2+ stem 182 cells. To permanently mark WNT-responsive cells and their descendants whilst 183 simultaneously marking SOX2+ PSCs, we combined the tamoxifen-inducible 184 Axin2CreERT2/+;ROSA26tdTomato/+ with the Sox2Egfp/+ strain, where cells expressing 185 SOX2 are labelled by EGFP (Axin2CreERT2/+;Sox2Egfp/+;ROSA26tdTomato/+). Following 186 tamoxifen administration from P21, tdTomato- and EGFP-labelled cells were 187 analysed by flow sorting after 72h, by which point all induced cells robustly express 188 tdTomato (Figure 2A, Figure 2 – figure supplement 1). Double-labelled cells 189 comprised 23.4% of the SOX2+ population (n=5 individual pituitaries) (Figure 2A, 190 arrowheads), with the majority of tdTomato+ cells found outside of the SOX2+ 191 compartment. It was previously shown that only around 2.5-5% of SOX2+ PSCs have 192 clonogenic potential through in vitro assays (Andoniadou et al., 2012; Andoniadou et 193 al., 2013; Perez Millan et al., 2016). To determine if WNT-responsive SOX2+ cells 194 are stem cells capable of forming colonies, we isolated double positive 195 tdTomato+;EGFP+ cells (i.e. INTRODUCTION 53 Axin2+;Sox2+) as well as the single-expressing 196 populations and plated these in equal numbers in stem cell-promoting media at clonal 197 densities (Figure 2B). Double positive tdTomato+;EGFP+ cells showed a significant 198 increase in the efficiency of colony formation compared to single-labelled EGFP+ 199 cells (average 9% compared to 5%, n=5 pituitaries, P=0.0226, Mann-Whitney U test 200 (two-tailed)), demonstrating WNT-responsive SOX2+ PSCs have a greater clonogenic 201 8 8 potential under these in vitro conditions, confirming in vivo data in Figure 1B. As 202 expected from previous work, none of the single-labelled tdTomato+ cells (i.e. SOX2 203 negative) were able to form colonies (Andoniadou et al., 2012). 204 potential under these in vitro conditions, confirming in vivo data in Figure 1B. As 202 expected from previous work, none of the single-labelled tdTomato+ cells (i.e. SOX2 203 negative) were able to form colonies (Andoniadou et al., 2012). 204 expected from previous work, none of the single-labelled tdTomato+ cells (i.e. SOX2 203 negative) were able to form colonies (Andoniadou et al., 2012). 204 To confirm that PSCs with active WNT signalling through -catenin have a 205 greater propensity to form colonies in vitro, we analysed postnatal pituitaries from 206 TCF/Lef:H2B-EGFP mice, reporting the activation of response to WNT signals. This 207 response is detected through expression of an EGFP-tagged variant of histone H2B, 208 which is incorporated into chromatin and diluted in descendants with cell division 209 (Ferrer-Vaquer et al., 2010). Therefore, cells responding to, or having recently 210 responded to WNT, as well as their immediate descendants will be EGFP+. At P21, 211 EGFP+ cells were abundant in all three lobes and particularly in the marginal zone 212 harbouring SOX2+ stem cells (Figure 2 – figure supplement 2A). Through double 213 mRNA in situ hybridisation against Egfp and Sox2 in TCF/Lef:H2B-EGFP pituitaries, 214 we confirmed that Sox2-expressing cells activate H2B-EGFP expression at this time 215 point (Figure 2 – figure supplement 2B). Isolation by fluorescence-activated cell 216 sorting and in vitro culture of the postnatal EGFP+ compartment revealed an 217 enrichment of cells with clonogenic potential in the EGFPHigh fraction compared to 218 EGFPLow or negative cells (Figure 2 – figure supplement 2C, n=5 pituitaries). 219 Together these results reveal that a proportion of postnatal SOX2+ stem cells respond 220 to WNTs through downstream -catenin/TCF/LEF signalling and that these cells have 221 greater clonogenic capacity in vitro. INTRODUCTION 53 222 To further address the role of the canonical WNT response in the activity of 223 To confirm that PSCs with active WNT signalling through -catenin have a 205 greater propensity to form colonies in vitro, we analysed postnatal pituitaries from 206 TCF/Lef:H2B-EGFP mice, reporting the activation of response to WNT signals. This 207 response is detected through expression of an EGFP-tagged variant of histone H2B, 208 which is incorporated into chromatin and diluted in descendants with cell division 209 (Ferrer-Vaquer et al., 2010). Therefore, cells responding to, or having recently 210 responded to WNT, as well as their immediate descendants will be EGFP+. At P21, 211 EGFP+ cells were abundant in all three lobes and particularly in the marginal zone 212 harbouring SOX2+ stem cells (Figure 2 – figure supplement 2A). Through double 213 mRNA in situ hybridisation against Egfp and Sox2 in TCF/Lef:H2B-EGFP pituitaries, 214 we confirmed that Sox2-expressing cells activate H2B-EGFP expression at this time 215 point (Figure 2 – figure supplement 2B). Isolation by fluorescence-activated cell 216 sorting and in vitro culture of the postnatal EGFP+ compartment revealed an 217 enrichment of cells with clonogenic potential in the EGFPHigh fraction compared to 218 EGFPLow or negative cells (Figure 2 – figure supplement 2C, n=5 pituitaries). 219 To confirm that PSCs with active WNT signalling through -catenin have a 205 greater propensity to form colonies in vitro, we analysed postnatal pituitaries from 206 TCF/Lef:H2B-EGFP mice, reporting the activation of response to WNT signals. This 207 response is detected through expression of an EGFP-tagged variant of histone H2B, 208 Together these results reveal that a proportion of postnatal SOX2+ stem cells respond 220 to WNTs through downstream -catenin/TCF/LEF signalling and that these cells have 221 greater clonogenic capacity in vitro. 222 Together these results reveal that a proportion of postnatal SOX2+ stem cells respond 220 to WNTs through downstream -catenin/TCF/LEF signalling and that these cells have 221 greater clonogenic capacity in vitro. INTRODUCTION 53 In vivo 232 genetic tracing of targeted cells over the 22-week period 233 (Sox2CreERT2/+;Ctnnb1LOF/+;ROSA26mTmG/+ compared to 234 Sox2CreERT2/+;Ctnnb1LOF/LOF;ROSA26mTmG/+ pituitaries) revealed that targeted 235 (Ctnnb1-deficient) SOX2+ PSCs were capable of giving rise to the three committed 236 lineages PIT1, TPIT and SF1 (Figure 2 – figure supplement 2D), indicating that the 237 loss of Ctnnb1 does not prevent differentiation of SOX2+ PSCs into the three 238 lineages. Downregulation of -catenin was confirmed by immunofluorescence in 239 SOX2+ (mGFP+) derivatives (Figure 2 – figure supplement 2E). Although limited by 240 a small sample size, we conclude that WNT/-catenin signalling is likely required 241 cell-autonomously in SOX2+ stem cells and their descendants (Figure 2E). 242 243 SOX2+ stem cells express WNT ligands. 244 Having established that WNT activation is responsible for promoting proliferation in 245 the AP, we next focused on identifying the source of WNT ligands. Axin2 expressing 246 cells from Axin2CreERT2/+;ROSA26mTmG/+ mice were labelled at P14 by tamoxifen 247 induction. Cells expressing Axin2 at the time of induction are labelled by GFP 248 expression in the membrane. Double immunofluorescence staining for GFP together 249 with SOX2 revealed that Axin2 expressing cells (mGFP+) are frequently located in 250 close proximity to SOX2+ PSCs (Figure 3A). Two-dimensional quantification of the 251 Sox2CreERT2/+;Ctnnb1LOF/LOF;ROSA26mTmG/+ pituitaries) revealed that targeted 235 (Ctnnb1-deficient) SOX2+ PSCs were capable of giving rise to the three committed 236 lineages PIT1, TPIT and SF1 (Figure 2 – figure supplement 2D), indicating that the 237 loss of Ctnnb1 does not prevent differentiation of SOX2+ PSCs into the three 238 lineages. Downregulation of -catenin was confirmed by immunofluorescence in 239 SOX2+ (mGFP+) derivatives (Figure 2 – figure supplement 2E). Although limited by 240 a small sample size, we conclude that WNT/-catenin signalling is likely required 241 cell-autonomously in SOX2+ stem cells and their descendants (Figure 2E). 242 INTRODUCTION 53 222 To further address the role of the canonical WNT response in the activity of 223 SOX2+ PSCs in vivo, we expressed a loss-of-function allele of -catenin specifically 224 in Sox2-expressing cells (Sox2CreERT2/+;Ctnnb1lox(ex2-6)/lox(ex2-6) hereby 225 To further address the role of the canonical WNT response in the activity of 223 SOX2+ PSCs in vivo, we expressed a loss-of-function allele of -catenin specifically 224 in Sox2-expressing cells (Sox2CreERT2/+;Ctnnb1lox(ex2-6)/lox(ex2-6) hereby 225 To further address the role of the canonical WNT response in the activity of 223 SOX2+ PSCs in vivo, we expressed a loss-of-function allele of -catenin specifically 224 in Sox2-expressing cells (Sox2CreERT2/+;Ctnnb1lox(ex2-6)/lox(ex2-6) hereby 225 Sox2CreERT2/+;Ctnnb1LOF/LOF) from P14. Twenty-two weeks following induction, at 226 9 P168, there was a substantial drop in the number of cycling cells in the pituitary of 227 Sox2CreERT2/+;Ctnnb1LOF/LOF mutants compared to Sox2+/+;Ctnnb1LOF/LOF controls 228 (Figure 2C, n=2 pituitaries per genotype). This was accompanied by anterior pituitary 229 hypoplasia following the loss of Ctnnb1 in SOX2+ PSCs (Figure 2D). Therefore, in 230 this small sample size, the proliferative capacity of Ctnnb1-deficient SOX2+ PSCs 231 and of their descendants was impaired long-term, leading to reduced growth. In vivo 232 genetic tracing of targeted cells over the 22-week period 233 (Sox2CreERT2/+;Ctnnb1LOF/+;ROSA26mTmG/+ compared to 234 Sox2CreERT2/+;Ctnnb1LOF/LOF;ROSA26mTmG/+ pituitaries) revealed that targeted 235 (Ctnnb1-deficient) SOX2+ PSCs were capable of giving rise to the three committed 236 lineages PIT1, TPIT and SF1 (Figure 2 – figure supplement 2D), indicating that the 237 loss of Ctnnb1 does not prevent differentiation of SOX2+ PSCs into the three 238 lineages. Downregulation of -catenin was confirmed by immunofluorescence in 239 SOX2+ (mGFP+) derivatives (Figure 2 – figure supplement 2E). Although limited by 240 a small sample size, we conclude that WNT/-catenin signalling is likely required 241 cell-autonomously in SOX2+ stem cells and their descendants (Figure 2E). 242 243 Sox2 ;Ctnnb1 mutants compared to Sox2 ;Ctnnb1 controls 228 (Figure 2C, n=2 pituitaries per genotype). This was accompanied by anterior pituitary 229 hypoplasia following the loss of Ctnnb1 in SOX2+ PSCs (Figure 2D). Therefore, in 230 this small sample size, the proliferative capacity of Ctnnb1-deficient SOX2+ PSCs 231 and of their descendants was impaired long-term, leading to reduced growth. SOX2+ stem cells express WNT ligands. 244 Having established that WNT activation is responsible for promoting proliferation in 245 the AP, we next focused on identifying the source of WNT ligands. Axin2 expressing 246 cells from Axin2CreERT2/+;ROSA26mTmG/+ mice were labelled at P14 by tamoxifen 247 induction. Cells expressing Axin2 at the time of induction are labelled by GFP 248 expression in the membrane. Double immunofluorescence staining for GFP together 249 with SOX2 revealed that Axin2 expressing cells (mGFP+) are frequently located in 250 close proximity to SOX2+ PSCs (Figure 3A). Two-dimensional quantification of the 251 10 two cell types revealed that over 50% of mGFP+ cells were in direct contact with 252 SOX2+ nuclei (n=3 pituitaries, >500 SOX2+ cells per gland, Figure 3A). The analysis 253 did not take into account the cellular processes of SOX2+ cells. These results led us to 254 speculate that SOX2+ PSCs may be a source of key WNT ligands promoting 255 proliferation of lineage-committed cells. 256 two cell types revealed that over 50% of mGFP+ cells were in direct contact with 252 SOX2+ nuclei (n=3 pituitaries, >500 SOX2+ cells per gland, Figure 3A). The analysis 253 did not take into account the cellular processes of SOX2+ cells. These results led us to 254 speculate that SOX2+ PSCs may be a source of key WNT ligands promoting 255 proliferation of lineage-committed cells. 256 proliferation of lineage-committed cells. 256 In order to determine if SOX2+ PSCs express WNT ligands, we carried out 257 gene expression profiling of SOX2+ and SOX2- populations at P14, through bulk 258 RNA-sequencing. Pure populations of Sox2-expressing cells excluding lineage- 259 committed populations, were isolated from Sox2Egfp/+ male and female pituitaries at 260 P14 based on EGFP expression as previously shown (Andoniadou et al., 2012) 261 (Figure 3B, Figure 3 – figure supplement 1A). Analysis of global gene expression 262 signatures using ‘Gene Set Enrichment Analysis’ (GSEA) (Subramanian et al., 2005) 263 identified a significant enrichment of molecular signatures related to EMT, adherens 264 and tight junctions in the EGFP+ fraction, characteristic of the SOX2+ population 265 (Figure 3 – figure supplement 1B). SOX2+ stem cells express WNT ligands. 244 Analysis of Wnt gene expression confirmed 284 enriched expression of Wnt2, Wnt5a and Wnt9a in SOX2+ PSCs, and the expression 285 of multiple additional Wnt genes by both fractions at lower levels (SOX2+ fraction: 286 Wnt5b, Wnt6, Wnt16; SOX2- fraction: Wnt2, Wnt2b, Wnt3, Wnt4, Wnt5a, Wnt5b, 287 fingerprint of key pathway genes including Lgr4, Znrf3, Rnf43 capable of regulating 277 WNT signal intensity in SOX2+ PSCs, as well as enriched expression of the receptors 278 Fzd1, Fzd3, Fzd4, Fzd6 and Fzd7 (Figure 3 – figure supplement 1D). The 279 fingerprint of key pathway genes including Lgr4, Znrf3, Rnf43 capable of regulating 277 WNT signal intensity in SOX2+ PSCs, as well as enriched expression of the receptors 278 Fzd1, Fzd3, Fzd4, Fzd6 and Fzd7 (Figure 3 – figure supplement 1D). The 279 predominant R-spondin gene expressed in the pituitary was Rspo4, specifically by the 280 EGFP-negative fraction (Figure 3 – figure supplement 1D). The gene profiling 281 revealed that Wls expression, encoding Gpr177/WLS, a necessary mediator of WNT 282 ligand secretion (Carpenter et al., 2010; Takeo et al., 2013; Wang et al., 2015), is 283 enriched in SOX2+ PSCs (Figure 3C). Analysis of Wnt gene expression confirmed 284 enriched expression of Wnt2, Wnt5a and Wnt9a in SOX2+ PSCs, and the expression 285 of multiple additional Wnt genes by both fractions at lower levels (SOX2+ fraction: 286 Wnt5b, Wnt6, Wnt16; SOX2- fraction: Wnt2, Wnt2b, Wnt3, Wnt4, Wnt5a, Wnt5b, 287 Wnt9a, Wnt10a, Wnt16) (Figure 3D). These results reveal that SOX2+ PSCs express 288 the essential components to regulate activation of the WNT pathway and express Wnt 289 genes as well as the necessary molecular machinery to secrete WNT ligands. 290 291 Wnt9a, Wnt10a, Wnt16) (Figure 3D). These results reveal that SOX2+ PSCs express 288 the essential components to regulate activation of the WNT pathway and express Wnt 289 genes as well as the necessary molecular machinery to secrete WNT ligands. 290 Wnt9a, Wnt10a, Wnt16) (Figure 3D). These results reveal that SOX2+ PSCs express 288 the essential components to regulate activation of the WNT pathway and express Wnt 289 genes as well as the necessary molecular machinery to secrete WNT ligands. 290 291 SOX2+ stem cells express WNT ligands. 244 The SOX2+ fraction also displayed enrichment for 266 genes associated with several signalling pathways known to be active in these cells, 267 including EGFR (Iwai-Liao et al., 2000), Hippo (Lodge et al., 2016; Lodge et al., 268 2019; Xekouki et al., 2019), MAPK (Haston et al., 2017), FGF (Higuchi et al., 2017), 269 Ephrin (Yoshida et al., 2015; Yoshida et al., 2017) and p53 (Gonzalez-Meljem et al., 270 2017) (Figure 3 – figure supplement 1C, Supplementary File 1). Additionally, PI3K, 271 TGF and BMP pathway genes were significantly enriched in the SOX2+ population 272 (Figure 3 – figure supplement 1C, Supplementary File 1). Query of the WNT- 273 associated genes did not suggest a global enrichment in WNT targets (e.g. enrichment 274 of Myc and Jun, but not of Axin2 or Lef1) (Figure 3 – figure supplement 1D, 275 Supplementary File 1). Instead, SOX2+ PSCs expressed a unique transcriptomic 276 In order to determine if SOX2+ PSCs express WNT ligands, we carried out 257 gene expression profiling of SOX2+ and SOX2- populations at P14, through bulk 258 RNA-sequencing. Pure populations of Sox2-expressing cells excluding lineage- 259 committed populations, were isolated from Sox2Egfp/+ male and female pituitaries at 260 P14 based on EGFP expression as previously shown (Andoniadou et al., 2012) 261 Supplementary File 1). Instead, SOX2+ PSCs expressed a unique transcriptomic 276 Supplementary File 1). Instead, SOX2+ PSCs expressed a unique transcriptomic 276 Supplementary File 1). Instead, SOX2+ PSCs expressed a unique transcriptomic 276 11 11 fingerprint of key pathway genes including Lgr4, Znrf3, Rnf43 capable of regulating 277 WNT signal intensity in SOX2+ PSCs, as well as enriched expression of the receptors 278 Fzd1, Fzd3, Fzd4, Fzd6 and Fzd7 (Figure 3 – figure supplement 1D). The 279 predominant R-spondin gene expressed in the pituitary was Rspo4, specifically by the 280 EGFP-negative fraction (Figure 3 – figure supplement 1D). The gene profiling 281 revealed that Wls expression, encoding Gpr177/WLS, a necessary mediator of WNT 282 ligand secretion (Carpenter et al., 2010; Takeo et al., 2013; Wang et al., 2015), is 283 enriched in SOX2+ PSCs (Figure 3C). Paracrine signalling from SOX2+ stem cells promotes WNT activation. 292 Paracrine signalling from SOX2+ stem cells promotes WNT activation. 292 We sought to conclusively determine if WNT secretion specifically from SOX2+ 293 PSCs drives proliferation of surrounding cells in the postnatal pituitary gland. We 294 proceeded to delete Wls only in the Sox2-expressing population (Sox2CreERT2/+;Wlsfl/fl) 295 from P14 by a series of tamoxifen injections. Due to morbidity, we limited analyses to 296 one week following induction. Pituitaries appeared mildly hypoplastic at P21 along 297 the medio-lateral axis (Figure 4 – figure supplement 1, n=4 controls and n=5 298 mutants). To determine if this is a result of reduced proliferation, we carried out 299 immunofluorescence using antibodies against Ki-67 and SOX2. This revealed 300 significantly fewer cycling cells in the SOX2- population of Sox2CreERT2/+;Wlsfl/fl 301 We sought to conclusively determine if WNT secretion specifically from SOX2+ 293 PSCs drives proliferation of surrounding cells in the postnatal pituitary gland. We 294 proceeded to delete Wls only in the Sox2-expressing population (Sox2CreERT2/+;Wlsfl/fl) 295 from P14 by a series of tamoxifen injections. Due to morbidity, we limited analyses to 296 one week following induction. Pituitaries appeared mildly hypoplastic at P21 along 297 the medio-lateral axis (Figure 4 – figure supplement 1, n=4 controls and n=5 298 mutants). To determine if this is a result of reduced proliferation, we carried out 299 immunofluorescence using antibodies against Ki-67 and SOX2. This revealed 300 significantly fewer cycling cells in the SOX2- population of Sox2CreERT2/+;Wlsfl/fl 301 12 12 mutant pituitaries compared to Sox2+/+;Wlsfl/fl controls (10.326% Ki-67 in control 302 (n=4) compared to 3.129% in mutant (n=5), P=0.0008, unpaired t-test) (Figure 4A). 303 Additionally, we observed a reduction of cycling cells within the SOX2+ population 304 (5.582% Ki-67 in control compared to 2.225% in induced Sox2CreERT2/+;Wlsfl/fl mutant 305 pituitaries, P=0.0121, unpaired t-test) (Figure 4A), resulting in a smaller SOX2+ cell 306 pool in mutants (23.425% SOX2+/total AP cells in Sox2+/+;Wlsfl/fl controls compared 307 to 19.166% SOX2+/total AP cells in induced Sox2CreERT2/+;Wlsfl/fl mutant pituitaries, 308 P=0.0238, Student’s t-test, n=5 mutants, 4 controls). To determine if reduced levels of 309 WNT activation accompanied this phenotype, we carried out double mRNA in situ 310 hybridisation using specific probes against Lef1 and Sox2. There was an overall 311 reduction in Lef1 expression in mutants compared to controls (n=4 per genotype), in 312 which we frequently observed robust expression of Lef1 transcripts in close proximity 313 to cells expressing Sox2 (arrows, Figure 4B). Paracrine signalling from SOX2+ stem cells promotes WNT activation. 292 Together, our data support a paracrine 314 role for SOX2+ pituitary stem cells in driving the expansion of committed progeny 315 through the secretion of WNT ligands (Figure 4C). 316 317 DISCUSSION 318 Emerging disparities between the archetypal stem cell model, exhibited by the 319 haematopoietic system, and somatic stem cells of many organs, have led to the 320 concept that stem cell function can be executed by multiple cells not fitting a typical 321 stem cell paradigm (Clevers and Watt, 2018). In organs with persistent populations 322 possessing typical functional stem cell properties yet contributing minimally to 323 turnover and repair, the necessity for such classical stem cells is questioned. Here we 324 show that WNT signalling is required for postnatal pituitary growth by both SOX2+ 325 PSCs as well as SOX2- committed progenitors. We identify an additional discreet 326 Paracrine signalling from SOX2+ stem cells promotes WNT activation. 292 Together, our data support a paracrine 314 role for SOX2+ pituitary stem cells in driving the expansion of committed progeny 315 through the secretion of WNT ligands (Figure 4C). 316 mutant pituitaries compared to Sox2+/+;Wlsfl/fl controls (10.326% Ki-67 in control 302 (n=4) compared to 3.129% in mutant (n=5), P=0.0008, unpaired t-test) (Figure 4A). 303 Additionally, we observed a reduction of cycling cells within the SOX2+ population 304 (5.582% Ki-67 in control compared to 2.225% in induced Sox2CreERT2/+;Wlsfl/fl mutant 305 pituitaries, P=0.0121, unpaired t-test) (Figure 4A), resulting in a smaller SOX2+ cell 306 pool in mutants (23.425% SOX2+/total AP cells in Sox2+/+;Wlsfl/fl controls compared 307 to 19.166% SOX2+/total AP cells in induced Sox2CreERT2/+;Wlsfl/fl mutant pituitaries, 308 P=0.0238, Student’s t-test, n=5 mutants, 4 controls). To determine if reduced levels of 309 WNT activation accompanied this phenotype, we carried out double mRNA in situ 310 hybridisation using specific probes against Lef1 and Sox2. There was an overall 311 reduction in Lef1 expression in mutants compared to controls (n=4 per genotype), in 312 which we frequently observed robust expression of Lef1 transcripts in close proximity 313 to cells expressing Sox2 (arrows, Figure 4B). Together, our data support a paracrine 314 role for SOX2+ pituitary stem cells in driving the expansion of committed progeny 315 through the secretion of WNT ligands (Figure 4C). 316 mutant pituitaries compared to Sox2+/+;Wlsfl/fl controls (10.326% Ki-67 in control 302 (n=4) compared to 3.129% in mutant (n=5), P=0.0008, unpaired t-test) (Figure 4A). 303 Additionally, we observed a reduction of cycling cells within the SOX2+ population 304 (5.582% Ki-67 in control compared to 2.225% in induced Sox2CreERT2/+;Wlsfl/fl mutant 305 pituitaries, P=0.0121, unpaired t-test) (Figure 4A), resulting in a smaller SOX2+ cell 306 pool in mutants (23.425% SOX2+/total AP cells in Sox2+/+;Wlsfl/fl controls compared 307 to 19.166% SOX2+/total AP cells in induced Sox2CreERT2/+;Wlsfl/fl mutant pituitaries, 308 P=0.0238, Student’s t-test, n=5 mutants, 4 controls). To determine if reduced levels of 309 WNT activation accompanied this phenotype, we carried out double mRNA in situ 310 hybridisation using specific probes against Lef1 and Sox2. There was an overall 311 reduction in Lef1 expression in mutants compared to controls (n=4 per genotype), in 312 which we frequently observed robust expression of Lef1 transcripts in close proximity 313 to cells expressing Sox2 (arrows, Figure 4B). DISCUSSION 318 Emerging disparities between the archetypal stem cell model, exhibited by the 319 haematopoietic system, and somatic stem cells of many organs, have led to the 320 concept that stem cell function can be executed by multiple cells not fitting a typical 321 stem cell paradigm (Clevers and Watt, 2018). In organs with persistent populations 322 possessing typical functional stem cell properties yet contributing minimally to 323 turnover and repair, the necessity for such classical stem cells is questioned. Here we 324 show that WNT signalling is required for postnatal pituitary growth by both SOX2+ 325 PSCs as well as SOX2- committed progenitors. We identify an additional discreet 326 Emerging disparities between the archetypal stem cell model, exhibited by the 319 haematopoietic system, and somatic stem cells of many organs, have led to the 320 concept that stem cell function can be executed by multiple cells not fitting a typical 321 stem cell paradigm (Clevers and Watt, 2018). In organs with persistent populations 322 possessing typical functional stem cell properties yet contributing minimally to 323 turnover and repair, the necessity for such classical stem cells is questioned. Here we 324 show that WNT signalling is required for postnatal pituitary growth by both SOX2+ 325 PSCs as well as SOX2- committed progenitors. We identify an additional discreet 326 13 13 function for SOX2+ PSCs, where these signal in a feedforward manner by secreting 327 WNT ligands as cues to stimulate proliferation and promote tissue growth. 328 Consistent with previous reports, our data support that SOX2+ PSCs 329 contribute, but do not carry out the majority of tissue expansion during the postnatal 330 period (Zhu et al., 2015); instead, new cells primarily derive from more committed 331 progenitors, which we show to be WNT-responsive. We demonstrate that this 332 population of lineage-restricted WNT-responsive cells rapidly expands and 333 contributes long-lasting clones from postnatal stages. It remains to be shown if cells 334 among the SOX2- lineage-committed populations may also fall under the classical 335 definition of a stem cell. Preventing secretion of WNT ligands from SOX2+ PSCs 336 reveals that far from being dispensable, paracrine actions of the SOX2+ population 337 that are inactive in their majority, are necessary for anterior lobe expansion from 338 lineage-committed populations. DISCUSSION 318 In the adrenal gland, R-spondins are necessary for 339 cortical expansion and zonation, where deletion of Rspo3, expressed by the capsule 340 which contains adrenocortical stem cells, results in reduced proliferation of the 341 underlying steroidogenic cells (Vidal et al., 2016). Corroborating a model where 342 committed pituitary progenitors depend on the paracrine actions of SOX2+ PSCs, Zhu 343 and colleagues observed that in pituitaries with reduced numbers of PSCs, 344 proliferation among PIT1+ cells was significantly impaired (Zhu et al., 2015). It 345 would be intriguing to see if there is a reduction in WNT signalling in this model, or 346 following genetic ablation of adult SOX2+ PSCs (Roose et al., 2017). 347 We show that a sub-population of SOX2+ PSCs in the postnatal gland are also 348 function for SOX2+ PSCs, where these signal in a feedforward manner by secreting 327 WNT ligands as cues to stimulate proliferation and promote tissue growth. 328 proliferation among PIT1+ cells was significantly impaired (Zhu et al., 2015). It 345 would be intriguing to see if there is a reduction in WNT signalling in this model, or 346 following genetic ablation of adult SOX2+ PSCs (Roose et al., 2017). 347 We show that a sub-population of SOX2+ PSCs in the postnatal gland are also 348 WNT-responsive and have greater in vitro colony-forming potential under defined 349 conditions. This colony-forming potential is normally a property of a minority of 350 SOX2+ PSCs at any given age and reflects their in vivo proliferative capacity 351 14 14 (Andoniadou et al., 2012; Rizzoti et al., 2013). A role for the WNT pathway in 352 promoting SOX2+ cell activity is supported by studies showing that pathogenic 353 overexpression of -catenin promotes their colony-forming ability (Sarkar et al., 354 2016), and their in vivo expansion (Andoniadou et al., 2012). Additionally, elevated 355 WNT pathway activation has been described for pituitary side-population cells, 356 enriched for SOX2+ stem cells from young, compared to old pituitaries (Gremeaux et 357 al., 2012). This is in line with our findings that the WNT pathway has an important 358 function in promoting the activation of SOX2+ PSCs. DISCUSSION 318 It remains to be shown if this 359 response relies on autocrine WNT-signalling as for other stem cells (Lim et al., 2013), 360 however our results reveal reduced proliferation among SOX2+ PSCs and reduced 361 SOX2+ cell numbers when WNT secretion from these cells is abolished, supportive of 362 either autocrine signalling, or paracrine signalling between different subsets of the 363 SOX2+ population. 364 The mechanism preventing the majority of SOX2+ PSCs from responding to 365 WNT signals remains elusive but points to heterogeneity among the population. Such 366 regulation could occur at the level of receptor signalling; we have shown by bulk 367 transcriptomic profiling that SOX2+ PSCs express the receptors required to respond to 368 the WNT pathway, but also express high levels of the frizzled inhibitor Znrf3, and the 369 R-spondin receptor Lgr4. One conceivable scenario is that high levels of Znrf3 inhibit 370 frizzled receptors in the absence of R-spondin under normal physiological conditions, 371 supressing a WNT response. In support of this, R-spondins have been shown to 372 promote pituitary organoid formation (Cox et al., 2019). Whether the R- 373 spondin/LGR/ZNRF3 module is active under physiological conditions needs to be 374 determined. Furthermore, well-described factors expressed in PSCs are known to 375 have inhibitory effects on -catenin-mediated transcription, such as YAP/TAZ 376 (Andoniadou et al., 2012; Rizzoti et al., 2013). A role for the WNT pathway in 352 promoting SOX2+ cell activity is supported by studies showing that pathogenic 353 overexpression of -catenin promotes their colony-forming ability (Sarkar et al., 354 2016), and their in vivo expansion (Andoniadou et al., 2012). Additionally, elevated 355 WNT pathway activation has been described for pituitary side-population cells, 356 enriched for SOX2+ stem cells from young, compared to old pituitaries (Gremeaux et 357 al., 2012). This is in line with our findings that the WNT pathway has an important 358 function in promoting the activation of SOX2+ PSCs. It remains to be shown if this 359 response relies on autocrine WNT-signalling as for other stem cells (Lim et al., 2013), 360 however our results reveal reduced proliferation among SOX2+ PSCs and reduced 361 SOX2+ cell numbers when WNT secretion from these cells is abolished, supportive of 362 either autocrine signalling, or paracrine signalling between different subsets of the 363 SOX2+ population. DISCUSSION 318 364 15 15 (Azzolin et al., 2014; Gregorieff et al., 2015) and SOX2 itself (Alatzoglou et al., 377 (Azzolin et al., 2014; Gregorieff et al., 2015) and SOX2 itself (Alatzoglou et al., 377 2011; Kelberman et al., 2008). 378 In summary, we demonstrate an alternative mechanism for stem cell 379 contribution to homeostasis, whereby these can act as paracrine signalling hubs to 380 promote local proliferation. Applicable to other organs, this missing link between 381 SOX2+ PSCs and committed cell populations of the anterior pituitary, is key for basic 382 physiological functions and renders stem cells integral to organ expansion. 383 16 MATERIALS AND METHODS 384 385 Mice 386 All procedures were performed under compliance of the Animals (Scientific 387 Procedures) Act 1986, Home Office License (P5F0A1579). KCL Biological Services 388 Unit staff undertook daily animal husbandry. Genotyping was performed on ear 389 biopsies taken between P11 and P15 by standard PCR using the indicated primers. 390 These experiments were not conducted at random and the experimenters were not 391 blind while conducting the animal handling and assessment of tissue. Images are 392 representative of the respective genotypes. For all studies, both male and female 393 animals were used and results combined. 394 animals were used and results combined. 394 The Sox2CreERT2/+ and Sox2Egfp/+ strains were kept on a CD-1 background. 395 Axin2CreERT2/+animals were kept on a mixed background of C57BL/6 backcrossed 396 onto CD-1 for 5 generations and were viable and fertile, with offspring obtained at the 397 expected Mendelian ratios. ROSA26mTmG/mTmG, ROSA26Confetti/Confetti, 398 ROSA26tdTomato/tdTomato, Wlsfl/fl, Ctnnb1fl(ex2-6)/ fl(ex2-6) and TCF/LEF:H2B-EGFP mice 399 were kept on a mixed background of 129/Sv backcrossed onto CD-1 for at least 3 400 generations. For lineage tracing studies, male Axin2CreERT2/+ or Sox2CreERT2/+ mice 401 were bred with homozygous ROSA26mTmG/mTmG or ROSA26Confetti/Confetti dams to 402 produce the appropriate allele combinations on the reporter background. Pups were 403 induced at P14 or P15 with a single dose of tamoxifen (resuspended to 20mg/ml in 404 Corn Oil with 10% ethanol) by intraperitoneal injection, at a concentration of 0.15mg 405 per gram of body weight. Pituitaries were harvested at the indicated time points post 406 induction and processed for further analysis as described below. Mice were harvested 407 from different litters for each time point at random. For litters in which there was a 408 The Sox2CreERT2/+ and Sox2Egfp/+ strains were kept on a CD-1 background. DISCUSSION 318 440 Dissected pituitaries were incubated in Enzyme Mix (0.5% w/v collagenase type 2 441 (Lorne Laboratories), 0.1x Trypsin (Gibco), 50g/ml DNase I (Worthington) and 442 2.5g/ml Fungizone (Gibco) in Hank’s Balanced Salt Solution (HBSS)(Gibco)) in a 443 cell culture incubator for up to 3 hours. 850ml of HBSS were added to each 444 Eppendorf in order to quench the reaction. Pituitaries were dissociated by agitation, 445 pipetting up and down 100x at first with a 1ml pipette, followed by 100x with a 200l 446 pipette. Cells were transferred to a 15ml Falcon tube and resuspended in 9ml of HBSS 447 and spun down at 200g for 5 minutes. The supernatant was aspirated, leaving behind 448 the cell pellet that was resuspended in PBS and spun down at 1000rpm for 5 minutes 449 before being resuspended in a Live/Dead cell stain (Life Technologies, L34975) 450 prepared to manufacturer’s instructions, for 30 minutes in the dark. Cells were 451 washed in PBS as above. The pellet was resuspended in FIX & PERM Cell 452 Permeabilization Kit (Life Technologies, GAS003) prepared as per manufacturer’s 453 instructions for 10 minutes at room temperature. Cells were washed as above, and the 454 pellet was resuspended in 500µl of FACS buffer (1% fetal calf serum (Sigma), 25mM 455 HEPES in PBS) and filtered through 70m filters (BD Falcon), into 5ml round 456 bottom polypropylene tubes (BD Falcon). 1 minute prior to analysis, 1µl of Hoechst 457 was added to the suspended cells and incubated. Samples were analysed on a BD 458 DISCUSSION 318 395 Axin2CreERT2/+animals were kept on a mixed background of C57BL/6 backcrosse 396 onto CD-1 for 5 generations and were viable and fertile, with offspring obtained at the 397 expected Mendelian ratios. ROSA26mTmG/mTmG, ROSA26Confetti/Confetti, 398 17 17 surplus of experimental mice, multiple samples were harvested for each required time 409 point. 410 For Wntless deletion studies, Sox2CreERT2/+;Wlsfl/+;ROSA26mTmG/mTmG males were bred 411 with Wlsfl/fl;ROSA26mTmG/mTmG dams, to produce 412 Sox2CreERT2/+;Wlsfl/+;ROSA26mTmG/mTmG, Sox2CreERT2/+;Wlsfl/fl;ROSA26mTmG/mTmG and 413 Wlsfl/fl;ROSA26mTmG/mTmG offspring. Pups of the indicated genotypes received 414 intraperitoneal injections of 0.15mg of tamoxifen/gram body weight on 4 consecutive 415 days, beginning at P14, and harvested 3 days after the final injection. 416 For the -catenin loss-of-function experiments, either Sox2CreERT2/+;Ctnnb1fl(ex2- 417 6)/+;ROSA26mTmG/mTmG or Axin2CreERT2/+;Ctnnb1fl(ex2-6)/+;ROSA26mTmG/mTmG males were 418 crossed with Ctnnb1fl(ex2-6)/fl(ex2-6);ROSA26mTmG/mTmG dams. Axin2CreERT2/+;Ctnnb1fl(ex2- 419 6)/fl(ex2-6);ROSA26mTmG/mTmG and Axin2CreERT2/+;Ctnnb1fl(ex2-6)/+;ROSA26mTmG/mTmG pups 420 were induced with a single dose of tamoxifen, at a concentration of 0.15mg per gram 421 of body weight and kept alive for 7 days before harvesting. Sox2CreERT2/+;Ctnnb1fl(ex2- 422 6)/+;ROSA26mTmG/mTmG and Sox2CreERT2/+;Ctnnb1fl(ex2-6)/fl(ex2-6);ROSA26mTmG/mTmG pups 423 received two intraperitoneal injections of tamoxifen, at a concentration of 424 0.15mg/gram body weight, on two consecutive days and were kept alive for the 425 indicated length of time before harvesting. 426 TCF/LEF:H2B-EGFP mice were culled and the pituitaries harvested at the indicated 427 ages for the respective experiments. For fluorescence-activated cell sorting 428 experiments, mice were harvested at 21 days of age. Axin2CreERT2/+;Sox2eGFP/+ males 429 were crossed with ROSA26tdTomat/tdTomato dams to produce 430 Axin2CreERT2/+;Sox2eGFP/+;ROSA26tdTomato/+ that were induced with single doses of 431 tamoxifen at 21 and 22 days of age and harvested three days after the first injection 432 for fluorescence-activated cell sorting experiments. 433 surplus of experimental mice, multiple samples were harvested for each required time 409 point. 410 surplus of experimental mice, multiple samples were harvested for each required time 409 point. 410 For Wntless deletion studies, Sox2CreERT2/+;Wlsfl/+;ROSA26mTmG/mTmG males were bred 411 18 434 434 Flow cytometry analysis of lineage traced pituitaries 435 For the quantification of cells by flow cytometry, anterior lobes of 436 Axin2CreERT2/+;ROSA26mTmG/+ mice dissected at the indicated time points. The 437 posterior and intermediate lobes were dissected from the anterior lobes under a 438 dissection microscope. Untreated ROSA26mTmG/+ and wild type pituitaries from age- 439 matched litters were used as tdTomato only and negative controls, respectively. Flow cytometry analysis of lineage traced pituitaries 435 Flow cytometry analysis of lineage traced pituitaries 435 For the quantification of cells by flow cytometry, anterior lobes of 436 Axin2CreERT2/+;ROSA26mTmG/+ mice dissected at the indicated time points. The 437 posterior and intermediate lobes were dissected from the anterior lobes under a 438 dissection microscope. Untreated ROSA26mTmG/+ and wild type pituitaries from age- 439 matched litters were used as tdTomato only and negative controls, respectively. 440 Dissected pituitaries were incubated in Enzyme Mix (0.5% w/v collagenase type 2 441 (Lorne Laboratories), 0.1x Trypsin (Gibco), 50g/ml DNase I (Worthington) and 442 2.5g/ml Fungizone (Gibco) in Hank’s Balanced Salt Solution (HBSS)(Gibco)) in a 443 cell culture incubator for up to 3 hours. 850ml of HBSS were added to each 444 For the quantification of cells by flow cytometry, anterior lobes of 436 Eppendorf in order to quench the reaction. Pituitaries were dissociated by agitation, 445 19 19 Fortessa, and gated according to negative and single fluorophore controls. Single cells 459 were gated according to SSC-A and SSC-W. Dead cells were excluded according to 460 DAPI (2ng/ml, incubated for 2 mins prior to sorting). GFP+, tdTomato+ and 461 GFP+;tdTomato+ cells were gated according to negative controls in the PE-A and 462 FITC-A channels. 463 464 Fluorescence Activated Cell Sorting for sequencing or colony forming assays 465 For fluorescence activated cell sorting, the anterior lobes from Sox2eGFP/+, 466 TCF/LEF:H2B-GFP or Axin2CreERT2/+;Sox2eGFP/+;ROSA26tdTomato/+ and their respective 467 controls were dissected and dissociated as above. After dissociation cells were spun 468 down at 200g in HBSS and the pellet was resuspended in 500µl FACS buffer. Using 469 an Aria III FACs machine (BD systems), samples were gated according to negative 470 controls, and where applicable single fluorophore controls. Experimental samples 471 were sorted according to their fluorescence, as indicated, into tubes containing either 472 RNAlater (Qiagen) for RNA isolation or 1ml of Pit Complete Media for culture ((Pit 473 Complete: 20ng/ml bFGF and 50ng/ml of cholera toxin in ‘Pit Basic’ media (DMEM- 474 F12 with 5% Fetal Calf Serum,100U/ml Penicillin and 100g/ml Streptomycin). Cells 475 were plated in 12-well plates at clonal density, approximately 500 cells/well. Colonies 476 were incubated for 7 days total before being fixed in 10% neutral buffered formalin 477 (NBF) (Sigma) for 10 minutes at room temperature, washed for five minutes, three 478 times, mins with PBS and stained with crystal violet in order for the number of 479 colonies to be quantified. Flow cytometry analysis of lineage traced pituitaries 435 480 481 Fortessa, and gated according to negative and single fluorophore controls. Single cells 459 were gated according to SSC-A and SSC-W. Dead cells were excluded according to 460 DAPI (2ng/ml, incubated for 2 mins prior to sorting). GFP+, tdTomato+ and 461 GFP+;tdTomato+ cells were gated according to negative controls in the PE-A and 462 FITC-A channels. 463 Fluorescence Activated Cell Sorting for sequencing or colony forming assay 465 Fluorescence Activated Cell Sorting for sequencing or colony forming assays 465 For fluorescence activated cell sorting, the anterior lobes from Sox2eGFP/+, 466 TCF/LEF:H2B-GFP or Axin2CreERT2/+;Sox2eGFP/+;ROSA26tdTomato/+ and their respective 467 controls were dissected and dissociated as above. After dissociation cells were spun 468 down at 200g in HBSS and the pellet was resuspended in 500µl FACS buffer. Using 469 an Aria III FACs machine (BD systems), samples were gated according to negative 470 controls, and where applicable single fluorophore controls. Experimental samples 471 were sorted according to their fluorescence, as indicated, into tubes containing either 472 RNAlater (Qiagen) for RNA isolation or 1ml of Pit Complete Media for culture ((Pit 473 Complete: 20ng/ml bFGF and 50ng/ml of cholera toxin in ‘Pit Basic’ media (DMEM- 474 F12 with 5% Fetal Calf Serum,100U/ml Penicillin and 100g/ml Streptomycin). Cells 475 were plated in 12-well plates at clonal density, approximately 500 cells/well. Colonies 476 were incubated for 7 days total before being fixed in 10% neutral buffered formalin 477 (NBF) (Sigma) for 10 minutes at room temperature, washed for five minutes, three 478 times, mins with PBS and stained with crystal violet in order for the number of 479 colonies to be quantified. 480 481 20 Total RNA was isolated from each sample and following poly-A selection, cDNA 483 libraries were generated using TruSeq (Clontech, 634925). Barcoded libraries were 484 then pooled at equal molar concentrations and sequenced on an Illumina Hiseq 4000 485 instrument in a 75 base pair, paired – end sequencing mode, at the Wellcome Trust 486 Centre for Human Genetics (Oxford, United Kingdom). Raw sequencing reads were 487 quality checked for nucleotide calling accuracy and trimmed accordingly to remove 488 potential sequencing primer contaminants. Following QC, forward and reverse reads 489 were mapped to GRCm38/mm10 using Hisat2 (Kim et al., 2015). Using a mouse 490 transcriptome specific GTF as a guide, FeatureCounts (Liao et al., 2014) was used to 491 generate gene count tables for every sample. These were utilised within the 492 framework of the Deseq2 (Love et al., 2014) and FPKM values (generated by FPKM 493 count (Wang et al., 2012)) were processed using the Cufflinks (Trapnell et al., 2012) 494 pipelines which identified statistically significant gene expression differences 495 between the sample groups. Fluorescence Activated Cell Sorting for sequencing or colony forming assay 465 Following identification of differentially expressed genes 496 (at an FDR < 0.05) we focused on identifying differentially expressed pathways using 497 a significance threshold of FDR < 0.05 unless otherwise specified. The gene lists used 498 for Gene Set Enrichment Analysis (GSEA) were as found on the BROAD institute 499 GSEA MSigDBv.7 ‘molecular signatures database’. The deposited dataset can be 500 accessed through the following link: 501 https://dataview.ncbi.nlm.nih.gov/object/PRJNA421806?reviewer=kr90aklsdtikh3gkh 502 3tdlpv30s 503 504 Immunofluorescence and microscopy 505 Freshly harvested pituitaries were washed in PBS for 10 minutes before being fixed in 506 10% NBF for 18 hours at room temperature. In short, embryos and whole pituitaries 507 between the sample groups. Following identification of differentially expressed genes 496 (at an FDR < 0.05) we focused on identifying differentially expressed pathways using 497 a significance threshold of FDR < 0.05 unless otherwise specified. The gene lists used 498 for Gene Set Enrichment Analysis (GSEA) were as found on the BROAD institute 499 GSEA MSigDBv.7 ‘molecular signatures database’. The deposited dataset can be 500 accessed through the following link: 501 21 were washed in PBS 3 times, before being dehydrated through a series of 1 hour 508 washes in 25%, 50%, 70%, 80%, 90%, 95% and 100% ethanol. Tissues were washed 509 in Neo-Clear (Sigma) at room temperature for 10 minutes, then in fresh preheated 510 Neo-Clear at 60 °C for 10 minutes. Subsequently, a mixture of 50% Neo-Clear:50% 511 paraffin wax at 60°C for 15 minutes followed by three changes of pure wax for a 512 minimum of 1 hour washes at 60°C, before being orientated to be sectioned in the 513 frontal plane. Embedded samples were sectioned at 5µm and mounted on to Super 514 Frost+ slides. 515 For immunofluorescence, slides were deparaffinised in Neo-Clear for three times ten 516 minutes, washed in 100% ethanol for three times five minutes, and rehydrated in a 517 series of five minute ethanol washes up to distilled water (95%, 90%, 80%, 70%, 518 50%, 25%, H2O). Heat induced epitope retrieval was performed with 1x DeClear 519 Buffer (citrate pH 6) in a Decloaking chamber NXGEN (Menarini Diagnostics) for 3 520 minutes at 110°C. Fluorescence Activated Cell Sorting for sequencing or colony forming assay 465 Slides were left to cool to room temperature before proceeding to 521 block for 1 hour at room temperature in Blocking Buffer (0.2% BSA, 0.15% glycine, 522 0.1% TritonX in PBS) with 10% serum (sheep or donkey, depending on secondary 523 antibodies). Primary antibodies were diluted in blocking buffer with 1% of the 524 appropriate serum and incubated overnight at 4°C. Slides were washed three times for 525 10 minutes with PBST. Slides were incubated with secondary antibodies diluted 526 1:400 in blocking buffer with 1% serum for one hour at room temperature. Slides 527 were washed three times with PBST as above. Where biotinylated secondary 528 antibodies were used, slides were incubated with streptavidin diluted 1:400 in 529 blocking buffer with 1% serum for one hour at room temperature. Finally, slides were 530 washed with PBST and mounted using Vectashield Antifade Mounting Medium 531 (Vector Laboratories, H-1000). 532 For immunofluorescence, slides were deparaffinised in Neo-Clear for three times ten 516 minutes, washed in 100% ethanol for three times five minutes, and rehydrated in a 517 series of five minute ethanol washes up to distilled water (95%, 90%, 80%, 70%, 518 22 The following antibodies, along with their dilutions and detection technique, were 533 used: GFP (1:400, Alexa Fluor-488 or -647 secondary), SOX2 raised in goat (1:200, 534 Alexa Fluor-488 secondary), SOX2 raised in rabbit (1:100, biotinylated secondary), 535 SOX9 (1:500, biotinylated secondary), PIT1 (1:500, biotinylated secondary), SF1 536 (1:300, biotinylated secondary), TPIT (1:200, biotinylated secondary), Ki-67 (1:100, 537 biotinylated secondary), pH-H3 (1:500, biotinylated secondary), GH (1:1000, 538 biotinylated secondary), TSH (1:1000, biotinylated secondary), PRL (1:1000, 539 biotinylated secondary), ACTH (1:400, Alexa Fluor-555 secondary), LH/FSH (1:300, 540 biotinylated secondary), ZO-1 (1:300, Alexa Fuor-488), E-Cadherin (1:300, Alexa 541 Fluor-488). Nuclei were visualized with Hoechst (1:1000). Images were taken on a 542 TCS SPS Confocal (Leica Microsystem) with a 20x objective for analysis. 543 544 mRNA In Situ Hybridisation 545 All mRNA in situ hybridisations were performed using the RNAscope singleplex or 546 duplex chromogenic kits (Advanced Cell Diagnostics) on formalin fixed paraffin 547 embedded sections processed as described in the above section. The protocol 548 followed the manufacturer’s instructions with slight modifications. ImmEdge 549 Hydrophobic Barrier PAP Pen (Vector Laboratories, H-4000) was used to draw a 550 barrier around section while air-drying following the first ethanol washes. Fluorescence Activated Cell Sorting for sequencing or colony forming assay 465 551 Pretreatment followed the standard length of time for pituitaries (twelve minutes), 552 while embryos were boiled for 10 minutes. For singleplex, the protocol proceeded to 553 follow the instructions exactly. For duplex, Amplification 9 was extended to one hour 554 and the dilution of the Green Detection reagent was increased to 1:30. For both 555 protocols, sections were counterstained with Mayer’s Haematoxylin (Vector 556 The following antibodies, along with their dilutions and detection technique, were 533 used: GFP (1:400, Alexa Fluor-488 or -647 secondary), SOX2 raised in goat (1:200, 534 Alexa Fluor-488 secondary), SOX2 raised in rabbit (1:100, biotinylated secondary), 535 SOX9 (1:500, biotinylated secondary), PIT1 (1:500, biotinylated secondary), SF1 536 (1:300, biotinylated secondary), TPIT (1:200, biotinylated secondary), Ki-67 (1:100, 537 biotinylated secondary), pH-H3 (1:500, biotinylated secondary), GH (1:1000, 538 biotinylated secondary), TSH (1:1000, biotinylated secondary), PRL (1:1000, 539 biotinylated secondary), ACTH (1:400, Alexa Fluor-555 secondary), LH/FSH (1:300, 540 biotinylated secondary), ZO-1 (1:300, Alexa Fuor-488), E-Cadherin (1:300, Alexa 541 Fluor-488). Nuclei were visualized with Hoechst (1:1000). Images were taken on a 542 TCS SPS Confocal (Leica Microsystem) with a 20x objective for analysis. 543 The following antibodies, along with their dilutions and detection technique, were 533 used: GFP (1:400, Alexa Fluor-488 or -647 secondary), SOX2 raised in goat (1:200, 534 Alexa Fluor-488 secondary), SOX2 raised in rabbit (1:100, biotinylated secondary), 535 SOX9 (1:500, biotinylated secondary), PIT1 (1:500, biotinylated secondary), SF1 536 (1:300, biotinylated secondary), TPIT (1:200, biotinylated secondary), Ki-67 (1:100, 537 biotinylated secondary), pH-H3 (1:500, biotinylated secondary), GH (1:1000, 538 biotinylated secondary), TSH (1:1000, biotinylated secondary), PRL (1:1000, 539 biotinylated secondary), ACTH (1:400, Alexa Fluor-555 secondary), LH/FSH (1:300, 540 biotinylated secondary), ZO-1 (1:300, Alexa Fuor-488), E-Cadherin (1:300, Alexa 541 Fluor-488). Nuclei were visualized with Hoechst (1:1000). Images were taken on a 542 TCS SPS Confocal (Leica Microsystem) with a 20x objective for analysis. 543 All mRNA in situ hybridisations were performed using the RNAscope singleplex or 546 duplex chromogenic kits (Advanced Cell Diagnostics) on formalin fixed paraffin 547 embedded sections processed as described in the above section. The protocol 548 followed the manufacturer’s instructions with slight modifications. ImmEdge 549 Hydrophobic Barrier PAP Pen (Vector Laboratories, H-4000) was used to draw a 550 barrier around section while air-drying following the first ethanol washes. 551 23 24 VectaMount Permanent Mounting Medium (Vector Laboratories, H-5000). Fluorescence Activated Cell Sorting for sequencing or colony forming assay 465 Slides 558 were scanned using a Nanozoomer-XR Digital Slide Scanner (Hamamatsu) and 559 processed using Nanozoomer Digital Pathology View (Hamamatsu). 560 Quantification of cells 561 Cell numbers were quantified in ImageJ using the cell counter plugin (Schindelin et 562 al., 2012). At a minimum, three sections per pituitary were quantified, spaced no les 563 than 100µM apart in the tissue. 564 565 Statistics 566 All statistical analyses were performed in GraphPad Prism. Data points in graphs 567 represent the mean values of recordings from a single biological replicate unless 568 otherwise stated. 569 570 571 ACKNOWLEDGEMENTS 572 This study has been supported by the Medical Research Council (MR/L016729/1, 573 MR/T012153/1) (C.L.A.), The Lister Institute of Preventive Medicine (C.L.A.), the 574 Deutsche Forschungsgemeinschaft (DFG German Research Foundation) (Project 575 Number 314061271 – TRR 205) (C.L.A.), the Howard Hughes Medical Institute 576 (R.N.), the Agence Nationale de la Recherche (ANR-18-CE14-0017) and Fondation 577 pour la Recherche Médicale (DEQ20150331732) (P.M.). J.P.R. was supported by a 578 Dianna Trebble Endowment Fund Dental Institute Studentship, E.J.L. by the King’s 579 Bioscience Institute and the Guy’s and St Thomas' Charity Prize PhD Programme in 580 Biomedical and Translational Science, Y.K. by a Project Support Grant from the 581 British Society for Neuroendocrinology. We thank Dr A.F. Parlow and the National 582 VectaMount Permanent Mounting Medium (Vector Laboratories, H-5000). Slides 558 were scanned using a Nanozoomer-XR Digital Slide Scanner (Hamamatsu) and 559 processed using Nanozoomer Digital Pathology View (Hamamatsu). 560 Quantification of cells 561 Cell numbers were quantified in ImageJ using the cell counter plugin (Schindelin et 562 al., 2012). At a minimum, three sections per pituitary were quantified, spaced no less 563 than 100µM apart in the tissue. 564 565 Statistics 566 All statistical analyses were performed in GraphPad Prism. Data points in graphs 567 represent the mean values of recordings from a single biological replicate unless 568 otherwise stated. 569 570 571 ACKNOWLEDGEMENTS 572 This study has been supported by the Medical Research Council (MR/L016729/1, 573 MR/T012153/1) (C.L.A.), The Lister Institute of Preventive Medicine (C.L.A.), the 574 Deutsche Forschungsgemeinschaft (DFG German Research Foundation) (Project 575 Number 314061271 – TRR 205) (C.L.A.), the Howard Hughes Medical Institute 576 (R N ) the Agence Nationale de la Recherche (ANR-18-CE14-0017) and Fondation 577 VectaMount Permanent Mounting Medium (Vector Laboratories, H-5000). Fluorescence Activated Cell Sorting for sequencing or colony forming assay 465 Slides 558 were scanned using a Nanozoomer-XR Digital Slide Scanner (Hamamatsu) and 559 processed using Nanozoomer Digital Pathology View (Hamamatsu). 560 VectaMount Permanent Mounting Medium (Vector Laboratories, H-5000). Slides 558 were scanned using a Nanozoomer-XR Digital Slide Scanner (Hamamatsu) and 559 processed using Nanozoomer Digital Pathology View (Hamamatsu). 560 This study has been supported by the Medical Research Council (MR/L016729/ 573 MR/T012153/1) (C.L.A.), The Lister Institute of Preventive Medicine (C.L.A.), the 574 Deutsche Forschungsgemeinschaft (DFG German Research Foundation) (Project 575 Number 314061271 – TRR 205) (C.L.A.), the Howard Hughes Medical Institute 576 (R.N.), the Agence Nationale de la Recherche (ANR-18-CE14-0017) and Fondation 577 pour la Recherche Médicale (DEQ20150331732) (P.M.). J.P.R. was supported by a 578 Dianna Trebble Endowment Fund Dental Institute Studentship, E.J.L. by the King’s 579 Bioscience Institute and the Guy’s and St Thomas' Charity Prize PhD Programme in 580 Biomedical and Translational Science, Y.K. by a Project Support Grant from the 581 British Society for Neuroendocrinology. We thank Dr A.F. Parlow and the National 582 24 24 Hormone and Peptide Program (Harbor–University of California, Los Angeles 583 Medical Center) for providing some of the antibodies used in this study and Prof. J. 584 Drouin and Prof. S. Rhodes for TPIT and PIT1 antibodies respectively. We thank the 585 High-Throughput Genomics Group at the Wellcome Trust Centre for Human 586 Genetics (funded by Wellcome Trust grant reference 090532/Z/09/Z) for the 587 generation of the Sequencing data. For flow sorting and analysis, this research was 588 supported by the National Institute for Health Research (NIHR) Biomedical Research 589 Centre based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College 590 London. 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Endocrine-related 797 cancer 26, 215-225. 798 Yan, K.S., Janda, C.Y., Chang, J., Zheng, G.X.Y., Larkin, K.A., Luca, V.C., Chia, 9 L.A., Mah, A.T., Han, A., Terry, J.M., et al. (2017). Non-equivalence of Wnt and R- 800 spondin ligands during Lgr5(+) intestinal stem-cell self-renewal. Nature 545, 238- 801 242. 802 Yoshida, S., Kato, T., Higuchi, M., Chen, M., Ueharu, H., Nishimura, N., and Kato, 803 Y. (2015). Localization of juxtacrine factor ephrin-B2 in pituitary stem/progenitor cell 804 niches throughout life. Cell Tissue Res 359, 755-766. Fluorescence Activated Cell Sorting for sequencing or colony forming assay 465 805 Yoshida, S., Kato, T., Kanno, N., Nishimura, N., Nishihara, H., Horiguchi, K., and 806 Kato, Y. (2017). Cell type-specific localization of Ephs pairing with ephrin-B2 in the 807 Yoshida, S., Kato, T., Kanno, N., Nishimura, N., Nishihara, H., Horiguchi, K., and 806 Yoshida, S., Kato, T., Kanno, N., Nishimura, N., Nishihara, H., Horiguchi, K., and 806 Kato, Y. (2017). Cell type-specific localization of Ephs pairing with ephrin-B2 in the 807 rat postnatal pituitary gland. Cell Tissue Res 370, 99-112. 808 Kato, Y. (2017). Cell type-specific localization of Ephs pairing with ephrin-B2 in the 807 rat postnatal pituitary gland. Cell Tissue Res 370, 99-112. 808 31 Zhu, X., Tollkuhn, J., Taylor, H., and Rosenfeld, M.G. (2015). Notch-Dependent 809 Pituitary SOX2(+) Stem Cells Exhibit a Timed Functional Extinction in Regulation of 810 the Postnatal Gland. Stem Cell Reports 5, 1196-1209. 811 812 Zhu, X., Tollkuhn, J., Taylor, H., and Rosenfeld, M.G. (2015). Notch-Dependent 809 Pituitary SOX2(+) Stem Cells Exhibit a Timed Functional Extinction in Regulation of 810 the Postnatal Gland. Stem Cell Reports 5, 1196-1209. 811 812 813 814 815 FIGURES 816 Figure 1. Axin2 expressing cells contribute to pituitary growth and expansion of 817 all lineages 818 A. Immunofluorescence staining against GFP (green) with markers of PSCs or 819 lineage commitment (magenta) in Axin2CreERT2/+; ROSA26mTmG/+ pituitaries 820 harvested from mice induced at P14 and lineage traced for 2 days (top panel) and 821 14 days (bottom panel). Scale bar 10m. 822 B. Quantification of lineage expansion between 2 and 14 days following induction at 823 P14. Graph shows that the proportion of lineage committed cells (either PIT1+, 824 TPIT+ or SF1+) and PSCs (SOX2+), i.e. that are transcription factor (TF)+ cells 825 that are GFP+ increases between 2 days (black bars) and 14 days (grey bars) post 826 induction. PIT1 P=0.000004, TPIT P=0.008 multiple t-tests. n=4 animals per time 827 point. 828 C. Immunofluorescence staining against GFP (green) in pituitaries harvested from 829 Axin2CreERT2/+;ROSA26mTmG/+ mice induced at P14 and lineage traced for 2 days, 2 830 weeks and 8 weeks. Bottom panel shows magnified fields of view of regions of 831 interest indicated by white boxes in panels above. Scale bars 50m. 832 D. Top panel showing the quantification of the proportion of all cells in 833 Zhu, X., Tollkuhn, J., Taylor, H., and Rosenfeld, M.G. (2015). Notch-Dependent 809 Zhu, X., Tollkuhn, J., Taylor, H., and Rosenfeld, M.G. (2015). Notch-Dependent 809 Pituitary SOX2(+) Stem Cells Exhibit a Timed Functional Extinction in Regulation of 810 the Postnatal Gland. Stem Cell Reports 5, 1196-1209. 811 C. Immunofluorescence staining against GFP (green) in pituitaries harvested from 829 Axin2CreERT2/+;ROSA26mTmG/+ mice induced at P14 and lineage traced for 2 days, 2 830 weeks and 8 weeks. Bottom panel shows magnified fields of view of regions of 831 interest indicated by white boxes in panels above. Scale bars 50m. 832 D. Top panel showing the quantification of the proportion of all cells in 833 Axin2CreERT2/+;ROSA26mTmG/+ pituitaries that are GFP+ at 2, 7, 14, 28 and 56 days 834 32 32 post induction as analysed by flow cytometry. Day 2 to 7 P<0.0001 unpaired t- 835 test. Data points show individual measurements from biological replicates, n=4-8 836 pituitaries per time point. Zhu, X., Tollkuhn, J., Taylor, H., and Rosenfeld, M.G. (2015). Notch-Dependent 809 Pituitary SOX2(+) Stem Cells Exhibit a Timed Functional Extinction in Regulation of 810 the Postnatal Gland. Stem Cell Reports 5, 1196-1209. 811 812 (Bottom) Graph of the absolute number of GFP+ cells 837 (green) and as a proportion of total cells (blue) at the time points indicated. 838 E. X-gal staining in Axin2CreERT2/+;ROSA26LacZ/+ pituitaries harvested from mice 839 induced at P14 and lineage traced for 8 weeks (left) and 1 year (right). Scale bars 840 500m. 841 F. Model summarising the major contribution of WNT-responsive progenitors of all 842 lineages to pituitary growth, in addition to that of SOX2+ PSCs. 843 844 845 33 Figure 2. Activation of WNT signalling in SOX2+ PSCs and their descendants is 846 necessary for long-term growth 847 A. Schematic of the experimental timeline used in panels A and B. Endogenous 848 expression of tdTomato (magenta, Axin2 targeted cells) and EGFP (green, Sox2 849 expressing cells) in Axin2CreERT2/+;Sox2Egfp/+;ROSA26tdTomato/+ pituitaries harvested 850 at P24 sectioned in the frontal plane. Nuclei are counterstained with Hoechst in 851 the merged panel. Scale bar 50m. 852 B. A representative culture plate showing colonies derived from Tomato+, EGFP+ or 853 Tomato+;EGFP+ cells that were isolated from 854 Axin2CreERT2/+;Sox2Egfp/+;ROSA26tdTomato/+ pituitaries by FACS plated in stem cell 855 promoting media at clonogenic densities and stained with crystal violet (left 856 panel). The proportion of colony-forming cells in each subpopulation were 857 quantified by counting the number of colonies per well (right panel). Each data 858 point indicates individual wells, n=5 separate pituitaries. P=0.0226, Mann- 859 Whitney U test (two-tailed). Scale bar 10mm. 860 C. Immunofluorescence staining against SOX2 (green) and Ki-67 (magenta) in 861 Sox2+/+Ctnnb1LOF/LOF (control) and Sox2CreERT2/+Ctnnb1LOF/LOF (mutant) pituitaries 862 from mice induced at P14 and analysed 22 weeks after induction (at P168) 863 (bottom panel). Scale bar 50m. 864 D. Dorsal view of whole mount pituitaries of Sox2+/+;Ctnnb1LOF/LOF (control) and 865 Sox2CreERT2/+;Ctnnb1LOF/LOF (mutant), 22 weeks after induction (i.e. P168). Scale 866 bars 1mm. 867 E. Model summarising the effect of Ctnnb1 deletion in SOX2+ PSCs. 868 PL, posterior lobe; IL, intermediate lobe; AL, anterior lobe. 869 E. Model summarising the effect of Ctnnb1 deletion in SOX2+ PSCs. 868 PL, posterior lobe; IL, intermediate lobe; AL, anterior lobe. 869 34 34 Figure 3. SOX2+ PSCs are as a source of WNT ligands in the pituitary 871 A. Immunofluorescence staining against GFP (green) and SOX2 (magenta) in 872 Axin2CreERT2/+; ROSA26mTmG/+ pituitaries 48 hours post induction. Zhu, X., Tollkuhn, J., Taylor, H., and Rosenfeld, M.G. (2015). Notch-Dependent 809 Pituitary SOX2(+) Stem Cells Exhibit a Timed Functional Extinction in Regulation of 810 the Postnatal Gland. Stem Cell Reports 5, 1196-1209. 811 812 Graph 873 representing a quantification of the proximity of individual GFP+ cells to the 874 nearest SOX2+ cell as quantified by the number of nuclei separating them. Plotted 875 data represents the proportion of GFP+ cells that fall in to each category of the 876 total GFP+ cells, taken from n=3 separate pituitaries. Scale bars 50m. 877 B. Experimental paradigm for RNA Seq analysis of Sox2 positive and negative cells. 878 C. Graphs representing the FPKM values of Wls and Porcupine in Sox2 positive and 879 negative cells (black and grey bars, respectively). mRNA in situ hybridisation for 880 Sox2 and for Wls on wild type sagittal pituitaries at P14, demonstrating strong Wls 881 expression in the marginal zone epithelium. Scale bars 250m. 882 D. Bar chart showing the FPKM values of Wnt genes in the Sox2+ and Sox2- 883 Figure 3. SOX2+ PSCs are as a source of WNT ligands in the pituitary 871 Figure 3. SOX2+ PSCs are as a source of WNT ligands in the pituitary 871 A. Immunofluorescence staining against GFP (green) and SOX2 (magenta) in 872 Axin2CreERT2/+; ROSA26mTmG/+ pituitaries 48 hours post induction. Graph 873 representing a quantification of the proximity of individual GFP+ cells to the 874 nearest SOX2+ cell as quantified by the number of nuclei separating them. Plotted 875 data represents the proportion of GFP+ cells that fall in to each category of the 876 total GFP+ cells, taken from n=3 separate pituitaries. Scale bars 50m. 877 B. Experimental paradigm for RNA Seq analysis of Sox2 positive and negative cells. 878 C. Graphs representing the FPKM values of Wls and Porcupine in Sox2 positive and 879 negative cells (black and grey bars, respectively). mRNA in situ hybridisation for 880 Sox2 and for Wls on wild type sagittal pituitaries at P14, demonstrating strong Wls 881 expression in the marginal zone epithelium. Scale bars 250m. 882 D. Bar chart showing the FPKM values of Wnt genes in the Sox2+ and Sox2- 883 fractions. Double mRNA in situ hybridisation against Wnt2, Wnt5a and Wnt9a 884 (blue) together with Sox2 (red) validating expression in the Sox2+ population. 885 Boxed regions through the marginal zone epithelium are magnified. Scale bars 886 100m and 50m in boxed inserts. 887 890 35 Figure 4. Paracrine secretion of WNTs from SOX2+ PSCs is necessary for 891 expansion of committed cells 892 A. Immunofluorescence staining against SOX2 (green) and Ki-67 (magenta) in 893 Sox2+/+;Wlsfl/fl (control) and Sox2CreERT2/+;Wlsfl/fl (mutant) pituitaries induced from 894 P14 and analysed after one week. Nuclei were counterstained with Hoechst. (i) 895 and (ii) represent magnified fields of view of regions indicated by white boxes in 896 top panels. Scale bars 50m. Graph of quantification of cycling cells marked by 897 Ki-67 among cells negative for SOX2. Values represent mean +/- SEM, 898 P=0.0008, unpaired t-test. Graph of quantification of cycling cells marked by Ki- 899 67 among SOX2-positive cells. Values represent mean +/- SEM, P=0.0121, 900 unpaired t-test. Each data point shows the mean of one biological replicate, n=4 901 pituitaries from controls and 5 pituitaries from mutants. 902 B. Double mRNA in situ hybridisation using specific probes against Lef1 (blue) and 903 Sox2 (red) in control and mutant pituitaries following tamoxifen induction from 904 P14 and tracing for 7 days. Scale bars 250m and 50m in boxed regions. 905 C. SUPPLEMENTARY INFORMATION 910 SUPPLEMENTARY INFORMATION 910 SUPPLEMENTARY INFORMATION 910 Supplementary File 1. Gene lists of Gene Set Enrichment Analyses 911 Gene lists generated from Gene Set Enrichment Analyses of bulk RNA-sequencing 912 data comparing Sox2+ and Sox2- cells. Associated with Figure 3 – figure supplement 913 1. 914 915 SUPPLEMENARY FIGURES 916 Figure 1 – figure supplement 1. Axin2 expressing cells contribute to pituitary 917 growth and expansion of all lineages 918 A. Schematic of the combined experimental timeline used in panels A, B and C. 919 Immunofluorescence staining against GFP (green) and markers of hormone- 920 secreting endocrine cells (GH (somatotrophs), ACTH (corticotrophs), PRL 921 (lactotrophs), TSH (thyrotrophs), FSH/LH (gonadotrophs)) in 922 Axin2CreERT2/+;Rosa26mTmG/+ pituitaries induced at P14 and lineage traced for 48 923 hours. Scale bar 10µm. 924 B. Graph of quantification of expansion of the WNT-responsive SF1+ population in 925 Axin2CreERT2/+;ROSA26mTmG/+ pituitaries induced at P14 and lineage traced for 2 or 926 28 days. There is a significant increase of GFP+;SF1+ cells as a proportion of the 927 total SF1+ cells at P28. P=0.0048, unpaired t-test (n=2 at 2 days, 3 at 28 days). 928 C. Immunofluorescence staining against GFP (green) and markers of hormone- 929 secreting endocrine cells of the PIT1 lineage (GH (somatotrophs), PRL 930 (lactotrophs), TSH (thyrotrophs)) in Axin2CreERT2/+;ROSA26mTmG/+ pituitaries 931 induced at P14 and lineage traced for 14 days. Scale bars 50µm. Graph showing 932 expansion of each of the Hormone+ cell types (Hormone+;GFP+) as a percentage 933 of the total Hormone+ population between 2 and 14 days post-induction. There is 934 Figure 3. SOX2+ PSCs are as a source of WNT ligands in the pituitary 871 Model summarising paracrine WNT secretion from SOX2+ PSCs to lineage- 906 committed progenitors and the effects of abolishing WNT secretion from SOX2+ 907 PSCs through the deletion of Wls. 908 909 P=0.0008, unpaired t-test. Graph of quantification of cycling cells marked by Ki- 899 67 among SOX2-positive cells. Values represent mean +/- SEM, P=0.0121, 900 unpaired t-test. Each data point shows the mean of one biological replicate, n=4 901 pituitaries from controls and 5 pituitaries from mutants. 902 B. Double mRNA in situ hybridisation using specific probes against Lef1 (blue) and 903 Sox2 (red) in control and mutant pituitaries following tamoxifen induction from 904 P14 and tracing for 7 days. Scale bars 250m and 50m in boxed regions. 905 C. Model summarising paracrine WNT secretion from SOX2+ PSCs to lineage- 906 committed progenitors and the effects of abolishing WNT secretion from SOX2+ 907 PSCs through the deletion of Wls. 908 909 36 Supplementary File 1. Gene lists of Gene Set Enrichment Analyses 911 B. Graph of quantification of expansion of the WNT-responsive SF1+ population in 925 Axin2CreERT2/+;ROSA26mTmG/+ pituitaries induced at P14 and lineage traced for 2 or 926 28 days. There is a significant increase of GFP+;SF1+ cells as a proportion of the 927 total SF1+ cells at P28. P=0.0048, unpaired t-test (n=2 at 2 days, 3 at 28 days). 928 C. Immunofluorescence staining against GFP (green) and markers of hormone- 929 secreting endocrine cells of the PIT1 lineage (GH (somatotrophs), PRL 930 (lactotrophs), TSH (thyrotrophs)) in Axin2CreERT2/+;ROSA26mTmG/+ pituitaries 931 induced at P14 and lineage traced for 14 days. Scale bars 50µm. Graph showing 932 expansion of each of the Hormone+ cell types (Hormone+;GFP+) as a percentage 933 of the total Hormone+ population between 2 and 14 days post-induction. There is 934 37 37 a significant increase in GH+ somatotrophs (P=0.000548), and TSH+ thyrotrophs 935 (P=0.0016), whilst there is no significance (ns) between PRL+ lactotroph 936 populations between the two time points. multiple t-test (n=3 at 48 h, n=4 at 14 937 days post-induction). 938 D. Clonal analysis of individual cells targeted in Sox2CreERT2/+;ROSA26Confetti/+ (left 939 panel) and Axin2CreERT2/+;ROSA26Confetti/+ pituitaries (right panel), induced at P14 940 and harvested after 4 weeks (P42). Arrows point to individual clones, numbered 941 for the number of cells in the clone. Scale bar 100µm. 942 943 Figure 1 – figure supplement 2. Axin2 expressing cells contribute to pituitary 944 growth and expansion of all lineages 945 A. Dorsal wholemount view of Axin2CreERT2/+; Ctnnb1LOF/+;ROSA26mTmG/+and 946 Axin2CreERT2/+; Ctnnb1LOF/LOF;ROSA26mTmG/+ pituitaries induced at P14 and 947 lineage traced for 5 days. Scale bars 500m. Immunofluorescence staining 948 against GFP (green) and pH-H3 (magenta) in Axin2CreERT2/+; 949 Ctnnb1LOF/+;ROSA26mTmG/+and Axin2CreERT2/+; Ctnnb1LOF/LOF;ROSA26mTmG/+ 950 pituitaries. Scale bar 50µm. Quantification of the contribution of lineage traced 951 cells in control and mutants. Each data point represents the mean from one 952 individual. P=0.0313, unpaired t-test (n=3). 953 B. Immunofluorescence staining against GFP (green) and PIT1, SF1 and ACTH 954 (magenta) in Axin2CreERT2/+; Ctnnb1LOF/+;ROSA26mTmG/+and 955 Axin2CreERT2/+;Ctnnb1LOF/LOF;ROSA26mTmG/+ pituitaries induced at P14 and lineag 956 traced for 5 days. Quantification of the percentage of GFP+ cells, double-positive 957 f h f h li k h i i ifi h f h li 958 a significant increase in GH+ somatotrophs (P=0.000548), and TSH+ thyrotrophs 935 (P=0.0016), whilst there is no significance (ns) between PRL+ lactotroph 936 populations between the two time points. Supplementary File 1. Gene lists of Gene Set Enrichment Analyses 911 multiple t-test (n=3 at 48 h, n=4 at 14 937 days post-induction). 938 38 between controls and mutants (Unpaired t-test, PIT1 P=0.1729, SF1 P=0.9488, 959 ACTH P=0.6186. n=4 controls, 2 mutants). Scale bars 50µm. 960 C. Immunofluorescence against GFP (green) and SOX2 (magenta) in Axin2CreERT2/+; 961 Ctnnb1LOF/+;ROSA26mTmG/+and Axin2CreERT2/+;Ctnnb1LOF/LOF;ROSA26mTmG/+ 962 induced at P14 and lineage traced for 5 days (n=4 controls, 2 mutants). Scale bars 963 50µm. 964 D. Immunofluorescence against GFP (green) and Cleaved Caspase-3 (magenta) in 965 Axin2CreERT2/+; Ctnnb1LOF/+;ROSA26mTmG/+and 966 Axin2CreERT2/+;Ctnnb1LOF/LOF;ROSA26mTmG/+ induced at P14 and lineage traced for 967 5 days (n=4 controls, 2 mutants). Scale bars 50m. 968 between controls and mutants (Unpaired t-test, PIT1 P=0.1729, SF1 P=0.9488, 959 39 Figure 2 – figure supplement 1. Activation of WNT signalling in SOX2+ PSCs 969 and their descendants is necessary for long-term growth 970 A-E Step-wise gating strategy to isolate WNT-responsive, SOX2-EGFP+ cells by flow 971 sorting. 972 A – B Single pituitary cells dissociated from 973 Axin2CreERT2/+;ROSA26tdTomato/+;Sox2eGFP/+ mice were gated to exclude debris (A) and 974 gated for single cells according to SSC-A and SSC-W (B). 975 C. Dead cells were excluded according to incorporation of DAPI. 976 D. Three populations of fluorescent cells were identified and sorted according to the 977 following profiles: GFP-;tdTomato+, GFP+;tdTomato+ or GFP+;tdTomato-. 978 E. Quantification of the number of GFP+ cells out of all gated cells (left, n=5 979 biological repeats), the proportion of all GFP+ cells that were found to be tdTomato+ 980 (right, n=5 biological repeats) and a representation of the gating used for 981 quantification (bottom). 982 983 Figure 2 – figure supplement 2. Activation of WNT signalling in SOX2+ PSCs 984 and their descendants is necessary for long-term growth 985 A. Confocal images of native GFP fluorescence in frontal sections from 986 TCF/Lef:H2B-EGFP pituitaries at P21. Scale bar 50m. 987 B. mRNA in situ hybridisation in TCF/Lef:H2B-EGFP pituitaries at P21, detecting 988 Egfp transcripts (red). Double mRNA in situ hybridisation showing overlap 989 between Sox2 (red) and Egfp (blue) transcripts in pituitaries at P21. White 990 arrowheads indicate double-positive staining. Scale bars 50m. 991 C. Immunofluorescence staining against SOX2 (magenta) and GFP (green) in 992 TCF/Lef:H2B-EGFP pituitaries harvested from P21 mice. White arrows indicate 993 Figure 2 – figure supplement 1. Supplementary File 1. Gene lists of Gene Set Enrichment Analyses 911 Activation of WNT signalling in SOX2+ PSCs 969 and their descendants is necessary for long-term growth 970 A-E Step-wise gating strategy to isolate WNT-responsive, SOX2-EGFP+ cells by flow 971 sorting. 972 A – B Single pituitary cells dissociated from 973 Axin2CreERT2/+;ROSA26tdTomato/+;Sox2eGFP/+ mice were gated to exclude debris (A) and 974 gated for single cells according to SSC-A and SSC-W (B). 975 C. Dead cells were excluded according to incorporation of DAPI. 976 D. Three populations of fluorescent cells were identified and sorted according to the 977 following profiles: GFP-;tdTomato+, GFP+;tdTomato+ or GFP+;tdTomato-. 978 E. Quantification of the number of GFP+ cells out of all gated cells (left, n=5 979 biological repeats), the proportion of all GFP+ cells that were found to be tdTomato+ 980 (right, n=5 biological repeats) and a representation of the gating used for 981 quantification (bottom). 982 983 Figure 2 – figure supplement 2. Activation of WNT signalling in SOX2+ PSCs 984 and their descendants is necessary for long-term growth 985 C. Immunofluorescence staining against SOX2 (magenta) and GFP (green) in 992 TCF/Lef:H2B-EGFP pituitaries harvested from P21 mice. White arrows indicate 993 40 double positive cells. Graph of quantification of the in vitro colony forming 994 potential of GFP cells isolated from P21 TCF/Lef:H2B-EGFP pituitaries by flow 995 sorting. Each data point represents single well replicates. Error bars show SEM, 996 P<0.001 (One-way ANOVA, n=3 individual pituitaries). Scale bar 50m. 997 Representative scatter plot showing gating used for fluorescence activated cell 998 sorting and population percentages in each gate. 999 D. Immunofluorescence staining against PIT1, TPIT and SF1 (magenta) in 1000 Sox2CreERT2/+;Ctnnb1LOF/+;ROSA26mTmG/+ and 1001 Sox2CreERT2/+;Ctnnb1LOF/LOF;ROSA26mTmG/+ pituitaries 22 weeks post-induction at 1002 P14 (age P24). Arrows indicate double positive cells. Scale bar 50µm. 1003 E. Immunofluorescence staining against β-catenin (magenta) and GFP (green) in 1004 Sox2CreERT2/+; Ctnnb1LOF/+;ROSA26mTmG/+ and Sox2CreERT2/+; 1005 Ctnnb1LOF/LOF;ROSA26mTmG/+ pituitaries 22 weeks post-induction. Arrowheads 1006 indicate double positive cells, arrows indicate GFP+ cells that have lost β-catenin 1007 expression in mutants. Scale bar 50µm. 1008 PL, posterior lobe; IL, Intermediate lobe; AL, anterior lobe; Inf, infundibulum; RP, 1009 Rathke’s pouch; Sph, sphenoid bone. 1010 1011 double positive cells. Graph of quantification of the in vitro colony forming 994 potential of GFP cells isolated from P21 TCF/Lef:H2B-EGFP pituitaries by flow 995 sorting. Each data point represents single well replicates. Error bars show SEM, 996 P<0.001 (One-way ANOVA, n=3 individual pituitaries). Scale bar 50m. Supplementary File 1. Gene lists of Gene Set Enrichment Analyses 911 997 Representative scatter plot showing gating used for fluorescence activated cell 998 sorting and population percentages in each gate. 999 D. Immunofluorescence staining against PIT1, TPIT and SF1 (magenta) in 1000 Sox2CreERT2/+;Ctnnb1LOF/+;ROSA26mTmG/+ and 1001 Sox2CreERT2/+;Ctnnb1LOF/LOF;ROSA26mTmG/+ pituitaries 22 weeks post-induction at 1002 P14 (age P24). Arrows indicate double positive cells. Scale bar 50µm. 1003 PL, posterior lobe; IL, Intermediate lobe; AL, anterior lobe; Inf, infundibulum; RP, 1009 Rathke’s pouch; Sph sphenoid bone 1010 41 Figure 3 – figure supplement 1. SOX2+ PSCs are as a source of WNT ligands in 1012 the pituitary 1013 A. Native EGFP protein expression in frontal cryosection of a P14 Sox2Egfp/+ 1014 pituitary. Schematic of the workflow used for bulk RNA-sequencing analysis of 1015 Sox2+ and Sox2- cells. Genome browser views of reads aligning to the Sox2 and 1016 Pit1 loci in the positive and negative fractions indicating good separation of the 1017 EGFP+ population. Scale bar 50µm. 1018 B. Sox2+ cells express a significant enrichment in markers associated with epithelial- 1019 to-mesenchymal transition (EMT), adherens and tight junctions, consistent with 1020 their epithelial nature. GSEA plots and immunofluorescence staining against E- 1021 Cadherin (adherens junction marker) and ZO1 (tight junction marker) in the 1022 marginal zone epithelium at P14. Scale bar 50µm. See Supplementary File 1 for 1023 full GSEA gene lists. 1024 C. Sox2+ cells express a significant enrichment in several signalling pathways, shown 1025 with respective GSEA plots. See Supplementary File 1 for full GSEA gene lists. 1026 D. Bar charts showing the FPKM values of components of the 1027 LGR/RNF43/ZNRF3/RSPONDIN module in the Sox2+ and Sox2- fractions and 1028 the distribution of the Frizzled receptors. GSEA plot for components of the WNT 1029 pathway. Validation of sequencing: (i) mRNA in situ hybridisation with specific 1030 probes against Lgr4 (blue) and Sox2 (red) in P14 pituitaries showing co- 1031 expression. (ii) Double mRNA in situ hybridisation against Fzd4 (blue) and Sox2 1032 (red) indicating co-expression in both the marginal zone epithelium and 1033 parenchymal Sox2+ cells. Boxed regions are magnified. Scale bars 250µm and 1034 50m in boxed inserts. (iii) mRNA in situ hybridisation against Rspo1, Rspo2, 1035 Rspo3 and Rspo4 in sagittal sections of wild type pituitaries at P14. Boxed regions 1036 Figure 3 – figure supplement 1. SOX2+ PSCs are as a source of WNT ligands in 1012 the pituitary 1013 A. are magnified, only Rspo4 is detected. Scale bars 250µm and 100m in boxed 1037 inserts. 1038 Supplementary File 1. Gene lists of Gene Set Enrichment Analyses 911 Native EGFP protein expression in frontal cryosection of a P14 Sox2Egfp/+ 1014 pituitary. Schematic of the workflow used for bulk RNA-sequencing analysis of 1015 Sox2+ and Sox2- cells. Genome browser views of reads aligning to the Sox2 and 1016 Pit1 loci in the positive and negative fractions indicating good separation of the 1017 EGFP+ population. Scale bar 50µm. 1018 B. Sox2+ cells express a significant enrichment in markers associated with epithelial- 1019 to-mesenchymal transition (EMT), adherens and tight junctions, consistent with 1020 their epithelial nature. GSEA plots and immunofluorescence staining against E- 1021 Cadherin (adherens junction marker) and ZO1 (tight junction marker) in the 1022 marginal zone epithelium at P14. Scale bar 50µm. See Supplementary File 1 for 1023 full GSEA gene lists. 1024 C. Sox2+ cells express a significant enrichment in several signalling pathways, shown 1025 with respective GSEA plots. See Supplementary File 1 for full GSEA gene lists. 1026 D. Bar charts showing the FPKM values of components of the 1027 LGR/RNF43/ZNRF3/RSPONDIN module in the Sox2+ and Sox2- fractions and 1028 the distribution of the Frizzled receptors. GSEA plot for components of the WNT 1029 Figure 3 – figure supplement 1. SOX2+ PSCs are as a source of WNT ligands in 1012 the pituitary 1013 A. Native EGFP protein expression in frontal cryosection of a P14 Sox2Egfp/+ 1014 pituitary. Schematic of the workflow used for bulk RNA-sequencing analysis of 1015 Sox2+ and Sox2- cells. Genome browser views of reads aligning to the Sox2 and 1016 Pit1 loci in the positive and negative fractions indicating good separation of the 1017 EGFP+ population. Scale bar 50µm. 1018 42 are magnified, only Rspo4 is detected. Scale bars 250µm and 100m in boxed 1037 inserts. 1038 1037 1039 43 Figure 4 – figure supplement 1. Paracrine secretion of WNTs from SOX2+ PSCs 1040 is necessary for expansion of committed cells 1041 A. Schematic of time points for induction by tamoxifen induction and tissue 1042 harvesting of control Sox2+/+;Wlsfl/fl and mutant Sox2CreERT2/+;Wlsfl/fl pituitaries. 1043 B. Whole mount, dorsal views of control Sox2+/+;Wlsfl/fl (top panel) and mutant 1044 Sox2CreERT2/+;Wlsfl/fl (bottom panel) pituitaries at P21, representative of n=4 1045 controls and n=5 mutants. Scale bars 500m. 1046 1047 Figure 4 – figure supplement 1. Paracrine secretion of WNTs from SOX2+ PSCs 1040 is necessary for expansion of committed cells 1041 A. Schematic of time points for induction by tamoxifen induction and tissue 1042 harvesting of control Sox2+/+;Wlsfl/fl and mutant Sox2CreERT2/+;Wlsfl/fl pituitaries. 1043 B. Whole mount, dorsal views of control Sox2+/+;Wlsfl/fl (top panel) and mutant 1044 Sox2CreERT2/+;Wlsfl/fl (bottom panel) pituitaries at P21, representative of n=4 1045 controls and n=5 mutants. Scale bars 500m. 1046 A. Schematic of time points for induction by tamoxifen induction and tissue 1042 harvesting of control Sox2+/+;Wlsfl/fl and mutant Sox2CreERT2/+;Wlsfl/fl pituitaries. 1043 B. Whole mount, dorsal views of control Sox2+/+;Wlsfl/fl (top panel) and mutant 1044 Sox2CreERT2/+;Wlsfl/fl (bottom panel) pituitaries at P21, representative of n=4 1045 controls and n=5 mutants. Scale bars 500m. 1046 A. Schematic of time points for induction by tamoxifen induction and tissue 1042 harvesting of control Sox2+/+;Wlsfl/fl and mutant Sox2CreERT2/+;Wlsfl/fl pituitaries. 1043 B. Whole mount, dorsal views of control Sox2+/+;Wlsfl/fl (top panel) and mutant 1044 Sox2CreERT2/+;Wlsfl/fl (bottom panel) pituitaries at P21, representative of n=4 1045 controls and n=5 mutants. Scale bars 500m. 1046 44 Key Resources Table Reagent type (species) or resource Designation Source or reference Identifiers Additional information genetic reagent (Mus musculus) Axin2CreERT2/+ Roel Nusse, Stanford University The Jackson Laboratory JAX:0188 67, RRID:IM SR_JAX:0 18867 genetic reagent (Mus musculus) Sox2CreERT2/+ (Andoniadou et al. 2013) PMID: 24094324 DOI: 10.1016/j.stem.2 013.07.004 MGI:551 2893 genetic reagent (Mus musculus) ROSA26mTmG/ mTmG The Jackson Laboratory JAX:0076 76, RRID:IM SR_JAX:0 07676 genetic reagent (Mus musculus) ROSA26Confetti/ Confetti The Jackson Laboratory JAX:01749 2, RRID:IMS R_JAX:01 7492 genetic reagent (Mus musculus) ROSA26tdTomat o/tdTomato The Jackson Laboratory JAX:0079 09, RRID:IM SR_JAX:0 07909 genetic reagent (Mus musculus) Ctnnb1fl(ex2-6)/ fl(ex2-6) (CtnnbLOF/LOF) The Jackson Laboratory JAX:0041 52, RRID:IM SR_JAX:0 04152 genetic reagent (Mus musculus) Wlsfl/fl The Jackson Laboratory JAX:0128 88, RRID:IM SR_JAX:0 12888 genetic reagent (Mus musculus) Sox2eGFP/+ (Ellis et al., 2004) PMID: MGI:3589 809 15711057 DOI: 10.1159/000082 134 genetic reagent (Mus musculus) TCF/Lef:H2B -GFP The Jackson Laboratory JAX:0137 52, RRID:IM SR_JAX:0 13752 cell line (Mus musculus) Primary anterior pituitary cells This paper N/A Freshly isolated from Mus musculus. antibody Anti-GFP, (Chicken Polyclonal) Abcam ab13970, RRID:AB _300798 IF(1:400) antibody Anti-SOX2, (Goat Polyclonal) Immune Systems Ltd GT15098, RRID:AB _2195800 IF(1:200) antibody Anti-SOX2, (Rabbit Monoclonal) Abcam ab92494, RRID:AB _1058542 8 IF(1:100) antibody Anti-SOX9, (Rabbit Monoclonal) Abcam ab185230, RRID:AB _2715497 IF(1:500) antibody Anti-POU1F1 (PIT1), (Rabbit Monoclonal) Gifted by Dr S. J. Figure 4 – figure supplement 1. Paracrine secretion of WNTs from SOX2+ PSCs 1040 is necessary for expansion of committed cells 1041 Rhodes (IUPUI, USA) 422_Rhod es, RRID:AB _2722652 IF(1:500) antibody Anti-SF1 (NR5A1, clone N1665), (Mouse Monoclonal) Thermo Fisher Scientific 434200, RRID:AB _2532209 IF(1:300) antibody Anti-TBX19 (TPIT), (Rabbit Polyclonal) Gifted by Dr J. Figure 4 – figure supplement 1. Paracrine secretion of WNTs from SOX2+ PSCs 1040 is necessary for expansion of committed cells 1041 Drouin (Montreal Clinical Research Institute, Canada) Ac1250 #71, RRID:AB _2728662 IF(1:200) antibody Anti-Ki67, (Rabbit Monoclonal) Abcam ab15580, RRID:AB _443209 IF(1:100) antibody Anti-pH-H3, (Rabbit Polyclonal) Abcam ab5176, RRID:AB _304763 IF(1:500) antibody Anti-GH, (Rabbit Polyclonal) National Hormone and Peptide Program (NHPP) AFP- 5641801 IF(1:1000) antibody Anti-TSH, (Rabbit Polyclonal) National Hormone and Peptide Program (NHPP) AFP- 1274789 IF(1:1000) antibody Anti-PRL, (Rabbit Polyclonal) National Hormone and Peptide Program (NHPP) AFP- 4251091 IF(1:1000) antibody Anti-ACTH, (Mouse Monoclonal) Fitzgerald 10C- CR1096M 1, RRID:AB _1282437 IF(1:400) antibody Anti-LH, (Rabbit Polyclonal) National Hormone and Peptide Program (NHPP) AFP- 697071P IF(1:300) antibody Anti-FSH, (Rabbit Polyclonal) National Hormone and Peptide Program (NHPP) AFP- HFS6 IF(1:300) antibody Anti-ZO-1, (Rat Monoclonal) Santa Cruz SC33725, RRID:AB _628459 IF(1:300) antibody Anti-E- CADHERIN, (Rabbit Monoclonal) Cell Signaling 3195S, RRID:AB _2291471 IF(1:300) antibody Anti-Rabbit 488, (Goat Polyclonal) Life Technologies A11008, RRID:AB _143165 IF(1:400) antibody Anti-Rabbit 555, (Goat Polyclonal) Life Technologies A21426, RRID:AB _1500929 IF(1:400) antibody Anti-Rabbit 633, (Goat Polyclonal) Life Technologies A21050, RRID:AB _141431 IF(1:400) antibody Anti-Goat 488, (Donkey Polyclonal) Abcam ab150133, RRID:AB _2832252 IF(1:400) antibody Anti-Chicken 488, (Goat Polyclonal) Life Technologies A11039, RRID:AB _142924 IF(1:400) antibody Anti-Chicken 647, (Goat Polyclonal) Life Technologies A21449, RRID:AB _1500594 IF(1:400) antibody Anti-Rat 555, (Goat Polyclonal) Life Technologies A21434, RRID:AB _141733 IF(1:400) antibody Anti-Mouse 555, (Goat Polyclonal) Life Technologies A21426, RRID:AB _1500929 IF(1:400) antibody Anti-Rabbit Biotinylated, (Donkey Polyclonal) Abcam ab6801, RRID:AB _954900 IF(1:400) antibody Anti-Rabbit Biotinylated, (Goat Polyclonal) Abcam ab207995 IF(1:400) antibody Anti-Mouse Biotinylated, (Goat Biotinylated) Abcam ab6788, RRID:AB _954885 IF(1:400) sequence- based reagent RNAscope probe M.musculus Axin2 Advanced Cell Diagnostics 400331 sequence- based reagent RNAscope probe M.musculus Lef1 Advanced Cell Diagnostics 441861 sequence- based reagent RNAscope probe M.musculus Wls Advanced Cell Diagnostics 405011 sequence- based reagent RNAscope probe M.musculus Rspo1 Advanced Cell Diagnostics 401991 sequence- based reagent RNAscope probe M.musculus Rspo2 Advanced Cell Diagnostics 402001 sequence- based reagent RNAscope probe M.musculus Rspo3 Advanced Cell Diagnostics 402011 sequence- based reagent RNAscope probe M.musculus Rspo4 Advanced Cell Diagnostics 402021 sequence- based reagent RNAscope probe M.musculus Lgr4 Advanced Cell Diagnostics 318321 sequence- based reagent RNAscope probe M.musculus Wnt9a Advanced Cell Diagnostics 405081 sequence- based reagent RNAscope probe M.musculus Wnt2 Advanced Cell Diagnostics 313601 sequence- based reagent RNAscope probe M.musculus Wnt5a Advanced Cell Diagnostics 316791 sequence- based reagent RNAscope probe eGFP Advanced Cell Diagnostics 400281 sequence- based reagent RNAscope probe M.musculus Jun Advanced Cell Diagnostics 453561 sequence- based reagent RNAscope probe M.musculus Axin2 (Channel 2) Advanced Cell Diagnostics 400331- C2 sequence- based reagent RNAscope probe M.musculus Sox2 (Channel 2) Advanced Cell Diagnostics 401041- C2 sequence- based reagent RNAscope probe eGFP (Channel 2) Advanced Cell Diagnostics 400281- C2 sequence- based reagent RNAscope probe M.musculus Sox2 Advanced Cell Diagnostics 401041 sequence- based reagent RNAscope probe M.musculus Pou1f1 Advanced Cell Diagnostics 486441 sequence- based reagent RNAscope probe Duplex Positive Control Ppib- C1, Polr2a- C2 Advanced Cell Diagnostics 321641 sequence- based reagent RNAscope probe Duplex Negative Control DapB (both channels) Advanced Cell Diagnostics 320751 sequence- based reagent RNAscope probe Singleplex Positive Control Ppib Advanced Cell Diagnostics 313911 sequence- based reagent RNAscope probe: Singleplex Negative Control DapB Advanced Cell Diagnostics 310043 peptide, recombinant protein Streptavidin 488 Life Technologies S11223 IF(1:400) peptide, recombinant protein Streptavidin 555 Life Technologies S32355 IF(1:400) peptide, recombinant protein Streptavidin 633 Life Technologies S21375 IF(1:400) commercial assay or kit RNAScope 2.5 HD Assay -RED Advanced Cell Diagnostics 322350 commercial assay or kit RNAScope 2.5 HD Duplex Assay Advanced Cell Diagnostics 322430 commercial assay or kit LIVE/DEAD Fixable Near IR-Dead Cell Life Technologies L34975 Stain Kit commercial assay or kit FIX and PERM Cell Permeabilizati on Kit Life Technologies GAS003 chemical compound, drug Tamoxifen Sigma T5648 chemical compound, drug Corn Oil Sigma C8267 chemical compound, drug Collagenase Type 2 Worthington 4178 chemical compound, drug 10X Trypsin Sigma 59418C chemical compound, drug Deoxyribonuc lease I Worthington LS002172 chemical compound, drug Fungizone Gibco 15290 chemical compound, drug Hank’s Balanced Salt Solution (HBSS) Gibco 14170 chemical compound, drug Fetal Bovine Serum Sigma F2442 chemical compound, drug HEPES Thermo Fisher 15630 chemical compound, drug bFGF R&D Systems 233-FB- 025 chemical compound, drug Cholera Toxin Sigma C8052 chemical compound, drug DMEM-F12 Thermo Fisher 31330 chemical compound, drug Penicillin/Stre ptomycin Gibco 15070-063 chemical compound, drug Neutral Buffered Formalin Sigma HT501128 chemical compound, drug Hoechst 33342 Thermo Fisher H3570 1:1000 chemical compound, drug Declere Sigma D3565 chemical compound, drug Neo-Clear Sigma 65351-M software, algorithm FlowJo FlowJo, LLC https://ww w.flowjo.c om/ RRID:SC R_008520 software, algorithm Prism 7 GraphPad Software https://ww w.graphpa d.com/ software, algorithm Image Lab Bio-Rad Laboratories http://ww w.bio- rad.com/ software, algorithm NDP View Hamamatsu Photonics https://ww w.hamama tsu.com/ software, algorithm HISAT v2.0.3 (Kim, Langmead, & Salzberg, 2015) https://gith ub.com/inf philo/hisat 2 RRID:SC R_015530 software, algorithm DESeq2 v2.11.38 (Love, Huber, & Anders, 2014) https://gith ub.com/Bi oconducto r- mirror/DE Seq2 RRID:SC R_015687 software, algorithm featureCounts v1.4.6p5 (Liao, Smyth, & Shi, 2014) http://subr ead.source forge.net/ RRID:SC R_012919 software, algorithm The Galaxy Platform (Afgan et al., 2016; Blankenberg et al., 2010; Goecks, Nekrutenko, & Taylor, 2010) https://use galaxu.org RRID:SC R_006281 software, algorithm Gene Set Enrichment Analysis (GSEA) (Subramanian et al, 2005) software.b roadinstitu te.org/gsea /index.jsp RRID:SC R_003199 software, algorithm Cufflinks (Trapnell et al., 2012) https://gith ub.com/co le- trapnell- lab/cufflin ks RRID:SC R_014597 other Deposited Data, RNA- Seq BioProject (NCBI) PRJNA42 1806 1
https://openalex.org/W4200533920	https://www.researchsquare.com/article/rs-1126390/latest.pdf	English	null	Investigation of Potential Environmental Impacts and Sustainable Management of Municipal Solid Waste using the Driving force-Pressure-State-Impact-Response-Outcome (DPSIRO) framework: Case of Bahir Dar, Ethiopia	Research Square (Research Square)	2,021	cc-by	10,517	Investigation of Potential Environmental Impacts and Sustainable Management of Municipal Solid Waste using the Driving force-Pressure-State- Impact-Response-Outcome (DPSIRO) framework: Case of Bahir Dar, Ethiopia Awoke Misganaw (  awokemisganaw12@gmail.com ) Debre Tabor University https://orcid.org/0000-0002-9859-0226 Banchamlak Akenaw Wolkite University Research Article Keywords: Sustainable management, Eutrophication potential, Greenhouse gas emission, Municipal solid waste, Framework, Ethiopia Posted Date: December 14th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-1126390/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License 1 Title: Investigation of potential environmental impacts and sustainable management of 1 municipal solid waste using the Driving force-Pressure-State-Impact-Response-Outcome 2 (DPSIRO) framework: Case of Bahir Dar, Ethiopia 3 Awoke Misganawa,, Banchamlak Akenaw b 4 aFaculty of Technology, Department of Chemical Engineering, Debre Tabor University, Debre 5 Tabor, Ethiopia: awokemisganaw12@gmail.com 6 bFaculty of Technology, Department of Chemical Engineering, Wolkite University, Wolkite, 7 Ethiopia: abesis44@gmail.com 8 Corresponding author address: 9 Email: awokemisganaw12@gmail.com 10 Mobile: +251-932-852-548 11 Posta 272 12 Abstract 13 The generation of MSW in urban areas in Ethiopia and elsewhere continues to increase and 14 poses a challenge to city governments and citizens if the wastes are not properly managed. 15 Applying an integrated system for managing MSW and recovering the material for use in new 16 products can reduce the negative impacts on the environment. The purpose of this study is to 17 apply the DPSIRO framework to develop a system that reduces the negative impacts of MSW in 18 Bahir Dar city in a sustainable way. The research started by identifying the main driving forces 19 that lead to the generation of MSW. Then, states because of pressures and the consequent 20 impacts were investigated. Finally, the appropriate responses and outcomes obtained from the 21 responses were dealt with. Methods used to quantify GHG emissions, leachate, and 22 eutrophication potential were applied. According to the findings, the waste disposal site emits an 23 Title: Investigation of potential environmental impacts and sustainable management of 1 municipal solid waste using the Driving force-Pressure-State-Impact-Response-Outcome 2 (DPSIRO) framework: Case of Bahir Dar, Ethiopia 3 Awoke Misganawa,, Banchamlak Akenaw b 4 aFaculty of Technology, Department of Chemical Engineering, Debre Tabor University, Debre 5 Tabor, Ethiopia: awokemisganaw12@gmail.com 6 bFaculty of Technology, Department of Chemical Engineering, Wolkite University, Wolkite, 7 Ethiopia: abesis44@gmail.com 8 Corresponding author address: 9 Email: awokemisganaw12@gmail.com 10 Mobile: +251-932-852-548 11 Posta 272 12 Abstract 13 The generation of MSW in urban areas in Ethiopia and elsewhere continues to increase and 14 poses a challenge to city governments and citizens if the wastes are not properly managed. Research Article 15 Applying an integrated system for managing MSW and recovering the material for use in new 16 products can reduce the negative impacts on the environment. The purpose of this study is to 17 apply the DPSIRO framework to develop a system that reduces the negative impacts of MSW in 18 Bahir Dar city in a sustainable way. The research started by identifying the main driving forces 19 that lead to the generation of MSW. Then, states because of pressures and the consequent 20 impacts were investigated. Finally, the appropriate responses and outcomes obtained from the 21 responses were dealt with. Methods used to quantify GHG emissions, leachate, and 22 eutrophication potential were applied. According to the findings, the waste disposal site emits an 23 Title: Investigation of potential environmental impacts and sustainable management of 1 municipal solid waste using the Driving force-Pressure-State-Impact-Response-Outcome 2 (DPSIRO) framework: Case of Bahir Dar, Ethiopia 3 1 Title: Investigation of potential environmental im 1 municipal solid waste using the Driving force-Pr 2 (DPSIRO) framework: Case of Bahir Dar, Ethiopia 3 Awoke Misganawa,, Banchamlak Akenaw b 4 aFaculty of Technology, Department of Chemical En 5 Tabor, Ethiopia: awokemisganaw12@gmail.com 6 bFaculty of Technology, Department of Chemical E 7 Ethiopia: abesis44@gmail.com 8 Corresponding author address: 9 Email: awokemisganaw12@gmail.com 10 Mobile: +251-932-852-548 11 Posta 272 12 Abstract 13 The generation of MSW in urban areas in Ethiopia 14 poses a challenge to city governments and citizens 15 Applying an integrated system for managing MSW a 16 products can reduce the negative impacts on the env 17 apply the DPSIRO framework to develop a system th 18 Bahir Dar city in a sustainable way. The research sta 19 that lead to the generation of MSW. Then, states 20 impacts were investigated. Finally, the appropriate r 21 responses were dealt with. Methods used to q 22 eutrophication potential were applied. According to th 23 Awoke Misganawa,*, Banchamlak Akenaw b 4 aFaculty of Technology, Department of Chemical Engineering, Debre Tabor University, Debre 5 Tabor, Ethiopia: awokemisganaw12@gmail.com 6 aFaculty of Technology, Department of Chemical Engineering, Debre Tabor University, Debre 5 Tabor, Ethiopia: awokemisganaw12@gmail.com 6 The generation of MSW in urban areas in Ethiopia and elsewhere continues to increase and 14 poses a challenge to city governments and citizens if the wastes are not properly managed. 15 Applying an integrated system for managing MSW and recovering the material for use in new 16 products can reduce the negative impacts on the environment. The purpose of this study is to 17 apply the DPSIRO framework to develop a system that reduces the negative impacts of MSW in 18 Bahir Dar city in a sustainable way. The research started by identifying the main driving forces 19 that lead to the generation of MSW. Then, states because of pressures and the consequent 20 impacts were investigated. Finally, the appropriate responses and outcomes obtained from the 21 responses were dealt with. Methods used to quantify GHG emissions, leachate, and 22 eutrophication potential were applied. According to the findings, the waste disposal site emits an 23 1 1 estimated 46Gg of greenhouse gases per year in 2020.The eutrophication capacity of organic 24 waste generated in the city was 0.0594 Kg N-equivalent or 59.4g N-equivalent. The waste 25 also contains an average of 1,112mm of leachate per day on an annual basis. The state of the 26 environment has an impact on human health and the ecosystem. Applying the circular economic 27 system, knowledge transfer, and waste management fees are the main responses suggested to 28 decision and policy makers. The responses correspond with balanced economic, social and 29 environmental situations. Outcomes were quantified in terms of organic fertilizer, income and 30 renewable energy (briquette) when the actions were taken. 31 Keywords: Sustainable management, Eutrophication potential, Greenhouse gas emission, 32 Municipal solid waste, Framework, Ethiopia 33 Keywords: Sustainable management, Eutrophication potential, Greenhouse gas emission, 32 Municipal solid waste, Framework, Ethiopia 33 Municipal solid waste, Framework, Ethiopia 33 1. Introduction 34 Citizens and businesses in most of the world’s cities consume a great deal of materials and 35 produce a great deal of waste (Makarichi et al., 2018). With rapid population and economic 36 growth and urbanization, solid waste output is increasing in urban areas around the world 37 (Farzadkia et al., 2020; Singh, 2019). According to a World Bank report (Franco et al., 2021; 38 Kaza et al., 2018) cities around the world generate roughly 2.01 billion tons of MSW per year, 39 and that number is projected to increase to 3.40 billion tons by 2050. Clearly the solid waste 40 problem is a major concern for national and local authorities in many cities throughout 41 developing and developed countries (Inghels et al., 2016; Noufal et al., 2020). Especially in 42 developing countries, the common solid waste management system that they use is landfill 43 because it is relatively less costly to implement and operate than other options. However, 44 landfills are widely regarded as the least preferable municipal solid waste management system 45 due to its high contamination potential, including water and soil pollution due to the leachate 46 2 seepage and greenhouse gases (GHGs) emissions resulting from the decomposition of 47 biodegradable waste (Adeleke et al., 2021; Wang et al., 2020), and the sequestration of 48 potentially reusable materials in lieu of recycling. 49 The negative impact of municipal solid waste due to its mismanagement in the cities covers from 50 local to global level. The release of odorous compounds from landfill has a very local (micro-) 51 impact as it affects the surrounding population. Discharge of pollutants into ground or surface 52 water due to landfilling can have an impact on the regional (meso-) scale, as eutrophication can 53 occur tens of kilometers away from disposal facilities unless fugitive leachate is collected and 54 properly treated. Emissions of greenhouse gases such as methane contribute to global warming, 55 which has an impact on the global population (macro impact) (Ellen et al., 2018; Matheus, 2018; 56 Mattos et al., 2020) . The increasing demand for goods due to increased urbanization and 57 population is causing solid waste generation in Bahir Dar city to steadily increase. 1. Introduction 34 Currently, the 58 majority of solid waste produced in the city is dumped into open dumpsites without any 59 processing and uncontrolled emissions lead to negative impacts on soil, water and air 60 environment; these emissions are expected to continue in the future until MSW management 61 systems are improved. 62 Sustainable MSW management necessitates a multifaceted approach including a wide range of 63 stakeholders (Salem et al., 2020). The aim of the study was to apply the Driving force-Pressure- 64 State-Impact-Response-Outcome (DPSIRO) framework to study the potential impact of 65 municipal solid waste on air, soil and water quality and develop sustainable indicators to reduce 66 the impact. The DPSIRO framework is an extension of the DPSIR framework and it is a cycle 67 which relates to a particular human need and accompanying activities(Misganaw & Teffera, 68 2021). The DPSIRO framework analysis describes that economic and social development, which 69 3 are common driving force (D), exert pressure (P) on the environment, and, as a result, the state 70 (S) of the environment such as depletion of natural resources and degradation of environmental 71 quality changes. These changes then have an impact (I) on the environment and human health. 72 Due to these impacts, society responds (R) to the driving force, the pressure, state or impact, and 73 then when society gives the response, the outcome (O) shows the expected result at each 74 response of the impact. 75 are common driving force (D), exert pressure (P) on the environment, and, as a result, the state 70 (S) of the environment such as depletion of natural resources and degradation of environmental 71 quality changes. These changes then have an impact (I) on the environment and human health. 72 Due to these impacts, society responds (R) to the driving force, the pressure, state or impact, and 73 then when society gives the response, the outcome (O) shows the expected result at each 74 response of the impact. 75 2.1. Overview of study area 77 The city of Bahir Dar was chosen for investigation of the possible negative impacts of MSW and 78 the development of sustainable indicators to reduce the impacts using the DPSIRO framework. 79 Bahir Dar is a town in Ethiopia's northwestern region, near the southern end of Lake Tana, at the 80 headwaters of the Blue Nile River that then flows through Sudan and Egypt. The city is located 81 at an elevation of approximately 1,820 meters above sea level, with geographic coordinates of 82 11036'N and 37° 25' E. The establishment of Bahir Dar can be traced back to the fourteenth 83 century, when Saint Kidane Miheret Church was built on the current site of Saint Giorgis 84 Church (Biruk, 2017; Kassie, 2016; Tirusew et al., 2013). 85 The city evolved from a monastery administration site as well as a market place to a rapidly 86 growing urban center. The Amhara National Regional State's administrative seat is now located 87 there. The city is made up of five sub-cities that share a common geographical location and 88 people's living habits. With the range of attractions on the nearby Lake Tana (Ethiopia's largest 89 lake and popular for churches and monasteries on the lake's 37 islands), it has become one of the 90 country's main tourist destinations. Currently, the city consists of more than 415,000 peoples. 91 4 4 2.2. Research Method 2.2. Research Method 92 In this study, the DPSIRO framework has been applied to assess the impact of municipal solid 93 waste on soil, water and the air environment in Bahir Dar city. Figure 1 illustrates the cause and 94 effect relation between factors of the DPSIRO framework. During the time of study, initially the 95 driving forces which lead to the generation of municipal solid waste as pressure ware 96 determined. Second, the environmental change that results from pressure has been investigated. 97 The state change of the environment was quantified in terms of greenhouse gas emission, 98 eutrophication potential and leachate using mathematical models. Using an intergovernmental 99 panel on climate change (IPCC) model, the total amount of CO2 and CH4 emitted from Bahir Dar 100 city waste was estimated (Misganaw & Teffera, 2021). According to IPCC guidelines, the 101 equation for measuring methane emissions from waste disposal sites was calculated using 102 equation1. 103 Methane emission ( Gg yr) = MSWT×MSWF×MCF×DOC×DOCF×F×(1−OX) 16 (12−R) ……………………….1 104 The abbreviations in the above equation represent, MSWT is total municipal solid waste 105 generated (Gg/yr.), MSWF represents the fraction of MSW disposed of in one or more solid 106 waste disposal sites, MCF is methane correction factor (fraction), DOC indicates degradable 107 organic carbon (fraction) (kg C/kg SW), DOCF illustrate fraction DOC dissimilated, F is fraction 108 of methane in waste dump gas, 16/12 is the conversion of carbon to methane, R represents 109 recovered methane (Gg/yr.) and OX is oxidation factor (fraction-IPCC default is 0). 110 The approach assumes that all future methane emissions occur in the same year that the waste is 111 discarded. The method is straightforward, and calculating emissions needs only the input of a 112 small number of parameters, for which the IPCC guidelines provide default values in cases 113 where country or city-specific quantities and data are unavailable. The amount of methane gas 114 5 released from the disposal site was estimated using the recommended degradable organic carbon 115 and decay rate value for waste disposal site. 116 released from the disposal site was estimated using the recommended degradable organic carbon 115 and decay rate value for waste disposal site. 2.2. Research Method The responses and outcomes form a balanced relationship with 142 economic, social and environmental situations. Finally, the overall scenario of the DPSIRO 143 framework was conceptualized holistically by forming a balanced relation between economic, 144 social and environmental situation. [Figure 1] 145 The symbols in equation 6 represented as, L is the volume of leachate, R is the volume of rainfall 138 and Ea is the volume of real evapotranspiration (or simpler evaporation from the earth level). 139 and Ea is the volume of real evapotranspiration (or simpler evaporation from the earth level). 139 Third, the impact that results from the change of soil, water and air quality was determined. Next 140 to the impact, the recommended responses and expected outcomes obtained from the possible 141 actions were determined. The responses and outcomes form a balanced relationship with 142 economic, social and environmental situations. Finally, the overall scenario of the DPSIRO 143 framework was conceptualized holistically by forming a balanced relation between economic, 144 social and environmental situation. [Figure 1] 145 2.2. Research Method 116 The emissions of carbon dioxide from municipal solid waste disposal sites without gas collection 117 systems were calculated using B = 𝐴× ( 1−𝐹 𝐹+ 𝑂𝑋) × 44 16 … … … … … … . … 2. 118 In equation 2 the variable B is CO2 emission (Gg/yr.), A represents the quantity of CH4 119 calculated from equation 1above (Gg CH4/yr.), F is fraction by volume of CH4in landfill gas, 120 generally assumed to be 0.5, OX illustrates soil oxidation fraction, typically 0.1 (fraction) and the 121 numerical values 44 and 16 represent molecular weight of CO2 (kg/kg-mol) and molecular 122 weight of CH4 (kg/kg-mol) respectively. 123 The eutrophication capacity of organic waste was calculated using equation 3, 4 and 5 after the 124 molecular formula of organic waste of Bahir Dar city determined. 125 EP = 𝑉/𝑀𝑊 𝑉𝑟𝑒𝑓/𝑀𝑊𝑟𝑒𝑓… … … … … 3, 126 V = 𝑃+ 𝑁 16 + 𝑇ℎ𝑂𝐷 138 … … . .4 and 127 ThOD = 𝐶+ 𝐻−3𝑁 4 + 𝑂 2 … … … . .5. 128 The variables in the above three consecutive equations represent, EP is eutrophication potential, 129 P indicates the number of phosphorus atoms in the molecule equals to 0 for all of our 130 compounds, N is the number of nitrogen atoms in the molecule, O illustrates the number of 131 oxygen atoms in the molecule, C is number of carbon atoms in the molecule, H represent number 132 of hydrogen atoms in the molecule, V ref is the V for phosphate anion, [PO4]3- and V ref equals 133 to 1, MW ref indicate the MW for phosphate anion, [PO4]3- and MW ref equals to 94.97 g/mol, 134 MW is the MW of the compound and ThOD represent theoretical oxygen demand. The volume 135 of leachate resulting from municipal solid waste in Bahir Dar city were quantified by using the 136 6 6 ..6. relation L=R-Ea ..................6. 137 The symbols in equation 6 represented as, L is the volume of leachate, R is the volume of rainfall 138 and Ea is the volume of real evapotranspiration (or simpler evaporation from the earth level). 139 Third, the impact that results from the change of soil, water and air quality was determined. Next 140 to the impact, the recommended responses and expected outcomes obtained from the possible 141 actions were determined. 2.3. Data Collection, Data Source and Data Type 146 Secondary data was gathered from bot 169 existing and unpublished sources for the study. 170 3. Result and Discussion 171 3.1. Deriving Force and Pressure 172 Three main driving forces: natural population growth rate, economic growth and rapi 173 urbanization are led to generation of municipal solid waste in case of Bahir Dar city. With 174 waste generation rate of 0.223kg per capita per day, the municipal solid waste produced in Bah 175 Dar city increased from 73.05 tons per day in 2007 to 148.13 tons per day in 2020 (Misganaw 176 Teffera, 2021). The average annual population growth rate of 6.6 percent is one of the ke 177 factors that determines the shift in urban solid waste generation rate (Biruk, 2017; Tirusew et al 178 2013). The study of municipal solid waste in Bahir Dar city revealed that as the city's populatio 179 grows, the amount of municipal solid waste generated grows as well. As shown in table 1 below 180 solid waste production is rising in all waste generation sources. Depending on the result and from 181 3. Result and Discussion 171 3.1. Deriving Force and Pressure 172 3. Result and Discussion 171 2.3. Data Collection, Data Source and Data Type 146 All of the data needed to complete the DPSIRO framework analysis for this study was gathered 147 at the same time from both primary and secondary data sources. The five cases found in the city 148 were grouped into three major groups once the analysis was completed. The three major classes 149 were: inner, middle, and outer. A grouping technique based on population density, geographical 150 location, population settlement, and land use pattern was employed to make data gathering from 151 sample sites homogeneous. Data was randomly collected from the inner, middle, and outer 152 classrooms to make the data representative at the time. 153 There are around seventeen Keble’s in the city's five Kefleketema. The core (inner) part includes 154 There are around seventeen Keble’s in the city's five Kefleketema. The core (inner) part includes 154 Keble 01, 02, 03, 04, 05, 06, and 12. 07, 08, 09, 10, 15, and 17 are the Keble’s who belong to the 155 middle part. The remaining Keble numbers 11, 13, 14, and 16 make up the outer group. This data 156 was obtained from the Bahir Dar city administration office, which falls under the Keble's level 157 category for 2016. From those categories, three Keble examples were chosen. Keble 05, Keble 158 10, and Keble 11 came from the center, middle, and peripheral, respectively. A total of 65, 96, 159 7 7 and 62 households were chosen at random to be sampled. The total number of households taken 160 from both groups was 223. 161 The number of samples collected from sampling households was 33, 36, and 27 for the central, 162 middle, and inner regions, respectively, with a total weight of 646.215kg from residential. The 163 events took place between January 18 and March 23, 2020. The data for this approach was 164 primarily gathered through solid waste characterization, focus group discussions, and face-to- 165 face interviews with responsible persons regarding solid waste management, industry 166 availability, and the population of Bahir Dar. Land observation was the other technique used. It 167 was carried out to determine the actual capacity of municipal solid waste collected in the area, as 168 well as their solid waste collection and sorting methods. Secondary data was gathered from both 169 existing and unpublished sources for the study. 170 p y p , well as their solid waste collection and sorting methods. 3.1. Deriving Force and Pressure 172 Rapid 187 urbanization and technology used for production process are also considered as driving forces 188 leading to waste generation in the city. Production, consumption and land use for solid waste 189 disposal sites are pressures in addition to waste generation. 190 the total waste generated, only 58% was properly collected and disposed of and 86% of the total 182 waste generated is degradable (Asmare & Alelign, 2019; Wegedie, 2018). [Table 1] 183 Based on the analysis of environmental sustainability of the DPSIRO model, the pressure of 184 municipal solid waste generation (P) is initiated by the driving force of municipal solid waste 185 generation (D) and can block the action of the responsible community®. A large quantity of 186 solid waste is generated in residential areas. It covers around 55% of the total waste. Rapid 187 urbanization and technology used for production process are also considered as driving forces 188 leading to waste generation in the city. Production, consumption and land use for solid waste 189 disposal sites are pressures in addition to waste generation. 190 The pressure resulting from the driving force leads to changes in the state of environmental 192 components, such as water, air and soil quality. This environmental degradation creates an 193 elevated threat to economic growth and development (Chapagain et al., 2020). Change in 194 environmental quality is a combination of physical, chemical and biological conditions. 195 3.1. Deriving Force and Pressure 172 Three main driving forces: natural population growth rate, economic growth and rapid 173 urbanization are led to generation of municipal solid waste in case of Bahir Dar city. With a 174 waste generation rate of 0.223kg per capita per day, the municipal solid waste produced in Bahir 175 Dar city increased from 73.05 tons per day in 2007 to 148.13 tons per day in 2020 (Misganaw & 176 Teffera, 2021). The average annual population growth rate of 6.6 percent is one of the key 177 factors that determines the shift in urban solid waste generation rate (Biruk, 2017; Tirusew et al., 178 2013). The study of municipal solid waste in Bahir Dar city revealed that as the city's population 179 grows, the amount of municipal solid waste generated grows as well. As shown in table 1 below, 180 solid waste production is rising in all waste generation sources. Depending on the result and from 181 8 8 the total waste generated, only 58% was properly collected and disposed of and 86% of the total 182 waste generated is degradable (Asmare & Alelign, 2019; Wegedie, 2018). [Table 1] 183 Based on the analysis of environmental sustainability of the DPSIRO model, the pressure of 184 municipal solid waste generation (P) is initiated by the driving force of municipal solid waste 185 generation (D) and can block the action of the responsible community®. A large quantity of 186 solid waste is generated in residential areas. It covers around 55% of the total waste. Rapid 187 urbanization and technology used for production process are also considered as driving forces 188 leading to waste generation in the city. Production, consumption and land use for solid waste 189 disposal sites are pressures in addition to waste generation. 190 the total waste generated, only 58% was properly collected and disposed of and 86% of the total 182 waste generated is degradable (Asmare & Alelign, 2019; Wegedie, 2018). [Table 1] 183 Based on the analysis of environmental sustainability of the DPSIRO model, the pressure of 184 municipal solid waste generation (P) is initiated by the driving force of municipal solid waste 185 generation (D) and can block the action of the responsible community®. A large quantity of 186 solid waste is generated in residential areas. It covers around 55% of the total waste. 3.2.1. Greenhouse Gas Emission (GHGs) 196 [Table 3] 224 in to the drains and waste not reaching in to dump site due to insufficient solid waste 204 management system. This insufficient management increases the transmission of diseases, causes 205 contamination of surface and ground water, greenhouse gas emissions, and ecosystem damage. 206 The total GHG emissions from dumpsite are calculated as the sum of the CO2 emissions and the 207 CH4 emissions (converted to CO2eq) (Misganaw & Teffera, 2021). The result obtained by using 208 this relation was 23.112Gg in 2007 and reached 46.873Gg in 2020. As the result indicated in 209 figure 2 the emission of greenhouse gases is increasing from time to time from 2007 to 2020. 210 [Figure 2] 211 in to the drains and waste not reaching in to dump site due to insufficient solid waste 204 management system. This insufficient management increases the transmission of diseases, causes 205 contamination of surface and ground water, greenhouse gas emissions, and ecosystem damage. 206 The total GHG emissions from dumpsite are calculated as the sum of the CO2 emissions and the 207 CH4 emissions (converted to CO2eq) (Misganaw & Teffera, 2021). The result obtained by using 208 this relation was 23.112Gg in 2007 and reached 46.873Gg in 2020. As the result indicated in 209 figure 2 the emission of greenhouse gases is increasing from time to time from 2007 to 2020. 210 [Figure 2] 211 3.2.1. Greenhouse Gas Emission (GHGs) 196 Air pollution is one of the world's most important challenges, with long- and short-term health 197 consequences in countries of all socioeconomic levels (Luiz et al., 2020; Maroosi et al., 2019). 198 GHGs, such as CH4 and CO2, are produced from the aerobic and anaerobic biodegradation of 199 municipal solid waste (Zhang et al., 2019). The amount of CH4 and CO2 emitted from Bahir Dar 200 city dump site was determined using the IPCC model. In this paper, municipal solid waste equals 201 the amount of urban waste transported to the disposal site, then the MSWF is equal to 58% and 202 the remaining 42% is assumed to be lost due to recycling, waste burning at source, waste thrown 203 9 9 in to the drains and waste not reaching in to dump site due to insufficient so 204 management system. This insufficient management increases the transmission of diseas 205 contamination of surface and ground water, greenhouse gas emissions, and ecosystem 206 The total GHG emissions from dumpsite are calculated as the sum of the CO2 emission 207 CH4 emissions (converted to CO2eq) (Misganaw & Teffera, 2021). The result obtained 208 this relation was 23.112Gg in 2007 and reached 46.873Gg in 2020. As the result ind 209 figure 2 the emission of greenhouse gases is increasing from time to time from 2007 210 [Figure 2] 211 3.2.2. Eutrophication Potential 212 Another state condition that occurs in a water body when the composition of organic w 213 during the generation of municipal solid waste is eutrophication. The chemical fo 214 organic waste produced in Bahir Dar city was determined to calculate the EP ( 215 biological activity of organisms due to over-nitrification) of municipal solid waste gen 216 the area. The organic waste generated in the city with percentage coverage was indicate 217 2. [Table 2] 218 Table 3 indicates the percentage chemical composition of organic wastes in terms o 219 hydrogen, oxygen, and nitrogen. The weight of carbon (C), hydrogen (H), oxygen 220 nitrogen (N) for each portion of organic waste was determined using the data in table 3 221 of determining the weight of elements found in organic waste was to determine th 222 chemical molecular formula in terms of carbon, hydrogen, oxygen, and nitrogen to 223 eutrophication potential. 3.2.2. Eutrophication Potential 212 Another state condition that occurs in a water body when the composition of organic waste rises 213 during the generation of municipal solid waste is eutrophication. The chemical formula of 214 organic waste produced in Bahir Dar city was determined to calculate the EP (excessive 215 biological activity of organisms due to over-nitrification) of municipal solid waste generated in 216 the area. The organic waste generated in the city with percentage coverage was indicated in table 217 2. [Table 2] 218 Table 3 indicates the percentage chemical composition of organic wastes in terms of carbon, 219 hydrogen, oxygen, and nitrogen. The weight of carbon (C), hydrogen (H), oxygen (O), and 220 nitrogen (N) for each portion of organic waste was determined using the data in table 3. The aim 221 of determining the weight of elements found in organic waste was to determine the waste's 222 chemical molecular formula in terms of carbon, hydrogen, oxygen, and nitrogen to determine 223 eutrophication potential. [Table 3] 224 Another state condition that occurs in a water body when the composition of organic waste rises 213 during the generation of municipal solid waste is eutrophication. The chemical formula of 214 organic waste produced in Bahir Dar city was determined to calculate the EP (excessive 215 biological activity of organisms due to over-nitrification) of municipal solid waste generated in 216 the area. The organic waste generated in the city with percentage coverage was indicated in table 217 2. [Table 2] 218 Table 3 indicates the percentage chemical composition of organic wastes in terms of carbon, 219 hydrogen, oxygen, and nitrogen. The weight of carbon (C), hydrogen (H), oxygen (O), and 220 nitrogen (N) for each portion of organic waste was determined using the data in table 3. The aim 221 of determining the weight of elements found in organic waste was to determine the waste's 222 chemical molecular formula in terms of carbon, hydrogen, oxygen, and nitrogen to determine 223 eutrophication potential. [Table 3] 224 10 The chemical composition of organic wastes was determined based on the composition in the 225 table above. The total percentage composition calculated in the organic part of municipal solid 226 waste of Bahir Dar city is indicated in table 4. [Table 4] 227 The chemical composition of organic wastes was determined based on the composition in the 225 table above. 3.2.2. Eutrophication Potential 212 The total percentage composition calculated in the organic part of municipal solid 226 waste of Bahir Dar city is indicated in table 4. [Table 4] 227 The eutrophication potential (EP) can be calculated for any compound that contains only C, H, N 228 and O. In order to determine the eutrophication potential of organic waste generated in Bahir Dar 229 city, obtaining the molecular formula of the organic waste is required. Then the molecular 230 formula of the waste can be obtained by dividing each component by its molecular weight by 231 excluding sulfur and ash. To obtain the general chemical formula that is present in organic 232 municipal solid waste of Bahir Dar city, use the lowest represented element nitrogen as the base. 233 After taking nitrogen as the base, then dividing each value of the element by the number of 234 moles of nitrogen and the result is indicated in table 5. [Table 5] 235 As indicated in table 5, the organic waste produced in the city has the chemical formula 236 C24H36O13N. The calculated value of ThOD and V were 38.75 and 0.343 respectively by using 237 the molecular formula of organic waste of the city and mathematical equations in the material 238 and method part. The eutrophication potential of organic waste produced in Bahir Dar city was 239 0.0594 Kg N-equivalent or 59.4g N-equivalent. 240 3.2.3. Leachate 241 Indicators in the impact 258 component of the DPSIRO framework are used to measure the change in state of air, soil and 259 water due to the pressure and the driving force caused by the state of air, soil and water 260 environment change. As indicated in figure 2, the emission of greenhouse gases increased from 261 23.112Gg to 46.873Gg in the years between 2007 and 2020 (Misganaw & Teffera, 2021). By 262 trapping heat, this rise contributes to climate change, as well as respiratory problems caused by 263 smog and pollution. The increment of GHG emissions induces genetic damage in animals, plants 264 and bacteria in addition to human health impact (Mussury & Rocha, 2020). Other implications of 265 climate change produced by greenhouse gases include extreme weather, food supply shortages, 266 and increasing wildfires. 267 In the DPSIRO framework, impacts are expressed in terms of the effect of change in the quality 255 of the environment (soil, air and water) on social and ecological systems. Ecosystem and human 256 welfare effects are both included in the impacts, however the former is focused on ecosystem 257 problems such as reducing water, soil and air quality (Cooper, 2013). Indicators in the impact 258 component of the DPSIRO framework are used to measure the change in state of air, soil and 259 water due to the pressure and the driving force caused by the state of air, soil and water 260 environment change. As indicated in figure 2, the emission of greenhouse gases increased from 261 23.112Gg to 46.873Gg in the years between 2007 and 2020 (Misganaw & Teffera, 2021). By 262 trapping heat, this rise contributes to climate change, as well as respiratory problems caused by 263 smog and pollution. The increment of GHG emissions induces genetic damage in animals, plants 264 and bacteria in addition to human health impact (Mussury & Rocha, 2020). Other implications of 265 climate change produced by greenhouse gases include extreme weather, food supply shortages, 266 and increasing wildfires. 267 The eutrophication potential of municipal solid waste in the city is high and this leads to toxic 268 poisons for human health that can be produced by harmful algal bloom species. 3.2.3. Leachate 241 Like greenhouse gas emission and eutrophication, leachate is another factor which changes the 242 state of a given environment. A number of factors influence the proportion of leachate generated, 243 including waste characteristics, structure, and compressed congestion; weather conditions, 244 average annual temperature, cell size, and gradual processing of the disposal region; and ground 245 water impacts. 246 Like greenhouse gas emission and eutrophication, leachate is another factor which changes the 242 state of a given environment. A number of factors influence the proportion of leachate generated, 243 including waste characteristics, structure, and compressed congestion; weather conditions, 244 average annual temperature, cell size, and gradual processing of the disposal region; and ground 245 water impacts. 246 11 11 Generally, leachate quantity is patterned or specified through an easy water equilibrium method, 247 considering the amount of water reaching the landfill and the level of water departing from the 248 landfill (that is water used in biochemical processes and evaporation). The average annual 249 evapotranspiration of Bahir Dar city is 307mm per day and the average annual temperature and 250 rainfall is 19.60C and 1419mm per day respectively (source: national metrology agency). Then 251 the average amount of leachate that is found from municipal solid waste of Bahir Dar became 252 1,112mm per day. 253 Generally, leachate quantity is patterned or specified through an easy water equilibrium method, 247 considering the amount of water reaching the landfill and the level of water departing from the 248 landfill (that is water used in biochemical processes and evaporation). The average annual 249 evapotranspiration of Bahir Dar city is 307mm per day and the average annual temperature and 250 rainfall is 19.60C and 1419mm per day respectively (source: national metrology agency). Then 251 the average amount of leachate that is found from municipal solid waste of Bahir Dar became 252 1,112mm per day. 253 In the DPSIRO framework, impacts are expressed in terms of the effect of change in the quality 255 of the environment (soil, air and water) on social and ecological systems. Ecosystem and human 256 welfare effects are both included in the impacts, however the former is focused on ecosystem 257 problems such as reducing water, soil and air quality (Cooper, 2013). 3.2.3. Leachate 241 Algal toxins can 269 The eutrophication potential of municipal solid waste in the city is high and this leads to toxic 268 poisons for human health that can be produced by harmful algal bloom species. Algal toxins can 269 12 build in shellfish and, more broadly, seafood, reaching harmful levels for human and animal 270 health. Shellfish poisoning can result in paralysis, neurotoxicity, or diarrhea. Eutrophication has 271 high ecological impact in addition to human health impact. Lake Tana water, which is the largest 272 lake in Ethiopia, is found in Bahir Dar city and has high fish products for the community. 273 Increased turbidity or reduced light penetrations into the lower depths of the water column are 274 both caused by phytoplankton development. This can stifle the growth of submerged aquatic 275 plants in lakes, affecting species that rely on them. 276 Leachate also leads to various human health impacts like greenhouse gas emission and 277 eutrophication. Sweating, bleeding, and stomach ailments can all be induced by drinking 278 contaminated leachate water, according to medical literature, as can blood disorders, congenital 279 defects, and even cancer. Solid wastes generate organic pollutants and taken by agricultural 280 plants and their accumulation in edible parts cause serious health problems to animals and 281 humans (Parlavecchia & Loffredo, 2020). 282 3.4. Responses 283 There are various groups of responses in order to protect the resulting impact by municipal solid 284 waste generation on water, air and soil environment. This response is taken by society to 285 environmental situation and an initiative intended in order to reduce at least one impact (Cooper, 286 2013; Neves et al., 2008). Accordingly, the responses that given during the time of study are: 287 sustainable with environment (i.e. friendly with nature in current time and future), 288 technologically feasible with adequate method and equipment, economically feasible, wanted by 289 the society live in the city, legally much with national as well as international legislations and 290 also administratively attainable. The responsible parties either individuals or groups present in 291 Bahir Dar could consider the following responses: shifting the policy from a linear to a circular 292 13 economy (i.e. applied the wastes produced from one sector to as an input for another one). There 293 are several studies that can be used as inputs to convert Bahir Dar's policy from a linear to a 294 circular economy system (Asmare & Alelign, 2019; Biruk, 2017). According to (Asmare & 295 Alelign, 2019), more than 74 percent of Bahir Dar City's municipal solid waste can be used to 296 make briquettes, and over 86 percent of the waste can be composted into organic fertilizer. 297 economy (i.e. applied the wastes produced from one sector to as an input for another one). There 293 are several studies that can be used as inputs to convert Bahir Dar's policy from a linear to a 294 circular economy system (Asmare & Alelign, 2019; Biruk, 2017). According to (Asmare & 295 Alelign, 2019), more than 74 percent of Bahir Dar City's municipal solid waste can be used to 296 make briquettes, and over 86 percent of the waste can be composted into organic fertilizer. 297 The application of user fees for waste management services is the next possible action. Because 298 it imposes fees on waste generators, this reaction is a very essential policy for reducing the city's 299 garbage creation from various sources. The goal of imposing trash charges is to reduce waste 300 generation both in terms of quantity and risk, as well as to improve waste recovery (Michel et al., 301 2018; Sanjeevi & Shahabudeen, 2015). 3.4. Responses 283 Transportation and garbage treatment fees are included in 302 the prices, which should be lower for separated waste than for mixed waste. The purpose of the 303 trash tax is to promote garbage recovery and reduce waste on the land (Misganaw & Teffera, 304 2021). Create awareness for community regarding with reducing, reusing and recycling should 305 be increased; suitable policies and frameworks regarding to solid waste management should 306 implement and monitor properly; built the ability of municipality in terms of economic, 307 personnel and technical aspects and transfer information for community regarding to health, 308 socio-economic and other harmful impacts of improper solid waste management of the city are 309 important responses to reduce the generation and impact of waste. 310 The reaction to the DPSIRO model resulted in high efficiency in research to assist decision 312 makers in developing and executing policies in favor of the city's municipal solid waste 313 management system (Misganaw & Teffera, 2021). The strategic actions taken by responsible 314 parties at various level of DPSIRO framework (driver, pressure, state and impact) leads to 315 14 significant outputs with related to social, environmental and economic sustainability. The 316 possible responses dealt in this research leads to various outcomes. From those out comes, the 317 three basic states that identified in soil, water and air (leachate, eutrophication and greenhouse 318 gas emission) can be reduced due to waste quantity reduction in waste disposal site. Currently 319 the waste disposal site of Bahir Dar city consumes around 22 hectare (Asmare & Alelign, 2019). 320 So, implementation of 3R policy in municipal solid waste management of Bahir Dar city results 321 in creating of lands for construction, farming and other purposes from waste disposal site. The 322 impacts resulting from inorganic fertilizer also can be reduced by substituting it by organic 323 fertilizer (compost) using compostable wastes as raw material. The city now produces 148.12 324 tons of municipal solid trash each day. With a waste density of 162.3 kg/m3, the annual volume 325 of waste is 333,291.6 m3 per year. A maximum of 0.6 m3 of solid waste is required for the 326 creation of one quintal (100kg) of compost, according to Dream Light PLC's expertise. This 327 indicates that 47,772 ton of compost can be made from 86 percent of biodegradable garbage per 328 year. 3.4. Responses 283 Most of 344 the waste generated in the city is organic with the molecular formula C24H36O13N. This waste 345 increases the eutrophication in the water body with the potential of 0.0594 Kg N-equivalent. The 346 average amount of leachate that is found from municipal solid waste of Bahir Dar is 1,112mm 347 per day with an average annual temperature and rainfall is 19.60C and 1419mm per day 348 respectively. 349 The DPSIRO framework indicated the overall scenario of impact of municipal solid waste on air, 338 water and soil impacts. The generation of municipal solid waste in Bahir Dar city is increasing 339 from time to time due to population growth and economic transformation as a driving force. This 340 leads to a change in the quality of the given environment by mismanagement of municipal solid 341 waste. Currently, a large portion of the waste that is generated in Bahir Dar city is disposed 342 directly into an open dumpsite without any sorting activity. The amount of greenhouse estimated 343 from waste disposal sites reaches 46Gg per year in terms of carbon dioxide equivalent. Most of 344 the waste generated in the city is organic with the molecular formula C24H36O13N. This waste 345 increases the eutrophication in the water body with the potential of 0.0594 Kg N-equivalent. The 346 average amount of leachate that is found from municipal solid waste of Bahir Dar is 1,112mm 347 per day with an average annual temperature and rainfall is 19.60C and 1419mm per day 348 respectively. 349 These greenhouse gas emissions, eutrophication potential and leachate are considered under state 350 factors of the DPSIRO framework. The waste generated in the city poses various impacts on 351 human health and wildlife. The scenario of the response indicated that Bahir Dar city should start 352 to implement reduction, reuse and recycling solid management options in order to reduce 353 potential environmental and human health impacts with economic benefits. The waste 354 composition is also used as an input in order to implement those management options. As the 355 output obtained indicates, if Bahir Dar city implements reduction, reuse and recycling, it is 356 possible to produce 47,772 ton of compost from 86 percent of biodegradable garbage per year. 357 The average total amount of money produced per year, according to this product also around 358 1,313,724 Birr. 3.4. Responses 283 This 340 leads to a change in the quality of the given environment by mismanagement of municipal solid 341 waste. Currently, a large portion of the waste that is generated in Bahir Dar city is disposed 342 directly into an open dumpsite without any sorting activity. The amount of greenhouse estimated 343 from waste disposal sites reaches 46Gg per year in terms of carbon dioxide equivalent. Most of 344 the waste generated in the city is organic with the molecular formula C24H36O13N. This waste 345 increases the eutrophication in the water body with the potential of 0.0594 Kg N-equivalent. The 346 average amount of leachate that is found from municipal solid waste of Bahir Dar is 1,112mm 347 per day with an average annual temperature and rainfall is 19.60C and 1419mm per day 348 respectively. 349 These greenhouse gas emissions, eutrophication potential and leachate are considered under state 350 factors of the DPSIRO framework. The waste generated in the city poses various impacts on 351 human health and wildlife. The scenario of the response indicated that Bahir Dar city should start 352 to implement reduction, reuse and recycling solid management options in order to reduce 353 potential environmental and human health impacts with economic benefits. The waste 354 composition is also used as an input in order to implement those management options. As the 355 output obtained indicates, if Bahir Dar city implements reduction, reuse and recycling, it is 356 possible to produce 47,772 ton of compost from 86 percent of biodegradable garbage per year. 357 The average total amount of money produced per year, according to this product also around 358 1,313,724 Birr. The responses identified in this study leads to environmental, social and 359 The DPSIRO framework indicated the overall scenario of impact of municipal solid waste on air, 338 water and soil impacts. The generation of municipal solid waste in Bahir Dar city is increasing 339 from time to time due to population growth and economic transformation as a driving force. This 340 leads to a change in the quality of the given environment by mismanagement of municipal solid 341 waste. Currently, a large portion of the waste that is generated in Bahir Dar city is disposed 342 directly into an open dumpsite without any sorting activity. The amount of greenhouse estimated 343 from waste disposal sites reaches 46Gg per year in terms of carbon dioxide equivalent. 3.4. Responses 283 The average total amount of money produced per year, according to this product, is around 329 1,313,724 Birr (Misganaw & Teffera, 2021). 330 significant outputs with related to social, environmental and economic sustainability. The 316 possible responses dealt in this research leads to various outcomes. From those out comes, the 317 three basic states that identified in soil, water and air (leachate, eutrophication and greenhouse 318 gas emission) can be reduced due to waste quantity reduction in waste disposal site. Currently 319 the waste disposal site of Bahir Dar city consumes around 22 hectare (Asmare & Alelign, 2019). 320 So, implementation of 3R policy in municipal solid waste management of Bahir Dar city results 321 in creating of lands for construction, farming and other purposes from waste disposal site. The 322 impacts resulting from inorganic fertilizer also can be reduced by substituting it by organic 323 fertilizer (compost) using compostable wastes as raw material. The city now produces 148.12 324 tons of municipal solid trash each day. With a waste density of 162.3 kg/m3, the annual volume 325 of waste is 333,291.6 m3 per year. A maximum of 0.6 m3 of solid waste is required for the 326 creation of one quintal (100kg) of compost, according to Dream Light PLC's expertise. This 327 indicates that 47,772 ton of compost can be made from 86 percent of biodegradable garbage per 328 year. The average total amount of money produced per year, according to this product, is around 329 1,313,724 Birr (Misganaw & Teffera, 2021). 330 In addition to the financial benefits, the environmental consequences of municipal solid waste 331 mishandling are mitigated. Knowledge transmission regarding municipal solid waste 332 management and the use of waste taxes and levies in society in the context of a circular economy 333 raises public awareness about trash income production and protects people's economy, 334 environment, and health. Figure 3 uses the DPSIRO framework to summarize the result of better 335 knowledge of the overarching concept of the research. [Figure 3] 336 4. Conclusion on Research Findings 337 15 The DPSIRO framework indicated the overall scenario of impact of municipal solid waste on air, 338 water and soil impacts. The generation of municipal solid waste in Bahir Dar city is increasing 339 from time to time due to population growth and economic transformation as a driving force. 3.4. Responses 283 The responses identified in this study leads to environmental, social and 359 16 economic balance. For more investigation, quantification of the other impacts associated with 360 municipal solid waste is recommended. 361 economic balance. For more investigation, quantification of the other impacts associated with 360 municipal solid waste is recommended. 361 Declaration of interests 362 The authors declare that they have no known competing financial interests or personal 363 relationships that could have appeared to influence the work reported in this paper. 364 The datasets generated during and/or analyzed during the current study are available from the 366 corresponding author on reasonable request. 367 The datasets generated during and/or analyzed during the current study are available from the 366 corresponding author on reasonable request. 367 Author’s contribution 368 Awoke Misganaw: Conceptualization, Methodology, and Software, Data curation, Writing- 369 Original draft preparation: Visualization, Investigation, Supervision, and Validation. 370 Banchamlak Akenaw: Methodology, Data curation, Visualization, Writing- Reviewing and 371 Editing. 372 Funding Information 373 Funding information is not applicable / No funding was received 374 References 375 Adeleke, O., Akinlabi, S. 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Sustainability, 16 April, 1–15. 468 469 21 21 Figures Figures Figure 1 The DPSIRO conceptual framework developed by authors Figure 1 The DPSIRO conceptual framework developed by authors Figure 2 Net annual GHGs emission (Gg per year) from 2007 to 2020 Figure 3 Figure 3 Scenario of DPSIRO framework for municipal solid waste management system of Bahir Dar city Scenario of DPSIRO framework for municipal solid waste management system of Bahir Dar city
https://openalex.org/W1997100298	https://kclpure.kcl.ac.uk/ws/files/52006129/Variations_in_alveolar_partial_pressure_for_carbon_dioxide_and_oxygen_have_additive_not_synergistic_acute_effects_on_human_pulmonary_vasoconstriction_GoldPub.pdf	English	null	Variations in Alveolar Partial Pressure for Carbon Dioxide and Oxygen Have Additive Not Synergistic Acute Effects on Human Pulmonary Vasoconstriction	PloS one	2,013	cc-by	9,499	Citation for published version (APA): Croft, Q. P. P., Formenti, F., Talbot, N. P., Lunn, D., Robbins, P. A., & Dorrington, K. L. (2013). Variations in Alveolar Partial Pressure for Carbon Dioxide and Oxygen Have Additive Not Synergistic Acute Effects on Human Pulmonary Vasoconstriction. PLoS ONE, 8(7), Article e67886. https://doi.org/10.1371/journal.pone.0067886 Citation for published version (APA): Croft, Q. P. P., Formenti, F., Talbot, N. P., Lunn, D., Robbins, P. A., & Dorrington, K. L. (2013). Variations in Alveolar Partial Pressure for Carbon Dioxide and Oxygen Have Additive Not Synergistic Acute Effects on Human Pulmonary Vasoconstriction. PLoS ONE, 8(7), Article e67886. https://doi.org/10.1371/journal.pone.0067886 Citing this paper Pl h C t g t s pape Please note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this may differ from the final Published version. If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you are again advised to check the publisher's website for any subsequent corrections. Citation for published version (APA): Croft, Q. P. P., Formenti, F., Talbot, N. P., Lunn, D., Robbins, P. A., & Dorrington, K. L. (2013). Variations in Alveolar Partial Pressure for Carbon Dioxide and Oxygen Have Additive Not Synergistic Acute Effects on Human Pulmonary Vasoconstriction. PLoS ONE, 8(7), Article e67886. https://doi.org/10.1371/journal.pone.0067886 Introduction alone. In relation to the mammalian carotid body a stimulus interaction in the responses of single afferent fibres to CO2 and O2 has been known since 1975 [7], and considerable attention has been directed at establishing at what cellular level of transduction this synergy might occur [8,9]. The important consequences of this stimulus interaction on the control of breathing in humans in a wide variety of conditions has been recognized for many years [10,11]. In comparison, responses of pulmonary vascular smooth muscle to the combined stimuli CO2 and O2 have received little attention, but are arguably of a similar importance for under- standing the behaviour of the lung in health and disease [12,13]. A i l i h id d l i di i f The human pulmonary vasculature constricts in response to both hypercapnia and hypoxia [1–4]. Sometimes, variations in CO2 and O2 are such as to work in synchrony on the vasculature. For example, this occurs in a poorly ventilated region of the lung where they both act to direct blood flow away from the region to better ventilated lung tissue, thereby enhancing the efficiency of gas exchange [5]. At other times, variations in CO2 and O2 are such as to act in opposition on the vasculature. An example is human exposure to high altitude, where the whole lung is exposed to coexisting hypoxia and hypocapnia [6], and the potentially harmful pressor effect of the alveolar hypoxia is obtunded by the dilatory effect of the alveolar hypocapnia. It is not known in what way a combination of the stimuli of hypercapnia and hypoxia affect the blood vessels in the human lung. It is unclear, therefore, whether the effects of the stimuli are additive or synergistic, that is to say, whether variations in O2 could potentially enhance the response to CO2 or vice-versa. Animal preparations have not provided a clear indication of what one might expect for the human lung. Most, but not all [14], preparations show vasomotor responses to both respiratory gases, with some degree of synergistic interaction between the effects of CO2 and O2 being common but variable [15–20]. 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Download date: 24. Oct. 2024 Abstract Competing Interests: The authors have declared that no competing interests exist. * E-mail: keith.dorrington@dpag.ox.ac.uk Variations in Alveolar Partial Pressure for Carbon Dioxide and Oxygen Have Additive Not Synergistic Acute Effects on Human Pulmonary Vasoconstriction P. P. Croft1, Federico Formenti1, Nick P. Talbot1, Daniel Lunn2, Peter A. Robbins1, Dorrington1* Quentin P. P. Croft1, Federico Formenti1, Nick P. Talbot1, Daniel Lunn2, Peter A. Robbins1, Keith L. Dorrington1* 1 Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom, 2 Department of Statistics, University of Oxford, Oxford, United Kingdom of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom, 2 Department of Statistics, University of Oxfo Citation: Croft QPP, Formenti F, Talbot NP, Lunn D, Robbins PA, et al. (2013) Variations in Alveolar Partial Pressure for Carbon Dioxide and Oxygen Have Additive Not Synergistic Acute Effects on Human Pulmonary Vasoconstriction. PLoS ONE 8(7): e67886. doi:10.1371/journal.pone.0067886 PLOS ONE \| www.plosone.org Abstract The human pulmonary vasculature constricts in response to hypercapnia and hypoxia, with important consequences for homeostasis and adaptation. One function of these responses is to direct blood flow away from poorly-ventilated regions of the lung. In humans it is not known whether the stimuli of hypercapnia and hypoxia constrict the pulmonary blood vessels independently of each other or whether they act synergistically, such that the combination of hypercapnia and hypoxia is more effective than the sum of the responses to each stimulus on its own. We independently controlled the alveolar partial pressures of carbon dioxide (PACO2) and oxygen (PAO2) to examine their possible interaction on human pulmonary vasoconstriction. Nine volunteers each experienced sixteen possible combinations of four levels of PACO2 (+6, +1, 24 and 29 mmHg, relative to baseline) with four levels of PAO2 (175, 100, 75 and 50 mmHg). During each of these sixteen protocols Doppler echocardiography was used to evaluate cardiac output and systolic tricuspid pressure gradient, an index of pulmonary vasoconstriction. The degree of constriction varied linearly with both PACO2 and the calculated haemoglobin oxygen desaturation (1-SO2). Mixed effects modelling delivered coefficients defining the interdependence of cardiac output, systolic tricuspid pressure gradient, ventilation, PACO2 and SO2. No interaction was observed in the effects on pulmonary vasoconstriction of carbon dioxide and oxygen (p.0.64). Direct effects of the alveolar gases on systolic tricuspid pressure gradient greatly exceeded indirect effects arising from concurrent changes in cardiac output. Citation: Croft QPP, Formenti F, Talbot NP, Lunn D, Robbins PA, et al. (2013) Variations in Alveolar Partial Pressure for Carbon Dioxide and Oxygen Have Additive Not Synergistic Acute Effects on Human Pulmonary Vasoconstriction. PLoS ONE 8(7): e67886. doi:10.1371/journal.pone.0067886 Received January 11, 2013; Accepted May 23, 2013; Published July 31, 2013 Copyright: 2013 Croft et al. This is an open-access article distributed under the terms of the Creative Commons Attributi unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ft et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the Dunhill Medical Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. July 2013 \| Volume 8 \| Issue 7 \| e67886 Ethics Statement The study was approved by the Oxfordshire Research Ethics Committee and performed in accordance with the Declaration of Helsinki. Informed written consent was obtained from all volunteers. Introduction Study of vasoconstrictor responses in the in vivo healthy human lung is made particularly difficult by the fact that changes in PACO2 and PAO2 induce changes in pulmonary artery pressure and pulmonary vascular resistance (PVR) that are a summation of a direct active effect of the gases on vascular smooth muscle and an indirect passive effect of concurrent changes in pulmonary blood flow and, The question of whether there is a synergy between the effects CO2 and O2 in the sensing mechanisms of the pulmonary vasculature is of broader interest than in the context of this tissue 1 PLOS ONE \| www.plosone.org July 2013 \| Volume 8 \| Issue 7 \| e67886 Human Pulmonary Vascular Responses to O2 & CO2 baseline): +6, +1, 24 and 29 mmHg. The levels of PETO2 used were 175, 100, 75 and 50 mmHg. This provided an opportunity to span the range from relative hyperoxia to the hypoxia used in other studies [23–25], and so cover the likely regional values for these variables encountered within the healthy lung at sea level [13,26]. potentially, ventilation [21]. The indirect effect may be quite small, because pulmonary vessels tend to be quite distensible, and thus accommodate large changes in flow with little rise in perfusion pressure and with a fall in resistance. This nevertheless makes it misleading to measure either pulmonary artery pressure or PVR as a sole index of pulmonary vascular smooth muscle constriction. The luxury available in animal preparations of being able to impose a constant pulmonary flow, and using pulmonary artery pressure or PVR as the index of vasoconstriction, has not been achieved in humans [22]. We address this problem by using mixed effects modelling to extract coefficients in direct and indirect pathways linking PACO2 and PAO2 with pulmonary artery pressure, and the relative contribution of each pathway. Direct effects of alveolar gases on pulmonary artery pressure are found to dominate. This approach also evaluates whether the gases have an additive or synergistic action; an additive action is observed, consistent with the approach adopted in an earlier model of feedback control of regional gas exchange in the human lung [13]. Gas control PETCO2 and PETO2 were controlled using an end-tidal forcing system as previously described [27–29]. Volunteers lay in a semi- left lateral position and breathed through a mouthpiece with the nose occluded. Ventilatory volumes and flows were measured by turbine and pneumotachograph respectively. Gases were sampled by a catheter close to the mouth and analysed continuously by mass spectrometry. Ventilation during the protocols conducted at PETCO2 values of 29 and 24 mmHg was achieved by voluntary hyperventilation. Volunteers controlled the frequency of breathing through the use of an audible metronome, and the depth of breathing through feedback presented on an oscilloscope connected to the output of the turbine measuring ventilatory flows. Ventilation during the protocols conducted at PETCO2 values of +6 and +1 mmHg was spontaneous. Each protocol consisted of 5 min of spontaneous ventilation, or voluntary hyperventilation, with end-tidal gases held constant at baseline values (100 mmHg PETO2 and the measured baseline PETCO2) followed by ten minutes with these gases at the specified levels for the protocol. For protocols involving hypocapnia, a constant combination of breathing depth and frequency was used throughout. Echocardiography The general approach adopted was to use non-invasive measurement of systolic pulmonary artery pressure as our index of pulmonary vasoconstriction, whilst at the same time taking into account the dependence of this pressure upon other variables: ventilation and cardiac output. This separation of direct and indirect influences of PACO2 and PAO2 on systolic pulmonary artery pressure was achieved using mixed effects modelling. In approximately 70% of healthy volunteers it is possible to detect with Doppler ultrasound a regurgitant blood flow from the right ventricle to the right atrium during ventricular systole. Measurement of the peak velocity (v) of this regurgitant jet affords an opportunity to estimate the systolic pressure difference DPmax between the right ventricle (where the pressure is close to pulmonary artery systolic pressure) and right atrial pressure. This relationship is given by the Bernoulli equation: DPmax = rv2/2, where r is blood density. The peak systolic tricuspid pressure gradient (DPmax) and cardiac output were measured using a GE Vivid-i ultrasound machine with a S4 transducer (2–4 MHz). Assessment of DPmax used Doppler echocardiography, via a 4- chamber view of the heart, to measure the peak pressure difference between the right ventricle and the right atrium during systole. Since right atrial pressure changes little during hypoxia, changes in DPmax reflect changes in systolic pulmonary arterial pressure [30,31]. The utility of measuring DPmax as an index of pulmonary vascular constriction in healthy humans has been shown during hypoxia [24,25], hypercapnia and hypocapnia [2,13]. Volunteers Nine healthy volunteers (5 women and 4 men), aged 2464 years and with BMI 22.562 kg/m2 (mean 6 S.D.), completed the study. Female volunteers were asked to participate only during the first 14 days of their menstrual cycle. Volunteers visited the laboratory before undergoing the experimental protocols in order to discuss the procedures and confirm that they were suitable for echocardiographic assessment of tricuspid regurgitation. Human Pulmonary Vascular Responses to O2 & CO2 spectral traces at or as near as possible to end-expiration were saved digitally for later analysis. between protocol and baseline values. DSO2DPETCO2 allows for possible interaction between the stimuli. The logarithm of V˙ E was required in the analysis instead of V˙ E itself so as to avoid giving undue dominance to a small number of high values of V˙ E. The coefficients preceding each term were obtained by fitting the model to the experimental data. Data analysis Ventilation (V˙ E) and end-tidal gases were assessed using 30 s averages of the values calculated from each breath. For DPmax and Q˙ , approximately five measurements of each variable were obtained each minute and then 2 min averages were calculated. Figure 1 shows the conceptual framework for our modelling approach. DPmax is viewed as primarily a measure of pulmonary vasoconstriction dependent upon a direct effect of alveolar gases on vascular smooth muscle, whilst also being a weak function of Q˙ and V˙ E. These in turn are functions of alveolar gases, and provide an indirect route via which alveolar gases can change DPmax. The modelling described below delivers mean values plus confidence intervals, expressed as standard error of these means, to the nine coefficients displayed in Figure 1, as well as assessing the significance of the interactive term DSO2DPETCO2 in Eq. 1. Baseline variables were the average of values recorded during the first five minutes of each protocol. Protocol variables were the average of the last six minutes of each protocol. The change in each variable was the difference between the protocol and baseline values. PETO2 values were converted to an equivalent fractional oxyhaemoglobin saturation (SO2) using the equation provided by Severinghaus [32]. Although the major stimulus to pulmonary vascular constriction is the partial pressure of the sensed gases, the response to oxygen is known to be markedly non-linear and the purpose of this sigmoid transformation was to permit us to use a virtual saturation in place of PO2 in our analysis, and thereby assess the suggestion of previous authors [33] that hypoxic constriction tends to be a linear function of SO2 whilst being a markedly curvilinear function of PO2. The data were analysed with linear mixed effects modelling to account for correlation within individual volunteers and for variability between volunteers. A two-level multilevel model with an exchangeable correlation structure was fitted. This statistical technique can be used for analysing data that occur as repeated measurements on each of a number of participants in order to identify and quantify responses common to all participants, taking into account individual variability, with no two individuals being the same. Models similar to that in Eq. 1 were derived for Q˙ and ln(V˙ E). Study design The pulmonary vascular response to four different levels of PCO2 was studied at each of four different levels of PO2. This led to 16 different combinations of PCO2 and PO2 overall, each called a protocol. Each protocol comprised a ten-minute exposure to the particular PCO2/PO2 combination which was preceded by 5 min of baseline conditions (see below). Cardiovascular and respiratory variables were measured throughout each protocol. Cardiac output (Q˙ ) was measured using Doppler echocardiog- raphy to assess non-turbulent flow through the centre of the left ventricular outflow tract (LVOT). The cross-sectional area of the LVOT was obtained by measuring the diameter of the aortic valve using a parasternal long-axis view of the heart. Flow through the LVOT was imaged using an apical five-chamber view of the heart and measured using the velocity-time integral. Systolic flow was multiplied by the cross-sectional area of the LVOT to provide an estimate of stroke volume. Heart rate was recorded simultaneous- ly. The stroke volume was multiplied by the heart rate to provide an estimate of cardiac output. Each volunteer completed the sixteen protocols in one of four different orders, determined by block randomization based on date of first contact. Volunteers completed these protocols in two batches of eight in two afternoons. Each protocol was preceded by at least ten minutes of quiet rest. The sixteen protocols were the sixteen combinations of four levels each of end-tidal partial pressures of CO2 (PETCO2) and O2 (PETO2). These end-tidal values were assumed to be equivalent to alveolar partial pressures. The following four levels of PETCO2 were chosen (relative to normal For both measurements, results depend to some extent upon the phase of the respiratory cycle, so end-expiration was chosen as the phase of that cycle giving minimal disturbance; images of the PLOS ONE \| www.plosone.org July 2013 \| Volume 8 \| Issue 7 \| e67886 2 Human Pulmonary Vascular Responses to O2 & CO2 Human Pulmonary Vascular Responses to O2 & CO2 Human Pulmonary Vascular Responses to O2 & CO2 explanatory power was added, the variability being explicable by one of the two factors. The constant in the model (which provides the y-axis intercept on a graph of the function) was always modelled as a random factor, and if it correlated well with another random factor then it was acting as a surrogate for that factor and the factor could be subsumed by the intercept factor. Secondly, if the residual deviance was not decreased by a large amount then the explanatory power was not enhanced, and the addition of a random factor was not necessary. explanatory power was added, the variability being explicable by one of the two factors. The constant in the model (which provides the y-axis intercept on a graph of the function) was always modelled as a random factor, and if it correlated well with another random factor then it was acting as a surrogate for that factor and the factor could be subsumed by the intercept factor. Secondly, if the residual deviance was not decreased by a large amount then the explanatory power was not enhanced, and the addition of a random factor was not necessary. Modelling and statistical analysis The experimental data were analysed using the following linear model: Data were analysed using ‘R’, open-source computer software for statistical analyses. R uses a penalised likelihood method to fit the data to a given model iteratively until no improvement in the residual deviance is achieved. Data were initially fitted to a model in which all of the possible contributing factors in Eq. 1 were considered. The model was then adjusted to exclude the least significant factor until all remaining factors showed significance with p,0.05. This provided individual coefficients for each contributing factor that define the linear relationships. Each coefficient was then fitted as a random variable, with the mean and standard deviation estimated from the data, retaining adjustments that enhanced the explanatory power of the model. This was judged by two methods: first, if the random factor correlated well with another random factor then no additional DPmax protocol value~azb(BDPmax)zg(BSO2) za(DSO2)zd(BPETCO2 )zb(DPETCO2 )z m(DSO2 DPETCO2 )zv(B Q : )zg(D Q : )z x(B ln V : E)zh(D ln V : E) ð1Þ za(DSO2)zd(BPETCO2 )zb(DPETCO2 )z m(DSO2 DPETCO2 )zv(B Q : )zg(D Q : )z ð1Þ ð1Þ x(B ln V : E)zh(D ln V : E) where BDPmax, BSO2, BPETCO2, BQ˙ and BlnV˙ E refer to baseline values of the respective variables DPmax, SO2, PETCO2, Q˙ and lnV˙ E, whilst DSO2, DPETCO2, DQ˙ and DlnV˙ E refer to the differences Figure 1. Diagram of the relationships involved in the study. DPmax is viewed as the primary measure of pulmonary vasoconstriction, influenced directly by alveolar gases (pathways a and b), whilst also being a weak function of cardiac output and possibly ventilation (pathways g and h). The latter two are also functions of alveolar gases (via the pathways c–f). Interactions are not represented. doi:10.1371/journal.pone.0067886.g001 Figure 1. Diagram of the relationships involved in the study. DPmax is viewed as the primary measure of pulmonary vasoconstriction, influenced directly by alveolar gases (pathways a and b), whilst also being a weak function of cardiac output and possibly ventilation (pathways g and h). The latter two are also functions of alveolar gases (via the pathways c–f). Interactions are not represented. doi:10.1371/journal.pone.0067886.g001 July 2013 \| Volume 8 \| Issue 7 \| e67886 PLOS ONE \| www.plosone.org 3 Results of statistical analysis A major objective of this study was to investigate whether the stimuli of hypercapnia and hypoxia constrict the pulmonary blood vessels independently of each other, or whether they act synergistically; in other words, evidence of an interaction DSO2DPETCO2 was sought. The final model fitted the following equation: DPmax protocol value~azb(BDPmax)z a(DSO2)zb(DPETCO2 )zg(D Q : ) ð2Þ The main analysis used the model given in Eq. 1. Of the included factors baseline Q˙ , baseline PETCO2, baseline V˙ E, baseline SO2 and DSO2DPETCO2 were all removed from the model sequentially, in that order, without significantly worsening the fit, suggesting that they had no significant role in determining ð2Þ where the coefficients are given in Table 3 as a value 6 standard error. The model that best explains the experimental data delivers Table 2. Individual changes in systolic tricuspid pressure gradient (DPmax) in response to sixteen combinations of end-tidal gas composition. Results At the 5% significance level, the study had a power of 80% for the detection of a 4% change in the DPmax response due to interaction; the power for detecting a 10% change in the response was close to 100%. Despite adequate power, no evidence of an interaction was identified. Results Protocols were conducted between August 2007 and June 2009. Figure 2 shows representative data from two protocols that illustrate spontaneous ventilation during hypercapnia and con- trolled ventilation to induce hypocapnia. The left panel shows a protocol involving hypoxia with hypercapnia, and the right panel shows a protocol involving hypocapnia with hyperoxia. The upper panels show the control of PETO2 and PETCO2 for the two protocols; gas control achieved a rapid (,1 min) step from euoxia and eucapnia to protocol values and little variation from target end- tidal values either side of the change. The middle panels show the ventilations and cardiac outputs achieved during the protocols and Figure 2. Example data from two protocols on different volunteers. In each protocol the end-tidal gases were held at normal euoxic and eucapnic values for the baseline period of 5 min and then stepped to individual target protocol values. These were as follows: left panels: volunteer 1714 with hypercapnia (PETCO2 = baseline+6 mmHg) and hypoxia (PETO2 = 50 mmHg) using spontaneous hyperventilation; right panels: volunteer 1719 with hypocapnia (PETCO2 = baseline29 mmHg) and hyperoxia (PETO2 = 175 mmHg) using voluntarily controlled constant hyperventilation. Upper panels: inspired oxygen (PIO2) and carbon dioxide (PICO2) partial pressures and end-tidal oxygen (PETO2) and carbon dioxide (PETO2) partial pressures. Middle panels: ventilation and cardiac output. Lower panels: DPmax. Respiratory data represent means of multiple measurements (one per breath) in each time period. doi:10.1371/journal.pone.0067886.g002 Figure 2. Example data from two protocols on different volunteers. In each protocol the end-tidal gases were held at normal euoxic and eucapnic values for the baseline period of 5 min and then stepped to individual target protocol values. These were as follows: left panels: volunteer 1714 with hypercapnia (PETCO2 = baseline+6 mmHg) and hypoxia (PETO2 = 50 mmHg) using spontaneous hyperventilation; right panels: volunteer 1719 with hypocapnia (PETCO2 = baseline29 mmHg) and hyperoxia (PETO2 = 175 mmHg) using voluntarily controlled constant hyperventilation. Upper panels: inspired oxygen (PIO2) and carbon dioxide (PICO2) partial pressures and end-tidal oxygen (PETO2) and carbon dioxide (PETO2) partial pressures. Middle panels: ventilation and cardiac output. Lower panels: DPmax. Respiratory data represent means of multiple measurements (one per breath) in each time period. doi:10.1371/journal.pone.0067886.g002 July 2013 \| Volume 8 \| Issue 7 \| e67886 July 2013 \| Volume 8 \| Issue 7 \| e67886 4 Human Pulmonary Vascular Responses to O2 & CO2 Table 1. Volunteers were exposed to each combination of end-tidal PO2 and PCO2 for 10 min, preceded by 5 min baseline breathing with end-tidal gases held close to baseline values (100 mmHg end-tidal PO2 and the measured baseline end-tidal PCO2). The change in peak systolic tricuspid pressure gradient (DPmax) was calculated as the difference between the mean baseline DPmax and the mean DPmax during the last 6 minutes of each protocol. Gas control was achieved by means of end-tidal forcing. doi:10.1371/journal.pone.0067886.t002 July 2013 \| Volume 8 \| Issue 7 \| e67886 Results Errors (mean and standard deviation) in control of end-tidal gases calculated as the measured end-tidal partial pressure minus the target end-tidal partial pressure for the four levels of CO2 and four levels of O2 used in the study. Table 1. Errors (mean and standard deviation) in control of end-tidal gases calculated as the measured end-tidal partial pressure minus the target end-tidal partial pressure for the four levels of CO2 and four levels of O2 used in the study. minus the target end tidal partial pressure for the four levels of CO2 and four levels of O2 used in the study. CO2 error (mmHg) Target PCO2 (mmHg) 29 24 1 6 Error 0.023 20.008 0.018 20.051 SD 0.325 0.256 0.255 0.352 O2 error (mmHg) Target PO2 (mmHg) 175 100 75 50 Error 21.315 21.201 0.610 0.813 SD 1.792 0.766 1.100 1.557 doi:10.1371/journal.pone.0067886.t001 doi:10.1371/journal.pone.0067886.t001 DPmax protocol value. The interactive term was insignificant at the level p.0.64. the bottom panels show the values of DPmax recorded during the protocols. Table 1 gives the accuracy to which the gas control was achieved for each of the four levels of PETCO2 and the four levels of PETO2 that were targeted in the protocols. It can be seen that for CO2 the errors in gas control are well below 0.1 mmHg, whilst for O2 the errors in gas control are around 1 mmHg. Table 2 gives the individual changes in DPmax for each of the sixteen protocols. To ensure the study had sufficient power to detect any interaction between the effects of hypoxia and hypercapnia, we calculated power as a function of the percentage change of the DPmax response attributable to the interaction term (DSO2* DPETCO2). At the 5% significance level, the study had a power of 80% for the detection of a 4% change in the DPmax response due to interaction; the power for detecting a 10% change in the response was close to 100%. Despite adequate power, no evidence of an interaction was identified. To ensure the study had sufficient power to detect any interaction between the effects of hypoxia and hypercapnia, we calculated power as a function of the percentage change of the DPmax response attributable to the interaction term (DSO2* DPETCO2). Results of statistical analysis Change in DPmax (mmHg) End-tidal PO2 (mmHg) 50 mmHg 75 mmHg 100 mmHg 175 mmHg Change in end-tidal PCO2 (mmHg) +6 +1 24 29 +6 +1 24 29 +6 +1 24 29 +6 +1 24 29 Subject 1662 12.7 10.4 3.1 2.5 10.6 3.7 2.0 0.8 2.6 1.4 1.0 21.2 5.6 0.1 0.1 20.5 Subject 1664 3.9 3.5 3.7 3.4 0.0 0.0 0.0 0.1 3.0 1.9 0.3 21.7 2.4 1.3 20.6 20.7 Subject 1701 6.7 6.1 5.1 2.5 3.6 0.9 0.6 1.1 3.0 1.3 20.5 20.8 1.7 20.2 20.2 0.4 Subject 1703 5.9 1.6 3.7 1.9 5.5 2.0 0.0 1.5 2.8 1.9 2.5 1.0 3.4 0.5 0.2 2.1 Subject 1714 9.7 10.7 6.6 7.3 7.8 1.8 2.3 20.8 5.8 0.1 20.2 0.6 2.4 20.7 20.4 0.1 Subject 1719 15.1 12.6 15.0 13.8 4.7 3.6 5.2 21.6 3.9 0.0 20.7 0.8 3.2 0.0 1.4 22.4 Subject 1730 4.2 6.0 3.8 2.0 2.9 1.1 21.7 2.2 3.6 1.2 1.0 1.1 0.8 20.7 22.6 21.8 Subject 1096 12.4 12.7 9.9 7.2 2.6 0.8 21.8 20.7 3.2 21.0 20.2 21.9 1.0 21.5 22.5 20.9 Subject 1751 6.8 7.9 4.7 5.4 1.2 20.1 1.5 1.3 20.7 0.6 1.5 20.8 0.1 20.1 20.7 21.3 Mean 8.6 7.9 6.2 5.1 4.3 1.6 0.9 0.4 3.0 0.8 0.5 20.3 2.3 20.1 20.6 20.6 SEM 1.3 1.3 1.3 1.3 1.1 0.5 0.7 0.4 0.6 0.3 0.4 0.4 0.6 0.3 0.4 0.4 Volunteers were exposed to each combination of end-tidal PO2 and PCO2 for 10 min, preceded by 5 min baseline breathing with end-tidal gases held close to baseline values (100 mmHg end-tidal PO2 and the measured baseline end-tidal PCO2). The change in peak systolic tricuspid pressure gradient (DPmax) was calculated as the difference between the mean baseline DPmax and the mean DPmax during the last 6 minutes of each protocol. Gas control was achieved by means of end-tidal forcing. doi:10.1371/journal.pone.0067886.t002 Table 2. Individual changes in systolic tricuspid pressure gradient (DPmax) in response to sixteen combinations of end-tidal gas composition Table 2. Individual changes in systolic tricuspid pressure gradient (DPmax) in response to sixteen comb composition. Volunteers were exposed to each combination of end-tidal PO2 and PCO2 for 10 min, preceded by 5 min baseline breathing with end-tidal gases held close to baseline values (100 mmHg end-tidal PO2 and the measured baseline end-tidal PCO2). Discussion coefficients b and b as fixed coefficients with a, a and g as coefficients that vary between individuals with normal distribu- tions and standard deviations of 0.59 mmHg, 0.26 mmHg/ %desaturation and 0.36 mmHg/l/min, respectively. The main finding of this study is that the effects of CO2 and O2 on human pulmonary artery pressure are additive rather than synergistic. Specifically, the retention in the model for systolic pulmonary artery pressure of a term incorporating the product of oxyhaemoglobin saturation and carbon dioxide partial pressure could not improve the predictive power of the model. An additional finding is that the direct effects of alveolar gases on pulmonary artery pressure via vasoconstriction dominate the indirect effects that come about via changes in ventilation and cardiac output. The usual linear regression assumptions of normality and constant variance are confirmed by plotting the residuals against the fitted values (Fig. 3A) and inspection of a normal residuals- quantile plot (Fig. 3B). The purpose of the former plot is to show whether variance changes throughout the range of data, which would appear as a trend for the residuals to deviate from 0 as a function of the fitted values. One or two outliers on a dataset of this size are to be expected and are not necessarily inconsistent with a good fit. The latter plot shows whether the data are approximately normal, an assumption which is violated to the extent that the plot deviates from being linear. p Methods for measuring pulmonary vasoconstriction in vivo are controversial. In reduced preparations, typically perfusions of non- human animal lungs or vessels in vitro, it is common to manipulate pulmonary flow to be constant and then use either the pressure drop across the pulmonary circulation or PVR as measures of vascular ‘tone’ or ‘constriction’ [34,35]. An alternative approach is to maintain perfusion pressure constant, and associate changes in vascular constriction with changes in blood flow [20,36]. In awake humans neither of these approaches has proved accessible, and measurements of pulmonary vasoconstriction are complicated by the fact that both pulmonary arterial pressure and pulmonary blood flow usually change in response to changes in alveolar gases. A common invasive strategy has been to measure PVR using a Swan-Ganz pulmonary artery catheter, whilst accepting that changes in PVR occur independently in response to changes in both cardiac output [37] and alveolar gas composition [38]. Discussion This study demonstrates that the non-invasive measurement of systolic pulmonary artery pressure using Doppler ultrasound is a useful tool to assess vasoconstriction in response to changes in alveolar gases, as long as account is taken, as with catheter measurements, of the separate effect of cardiac output on this variable. The independent effects of altered PETCO2 and SO2 on Q˙ were modelled using the same approach. The analysis fitted the equation: Q : protocol value~cze(B Q : )zi(D ln V : E)z e(DSO2)zf(DPETCO2 ) ð3Þ ð3Þ where the coefficients are given in Table 3. The model delivered e, i, e and f as fixed coefficients, whilst c was taken to be normally distributed with a standard deviation of 0.08 l/min. A similar approach was used for lnV˙ E. Data from protocols involving hypocapnia were excluded from this analysis because V˙ E was consciously controlled in these protocols in order to achieve hypocapnia. The final model for V˙ E derived the following equation: ln (V : E) protocol value~lzf(B ln (V : E)) zc(DSO2)zd(DPETCO2 ) ð4Þ ð4Þ Results of statistical analysis The change in peak systolic tricuspid pressure gradient (DPmax) was calculated as the difference between the mean baseline DPmax and the mean DPmax during the last 6 minutes of each protocol. Gas control was achieved by means of end-tidal forcing. doi:10.1371/journal.pone.0067886.t002 July 2013 \| Volume 8 \| Issue 7 \| e67886 July 2013 \| Volume 8 \| Issue 7 \| e67886 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 5 Human Pulmonary Vascular Responses to O2 & CO2 Table 3. Model coefficients for interdependence of systolic tricuspid pressure gradient (DPmax), cardiac output (Q˙), ventilation (expressed as the natural logarithm of ventilation, ln(V˙E)), end-tidal partial pressure of CO2 (PETCO2) and end-tidal oxygen level expressed as equivalent haemoglobin saturation (SO2). Protocol valueIntercept Baseline DSO2 DPETCO2 DQ˙ DlnV˙ E DPmax a b a b g h 3.461.5 0.8960.06 0.4360.09 0.1860.03 0.6660.32 0 mmHg mmHg/%desat mmHg/l/min mmHg/ln(l/min) Q˙ c e e f i 1.160.3 0.7960.06 0.0660.01 0.0260.01 0.3360.14 l/min l/min/%desat l/min/mmHg l/min/ln(l/min) ln(V˙E) l f c d 1.2 60.2 0.5260.08 0.03960.004 0.09960.009 ln(l/min) ln(l/min)/%desat ln(l/min)/mmHg Roman alphabet coefficients are depicted in Fig. 1. Roman and Greek coefficients are defined in Eqs. 2, 3 and 4. Coefficients are given as a value 6 standard error. doi:10.1371/journal.pone.0067886.t003 Table 3. Model coefficients for interdependence of systolic tricuspid pressure gradient (DPmax), cardiac output (Q˙), ventilation (expressed as the natural logarithm of ventilation, ln(V˙E)), end-tidal partial pressure of CO2 (PETCO2) and end-tidal oxygen level expressed as equivalent haemoglobin saturation (SO2). Protocol valueIntercept Baseline DSO2 DPETCO2 DQ˙ DlnV˙ E DPmax a b a b g h 3.461.5 0.8960.06 0.4360.09 0.1860.03 0.6660.32 0 mmHg mmHg/%desat mmHg/l/min mmHg/ln(l/min) Q˙ c e e f i 1.160.3 0.7960.06 0.0660.01 0.0260.01 0.3360.14 l/min l/min/%desat l/min/mmHg l/min/ln(l/min) ln(V˙E) l f c d 1.2 60.2 0.5260.08 0.03960.004 0.09960.009 ln(l/min) ln(l/min)/%desat ln(l/min)/mmHg Roman alphabet coefficients are depicted in Fig. 1. Roman and Greek coefficients are defined in Eqs. 2, 3 and 4. Coefficients are given as a value 6 standard error. doi:10.1371/journal.pone.0067886.t003 Roman alphabet coefficients are depicted in Fig. 1. Roman and Greek coefficients are defined in Eqs. 2, 3 and 4. Coefficients are given as a value 6 standard error. doi:10.1371/journal.pone.0067886.t003 July 2013 \| Volume 8 \| Issue 7 \| e67886 Comparison of pulmonary vascular response with previous human studies where the coefficients are given in Table 3. The model delivered f, c and d as fixed coefficients, whilst l was taken to be normally distributed with a standard deviation of 0.22 ln(l/min). Fig. 4(B) suggests for this study that 10–15% of the effect of alveolar gases on DPmax occurs via indirect pathways. Two such pathways have been identified here: changes in cardiac output induced by changes in ventilation alone, and changes in cardiac output induced by CO2 and O2 in the absence of changes in ventilation. Few data are available from the literature for comparison. A study focusing on longer durations of hypoxia Figure 4 gives the results for the coefficients defined in Fig. 1, and summarizes direct and indirect pathways via which O2 and CO2 influence DPmax. For both gases, the direct pathway dominates. July 2013 \| Volume 8 \| Issue 7 \| e67886 PLOS ONE \| www.plosone.org 6 Human Pulmonary Vascular Responses to O2 & CO2 Human Pulmonary Vascular Responses to O2 & CO2 Figure 3. Plots of residuals for DPmax associated with model in Eq. 1. (A) Residuals for DPmax plotted against the values for DPmax fitted to the model in Eq. 1. A skewed plot would show that the assumption of constant variance had been violated. No such pattern is discernible in this plot. (B) Residuals for DPmax plotted against the standardized expected quantiles (units of standard deviation) fitted to the model in Eq. 1. The linear relationship demonstrates that the residual deviances map on to a Normal distribution. doi:10.1371/journal.pone.0067886.g003 Human Pulmonary Vascular Responses to O2 & CO2 Figure 3. Plots of residuals for DPmax associated with model in Eq. 1. (A) Residuals for DPmax plotted against the values for DPmax fitted to the model in Eq. 1. A skewed plot would show that the assumption of constant variance had been violated. No such pattern is discernible in this plot. (B) Residuals for DPmax plotted against the standardized expected quantiles (units of standard deviation) fitted to the model in Eq. 1. The linear relationship demonstrates that the residual deviances map on to a Normal distribution. doi:10.1371/journal.pone.0067886.g003 July 2013 \| Volume 8 \| Issue 7 \| e67886 Limitations of the study doi:10.1371/journal.pone.0067886.g004 Human Pulmonary Vascular Responses to O2 & CO2 Human Pulmonary Vascular Responses to O2 & CO2 Figure 4. Coefficients obtained from modelling studies. (A) Results for the coefficients from Fig. 1 obtained by mixed effects modelling, given as mean 6 standard deviation. (B) Components of direct and indirect pathways whereby alveolar oxygen and carbon dioxide influence DPmax, with comparisons with earlier studies [13,40]. For both gases the direct pathway dominates. doi:10.1371/journal.pone.0067886.g004 Figure 4. Coefficients obtained from modelling studies. (A) Results for the coefficients from Fig. 1 obtained by mixed effects modelling, given as mean 6 standard deviation. (B) Components of direct and indirect pathways whereby alveolar oxygen and carbon dioxide influence DPmax, with comparisons with earlier studies [13,40]. For both gases the direct pathway dominates. doi:10.1371/journal.pone.0067886.g004 they are secondary to the associated change in pH. This question has been addressed in animal studies, some of which suggest an effect of hypercapnia per se in the pulmonary vasculature [42,43], but further studies would be needed to explore this issue in humans. estimates from previous studies for comparison with coefficients b and f in Eqs. 2 & 3; this may be why it is these coefficients that agree least well with estimates from previous studies. With regard to the coefficients relating to the cardiopulmonary responses to oxygen, values for a, g, & e show fair agreement with published values obtained from well-defined steady-state measurements. Limitations of the study (0.5–8 h) found that approximately 5% of the rise in DPmax with hypoxia could be attributed to indirect effects via cardiac output [39]. ˙ The study measured changes in cardiopulmonary variables between 4 and 10 min after induction of new values of alveolar gases. A maximum exposure of 10 min to the perturbation in alveolar gas composition was chosen in part because of the difficulty experienced by volunteers in tolerating longer exposure to extremes such as combined hypoxia (PETO2 = 50 mmHg) and hypercapnia (PETCO2 = +6 mmHg). Previous work has suggested that this is a sufficiently long period in which to capture the initial acute phase of human hypoxic pulmonary vasoconstriction and the hypoxic increase in cardiac output, in which the time constants of the responses are around 2 min [40,41]. Recent work has found, however, that the time courses of the acute human cardiopulmonary responses to euoxic hypercapnia and hypocap- nia have time constants in the range 4–10 min [13], suggesting that the present experiments have measured a substantial but partial component of the acute changes in DPmax and Q˙ to changes in PACO2. It is consequently difficult to obtain reliable The sensitivity of DPmax to acute changes in Q is defined by coefficient g in Eq. 2 and Fig. 1. The contribution of Q˙ alone is defined by g = 0.66 mmHg/l/min. A previous study [39] observed spontaneous concurrent changes in DPmax with changes in Q˙ during air breathing in the absence of changes in alveolar gas composition and found a value for g of 0.60 mmHg/l/min, in good agreement with that found here. Other coefficients accessible from previous studies on similar human volunteers permit estimates for e (0.06 l/min/%desat from hypoxic exposures [13,40]; here identically 0.06 l/min/%desat) and f (0.04 l/min/mmHg from hypocapnic exposures at constant ventilation, [13]; here 0.02 l/min/mmHg). PLOS ONE \| www.plosone.org July 2013 \| Volume 8 \| Issue 7 \| e67886 July 2013 \| Volume 8 \| Issue 7 \| e67886 7 Figure 4. Coefficients obtained from modelling studies. (A) Results for the coefficients from Fig. 1 obtained by mixed effects modelling, given as mean 6 standard deviation. (B) Components of direct and indirect pathways whereby alveolar oxygen and carbon dioxide influence DPmax, with comparisons with earlier studies [13,40]. For both gases the direct pathway dominates. PLOS ONE \| www.plosone.org Human Pulmonary Vascular Responses to O2 & CO2 Human Pulmonary Vascular Responses to O2 & CO2 tions in end-tidal gas composition that might lead to global autonomic effects on the pulmonary circulation that would not occur with perturbations limited to small regions of lung tissue. Even on the assumption of additive, rather than interactive, effects of the two stimuli recent calculations suggest that CO2 may play a more substantial role than O2 in ventilation-perfusion matching in the healthy lung at sea level [13]. Under conditions of therapeutic artificial ventilation, clinicians recognize the potential adverse effect on oxygenation of the patient of a low PACO2 in a hyperventilated hypoxic lung leading to inhibition or elimination of hypoxic vasoconstriction in that lung [46,47], but the relative contributions of the stimuli have remained unclear. intense responses to more sustained combinations of CO2 and O2 stimuli, such as those occurring over hours and days at high altitude, combine in a similar additive manner. A novel finding from the study has been the possibility of obtaining a quantitative estimate of the effect of V˙ E on Q˙ that is independent of the effects of alveolar gases, namely the coefficient i. The value of i = 0.33 l/min/ln(l/min) suggests a 0.33 l/min rise in cardiac output attributable to a 2.72-fold rise in ventilation. Another interpretation, assuming linearity over a broad range of ventilation, is that a rise in ventilation from a resting value of about 4.5 l/min to a twenty-fold value of 90 l/min associated with very vigorous exercise might contribute a rise in cardiac output of ,1 litre/min from the direct effect of ventilation on the cardiovascular system alone. Interestingly, ventilation alone appears to have no direct effect upon DPmax (i.e. h = 0). Further studies are required to establish the magnitude of these interre- lationships over wider ranges of physiological disturbance. Pulmonary hypertension at high altitude is associated with global hypoxia with hypocapnia throughout the lung [10] and appears to be responsible for high altitude pulmonary edema in patients who have an exaggerated vasoconstrictor response [48]. It remains uncertain to what extent in affected individuals a weak vasodilatory effect of hypocapnia might inadequately ameliorate the pulmonary hypertension that results from a strong vasocon- strictor effect of hypoxia, because these stimuli have not been examined separately in this setting [49]. References 1. Viswanathan R, Lodi ST, Subramanian S, Radha TG (1976) Pulmonary vascular response to ventilation hypercapnia in man. Respiration 33: 165–178. 2. Balanos GM, Talbot NP, Dorrington KL, Robbins PA (2003) Human pulmonary vascular response to 4 h of hypercapnia and hypocapnia measured using Doppler echocardiography. J Appl Physiol 94: 1543–1551. 17. Viles PH, Shepherd JT (1968) Relationship between pH, PO2, and PCO2 on the pulmonary vascular bed of the cat. Am J Physiol 215: 1170–1176. 1. Viswanathan R, Lodi ST, Subramanian S, Radha TG (1976) Pulmonary vascular response to ventilation hypercapnia in man. Respiration 33: 165–178. 1. Viswanathan R, Lodi ST, Subramanian S, Radha TG (1976) Pulmonary vascular response to ventilation hypercapnia in man. Respiration 33: 165–178. 18. Von Euler US, Liljestrand G (1946) Observations on the pulmonary arterial pressure in the cat. Acta Physiol Scand 12: 301–320. 2. Balanos GM, Talbot NP, Dorrington KL, Robbins PA (2003) Human pulmonary vascular response to 4 h of hypercapnia and hypocapnia measured using Doppler echocardiography. J Appl Physiol 94: 1543–1551. 19. Shirai M, Sada K, Ninomiya I (1986) Effects of regional alveolar hypoxia and hypercapnia on small pulmonary vessels in cats. J Appl Physiol 61: 440–448. 3. Motley H, Cournand A, Werko L, Himmelstein A, Dresdale D (1947) The influence of short periods of acute anoxia upon pulmonary arterial pressure in man. Am J Physiol 150: 315–320. 20. Sheehan DW, Farhi LE (1993) Local pulmonary blood flow: control and gas exchange. Respir Physiol 94: 91–107. 21. Dorrington KL, Talbot NP (2004) Human pulmonary vascular responses to hypoxia and hypercapnia. Pflu¨gers Arch 449: 1–15. 4. Carlsson AJ, Bindslev L, Santesson J, Gottlieb I, Hedenstierna G (1985) Hypoxic pulmonary vasoconstriction in the human lung: the effect of prolonged unilateral hypoxic challenge during anaesthesia. Acta Anaesthesiol Scand 29: 346–351. 22. Sylvester JT, Shimoda LA, Aaronson PI, Ward JP (2005) Hypoxic pulmonary vasoconstriction. Physiol Rev 92: 367–520. 5. Naeije R, Brimioulle S (2001) Physiology in medicine: importance of hypoxic pulmonary vasoconstriction in maintaining arterial oxygenation during acute respiratory failure. Crit Care 5: 67–71. 23. Liu C, Smith TG, Balanos GM, Brooks J, Crosby A, et al. (2007) Lack of involvement of the autonomic nervous system in early ventilatory and pulmonary vascular acclimatization to hypoxia in humans. J Physiol 579: 215–225. 6. Dehnert C, Berger MM, Mairba¨url H, Ba¨rtsch P (2007) High altitude pulmonary edema: a pressure-induced leak. Respir Physiol Neurobiol 158: 266–273. 24. Acknowledgments We thank Mr David O’Connor and Dr Marzieh Fatemian for their technical assistance and the volunteers for their participation. References Smith TG, Balanos GM, Croft QP, Talbot NP, Dorrington KL, et al. (2008) The increase in pulmonary arterial pressure caused by hypoxia depends on iron status. J Physiol 586: 5999–6005. 7. Lahiri S, DeLaney RG (1975) Stimulus interaction in the responses of carotid body chemoreceptor single afferent fibers. Respir Physiol 24: 249–266. J y 25. Smith TG, Brooks JT, Balanos GM, Lappin TR, Layton DM, et al. (2006) Mutation of von Hippel-Lindau tumour suppressor and human cardiopulmo- nary physiology. PLoS Med 3: e290. 8. Roy A, Rozanov C, Mokashi A, Lahiri S (2000) P(O(2))-P(CO(2)) stimulus interaction in [Ca(2+)](i) and CSN activity in the adult rat carotid body. Respir Physiol 122: 15–26. y p y gy 26. West JB (1990) Ventilation/Blood Flow and Gas Exchange. Oxford: Blackwell. 26. West JB (1990) Ventilation/Blood Flow and Gas Exchange. Oxford: Blackwell. 27 Robbins PA Swanson GD Howson MG (1982) A prediction correction scheme y 9. Peers C (2004) Interactions of chemostimuli at the single cell level: studies in a model system. Exp Physiol 89: 60–65. 27. Robbins PA, Swanson GD, Howson MG (1982) A prediction-correction scheme for forcing alveolar gases along certain time courses. J Appl Physiol 52: 1353– 1357. 10. Milledge JS, West JB, Schoene RB (2007) High Altitude Medicine & Physiology London: Hodder Arnold. 28. Robbins PA, Swanson GD, Micco AJ, Schubert WP (1982) A fast gas-mixing system for breath-to-breath respiratory control studies. J Appl Physiol 52: 1358– 1362. 11. Lloyd BB, Jukes MGM, Cunningham DJC (1958) The relation between alveolar oxygen pressure and the respiratory response to carbon dioxide in man. Quarterly J Exp Physiol 43: 214–227. 29. Howson MG, Khamnei S, McIntyre ME, O’Connor DF, Robbins PA (1987) A rapid computer-controlled binary gas-mixing system for studies in respiratory control. J Physiol 394: 7P. Q y J p y 12. Howell K, Ooi H, Preston R, McLoughlin P (2004) Structural basis of hypoxic pulmonary hypertension: the modifying effect of chronic hypercapnia. Exp Physiol 89: 66–72. 30. Peacock AJ, Challenor V, Sutherland G (1990) Estimation of pulmonary artery pressure by Doppler echocardiography in normal subjects made hypoxic. Respir Med 84: 335–337. y 13. Dorrington KL, Balanos GM, Talbot NP, Robbins PA (2010) Extent to which pulmonary vascular responses to PCO2 and PO2 play a functional role within the healthy human lung. J Appl Physiol 108: 1084–1096. 31. Stevenson JG (1989) Comparison of several noninvasive methods for estimation of pulmonary artery pressure. Human Pulmonary Vascular Responses to O2 & CO2 The human lung shows considerable potential to dilate in response to sustained hypocap- nia [2], and it would clearly be beneficial at altitude for there to be a balance between the vasodilatory effects of hypocapnia and the constriction brought about by hypoxia. The present experiments have quantified the extent of this balance for very acute responses in the period 4–10 min following a step change of alveolar gases. Further work is required to find whether the considerably more Author Contributions Conceived and designed the experiments: QPPC NPT PAR KLD. Performed the experiments: QPPC FF NPT. Analyzed the data: QPPC DL PAR KLD. Wrote the paper: QPPC PAR KLD. Conceived and designed the experiments: QPPC NPT PAR KLD. Performed the experiments: QPPC FF NPT. Analyzed the data: QPPC DL PAR KLD. Wrote the paper: QPPC PAR KLD. Physiological significance of the findings A second limitation of the study arises from the requirement to establish voluntarily controlled ventilation for half of all measure- ments made, in order to achieve hypocapnia. The resulting halving of the number of data pertaining to the coefficients linking to ventilation in Fig. 1 will have reduced the precision with which coefficient i in Eq. 3 could be estimated and reduced the probability of detecting a small but non-zero value of the coefficient h linking DPmax directly with V˙ E. An accurate appreciation of the way in which the stimuli CO2 and O2 work together on pulmonary vessels is of importance to the understanding of situations in which they act in synchrony or in opposition. The spontaneous matching of perfusion to ventilation in the lung is usually modelled as being achieved solely by the vasoconstrictor effects of hypoxia on small pulmonary arteries [44,45], but the local vasoconstrictor effect of hypercapnia has the potential to enhance this matching [1,36]. It remains a possibility that the effects of hypoxia and hypercapnia acting only within an isolated small region of lung tissue might display a different, possibly interactive, relationship from the global effects on all lung tissue studied here. One possible reason for this is that the experiments subjected volunteers to relatively stressful perturba- Thirdly, this study did not seek to understand the cellular basis for any interaction between CO2 and O2 in the pulmonary circulation, but instead to understand the effects of alveolar gas composition at the integrative level in humans. For example, we did not address the question of whether changes in pulmonary vascular tone result directly from alterations in PCO2, or whether PLOS ONE \| www.plosone.org July 2013 \| Volume 8 \| Issue 7 \| e67886 8 Human Pulmonary Vascular Responses to O2 & CO2 Human Pulmonary Vascular Responses to O2 & CO2 35. Weissmann N, Akkayagil E, Quanz K, Schermuly RT, Ghofrani HA, et al. (2004) Basic features of hypoxic pulmonary vasoconstriction in mice. Respir Physiol Neurobiol 139: 191–202. 42. Viles PH, Shepherd JT (1968) Evidence for a dilator action of carbon dioxide on the pulmonary vessels of the cat. Circ Res 22: 325–332. 43. Ketabchi F, Egemnazarov B, Schermuly RT, Ghofrani HA, Seeger W, et al. (2009) Effects of hypercapnia with and without acidosis on hypoxic pulmonary vasoconstriction. Am J Physiol 297: L977–983. y 36. Barer GR, Howard P, Shaw JW (1970) Stimulus-response curves for the pulmonary vascular bed to hypoxia and hypercapnia. J Physiol 211: 139–155. p y yp yp p J y 37. Kovacs G, Olschewski A, Berghold A, Olschewski H (2012) Pulmonary vascular resistances during exercise in normal subjects: a systematic review. Eur Respir J 39: 319–328. y 44. Marshall BE, Hanson CW, Frasch F, Marshall C (1994) Role of hypoxic pulmonary vasoconstriction in pulmonary gas exchange and blood flow distribution. 2. Pathophysiology. Intensive Care Med 20: 379–389. 38. Groves BM, Reeves JT, Sutton JR, Wagner PD, Cymerman A, et al. (1987) Operation Everest II: Elevated high-altitude pulmonary resistance unresponsive to oxygen. J Appl Physiol 63: 521–530. p y gy 45. Brimioulle S, LeJeune P, Naeije R (1996) Effects of hypoxic pulmonary vasoconstriction on pulmonary gas exchange. J Appl Physiol 81: 1535–1543. 46. Bindslev L, Jolin-Carlsson A, Santesson J, Gottlieb I (1985) Hypoxic pulmonary vasoconstriction in man: effects of hyperventilation. Acta Anaesthesiol Scand 29: 547–551. 39. Balanos GM, Talbot NP, Robbins PA, Dorrington KL (2005) Separating the direct effect of hypoxia from the indirect effect of changes in cardiac output on the maximum pressure difference across the tricuspid valve in healthy humans. Pflu¨gers Arch 450: 372–380. 47. Noble WH, Kay JC, Fisher JA (1981) The effect of PCO2 on hypoxic pulmonary vasoconstriction. Can Anaesth Soc J 28: 422–430. 40. Talbot NP, Balanos GM, Dorrington KL, Robbins PA (2005) Two temporal components within the human pulmonary vascular response to approximately 2 h of isocapnic hypoxia. J Appl Physiol 98: 1125–1139. 48. Ba¨rtsch P, Mairba¨url H, Maggiorini M, Swenson ER (2005) Physiological aspects of high-altitude pulmonary edema. J Appl Physiol 98: 1101–1110. 49. Gru¨nig E, Mereles D, Hildebrandt W, Swenson ER, Ku¨bler W, et al. (2000) Stress doppler echocardiography for identification of susceptibility to high altitude pulmonary edema. 47. Noble WH, Kay JC, Fisher JA (1981) The effect of PCO2 on hypoxic pulmonary vasoconstriction. Can Anaesth Soc J 28: 422–430. 43. Ketabchi F, Egemnazarov B, Schermuly RT, Ghofrani HA, Seeger W, et al. (2009) Effects of hypercapnia with and without acidosis on hypoxic pulmonary vasoconstriction. Am J Physiol 297: L977–983. 49. Gru¨nig E, Mereles D, Hildebrandt W, Swenson ER, Ku¨bler W, et al. (2000) Stress doppler echocardiography for identification of susceptibility to high altitude pulmonary edema. J Am Coll Cardiol 35: 980–987. 48. Ba¨rtsch P, Mairba¨url H, Maggiorini M, Swenson ER (2005) Physiological aspects of high-altitude pulmonary edema. J Appl Physiol 98: 1101–1110. 42. Viles PH, Shepherd JT (1968) Evidence for a dilator action of carbon dioxide on the pulmonary vessels of the cat. Circ Res 22: 325–332. 46. Bindslev L, Jolin-Carlsson A, Santesson J, Gottlieb I (1985) Hypoxic pulmonary vasoconstriction in man: effects of hyperventilation. Acta Anaesthesiol Scand 29: 547–551. J y 44. Marshall BE, Hanson CW, Frasch F, Marshall C (1994) Role of hypoxic pulmonary vasoconstriction in pulmonary gas exchange and blood flow distribution. 2. Pathophysiology. Intensive Care Med 20: 379–389. References J Am Soc Echocardiogr 2: 157–171. 14. Grant BJ, Davies EE, Jones HA, Hughes JM (1976) Local regulation of pulmonary blood flow and ventilation-perfusion ratios in the coatimundi. J Appl Physiol 40: 216–228. 32. Severinghaus JW (1979) Simple, accurate equations for human blood O2 dissociation computations. J Appl Physiol 46: 599–602. dissociation computations. J Appl Physiol 46: 599–602. 15. Brimioulle S, Lejeune P, Vachiery JL, Leeman M, Melot C, et al. (1990) Effects of acidosis and alkalosis on hypoxic pulmonary vasoconstriction in dogs. Am J Physiol 258: H347–353. 33. Marshall C, Marshall B (1983) Site and sensitivity for stimulation of hypoxic pulmonary vasoconstriction. J Appl Physiol 55: 711–716. 34. Kiss L, Schutte H, Mayer K, Grimm H, Padberg W, et al. (2000) Synthesis of arachidonic acid-derived lipoxygenase and cytochrome P450 products in the intact human lung vasculature. Am J Respir Crit Care Med 161: 1917–1923. 16. Benumof JL, Wahrenbrock EA (1975) Blunted hypoxic pulmonary vasocon- striction by increased lung vascular pressures. J Appl Physiol 38: 846–850. 9 PLOS ONE \| www.plosone.org July 2013 \| Volume 8 \| Issue 7 \| e67886 July 2013 \| Volume 8 \| Issue 7 \| e67886 Human Pulmonary Vascular Responses to O2 & CO2 J Am Coll Cardiol 35: 980–987. p yp J pp y 41. Morrell NW, Nijran KS, Biggs T, Seed WA (1995) Magnitude and time course of acute hypoxic pulmonary vasoconstriction in man. Respir Physiol 100: 271– 281. PLOS ONE \| www.plosone.org July 2013 \| Volume 8 \| Issue 7 \| e67886 10 PLOS ONE \| www.plosone.org
https://openalex.org/W4382986610	http://archaeologie.pro/download/44/4.pdf	Russian	null	Burial Rite of the Shchukinsky Burial Ground of the Lomovatovo Archaeological Culture	Povolžskaâ arheologiâ	2,023	cc-by-sa	9,962	ПОВОЛЖСКАЯ АРХЕОЛОГИЯ e-ISSN 2500-2856 № 2 (44) 2023 ПОВОЛЖСКАЯ АРХЕОЛОГИЯ Редакционный совет: Б.А. Байтанаев – академик НАН РК, доктор исторических наук (Алматы, Казахстан) (председатель), Х.А. Амирханов – академик РАН, доктор исторических наук, профессор (Москва, Россия), С.Г. Бочаров – кандидат исторических наук (Севастополь, Россия), П. Георгиев – доктор наук, доцент (Шумен, Болгария), Е.П. Казаков – доктор истори- ческих наук (Казань, Россия), Н.Н. Крадин – член-корреспондент РАН, доктор истори- ческих наук, профессор (Владивосток, Россия), А. Тюрк – Ph.D. (Будапешт, Венгрия), А.А. Тишкин – доктор исторических наук профессор (Барнаул, Россия), В.С. Синика – кандидат исторических наук (Тирасполь, Молдова), Б.В. 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Leont’ev – Doctor of Historical Sciences (Institute of Archaeology of the Russian Academy of Sciences, Moscow, Russian Federation) Т. B. Nikitina – Doctor of Historical Sciences (Mari Research Institute of Language, Literature and History named after V. M. Vasilyev, Yoshkar-Ola, Russian Federation) A.A. Chizhevsky – Candidate of Historical Sciences (Institute of Archaeology named after A. Kh. Khalikov, Kazan, Russian Federation) Responsible for Issue F. Sh. Khuzin – Corresponding Member of the Tatarstan Academy of Sciences, Doctor of Historical Sciences Editorial Ofﬁ ce Address: Butlerov St., 30, Kazan, 420012, Republic of Tatarstan, Russian Federation Telephone: (843) 236-55-42 E-mail: arch.pov@mail.ru http://archaeologie.pro Publishing House “Fän” Kazan, Tatarstan POVOLZHSKAYA ARKHEOLOGIYA THE VOLGA RIVER REGION ARCHAEOLOGY e-ISSN 2500-2856 № 2 (44) 2023 Editor-in-Chief: Academician of the Tatarstan Academy of Sciences, Doctor of Historical Sciences A. G. Sitdikov Deputy Chief Editors: Corresponding Member of the Tatarstan Academy of Sciences, Doctor of Historical Sciences F. Sh. Khuzin Doctor of Historical Sciences Yu. A. Zeleneev Executive Secretary – Candidate of Veterinary Sciences G. Sh. Asylgaraeva Deputy Chief Editors: Corresponding Member of the Tatarstan Academy of Sciences, Doctor of Historical Sciences F. Sh. Khuzin Doctor of Historical Sciences Yu. A. Zeleneev Executive Secretary – Candidate of Veterinary Sciences G. Sh. Asylgaraeva Editorial Board: ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 2 (44) 2023 СОДЕРЖАНИЕ СОДЕРЖАНИЕ Финно-угорская археология Поволжья Брюхова Н.Г., Подосёнова Ю.А., Смертин А.Р. (Пермь, Россия) Железная поясная гарнитура из погребений Плотниковского могильника (родановская культура, Пермский край) .........8 Никитина Т.Б. (Йошкар-Ола, Россия), Тюрк А. (Будапешт, Венгрия), Буршнева С.Г. (Казань, Россия), Кишне Бэндэфи М., Вархедьи Ж., Харанги Ф., Янчик Б. (Будапешт, Венгрия), Богатова Л.Ф., Шайхутдинова Е.Ф., Федан П.В. (Казань, Россия) Комплексное изучение естественнонаучными методами и реставрация сумочки из Красногорского могильника ...............................22 Третьяков Е.А. (Тюмень, Россия), Тюрк А. (Будапешт, Венгрия) Вещи «Мадьярского круга» из памятников Зауралья: артефакты и контекст .......................................................................................38 Моряхина К.В. (Пермь, Россия) Погребальный обряд Щукинского могильника ломоватовской археологической культуры .....................................................51 Валеев Р.М. (Казань, Россия) Торговые контакты Волжской Болгарии с мордвой в X – начале XV века ......63 Средневековая археология Центральной Азии Кубарев Г.В. (Новосибирск, Россия), Нускабай А. (Астана, Республика Казахстан) Раннесредневековые поминальные оградки Семиречья: новые данные ...................................................................................................72 Серегин Н.Н. (Барнаул, Россия), Монгуш К.М. (Кызыл, Россия) Раннетюркские оградки комплекса Ак-Даг .........................................................87 Акымбек Е.Ш., Железняков Б.А. (Алматы, Республика Казахстан) Бронзовые кувшины мастерской Ахмада XI в. из Юго-Восточного Казахстана .....................................................................101 Байтанаев Б.А. (Алматы, Республика Казахстан), Ергешбаев А.А. (Шымкент, Республика Казахстан), Шаяхметов А.Х. (Алматы, Республика Казахстан) Хаммам из Ханкургана .......................................................................................114 Sizdikov B.S. (Turkestan, Republic of Kazakhstan), Baitanayev B.A. (Almaty, Republic of Kazakhstan), Murgabayev S.S., Bakhtybayev M.M. (Turkestan, Republic of Kazakhstan), Arynov K.S. (Almaty, Republic of Kazakhstan), Gursoy M. (Turkestan, Republic of Kazakhstan), Seraliyev A.A. (Astana, Republic of Kazakhstan) Mausoleums in the Medieval City of Syganak ......................................................131 Варфоломеев В.В. (Караганда, Республика Казахстан) Каменная печать с реки Каракенгир...................................................................145 Финно-угорская археология Поволжья POVOLZHSKAYA ARKHEOLOGIYA № 2 (44) 2023 Средневековые памятники Центральной России Точилова Н.Н. (Санкт-Петербург, Россия) Роговые обоймицы из раскопок в Великом Новгороде и Смоленске ...............................................................150 Медведь А.Н. (Москва, Россия) Фортификация Московского государства XVI в. и плетневые конструкции Центральной Европы .........................................160 Колоколов А.М. (Тула, Россия) Поминальный комплекс второй половины VII–VIII вв. у д. Гора Услань .....173 Археологические исследования золотоордынских памятников Волков И.В., Лопан О.В., Ситдиков А.Г. (Казань, Россия) Исследования на раскопе CXCIV в юго-восточной части Болгарского городища ............................................189 Пигарёв Е.М. (Йошкар-Ола, Россия), Ситдиков А.Г. (Казань, Россия) Мавзолейный комплекс у с. Лапас Астраханской области (из полевого дневника В.В. Дворниченко). ..................................................209 Бабенко В.А., Колесникова М.Е. (Ставрополь, Россия) Золотоордынские памятники Ставрополья в научном творчестве А.А. Спицына ............................................................221 Памятники нового времени Татауров Ф.С., Татаурова Л.В. (Омск, Россия) Археологические свидетельства русской цивилизации в культурах коренных народов Западной Сибири XVI–XVIII вв. .................................236 Список сокращений .............................................................................................248 Правила для авторов ............................................................................................250 ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 2 (44) 2023 CONTENT Finno-Ugric archaeology of the Volga region Bryukhova N.G., Podosenova Yu.A., Smertin A.R. (Perm, Russian Federation) Iron Belt Set from the Burials of the Plotnikovo Burial Ground (Rodanovo Culture, Perm Region) .......................................................................8 Nikitina T.B. (Yoshkar-Ola, Russian Federation), Turk A. (Budapest, Hungary), S.G. Burshneva (Kazan, Russian Federation), Kishne Bandeﬁ M., Varhedyi J., Harangi F., B. Yanchik (Budapest, Hungary), Bogatova L.F., Shaikhutdinova E.F., Fedan P.V. (Kazan, Russian Federation) Comprehensive Study of Natural Science Methods and Conservation of a Handbag from Krasnogorskiy Burial Ground .............................................22 Tretyakov E.A. (Tyumen, Russian Federation), Turk A. (Budapest, Hungary) Products of «Hungarian Style» from Archaeological Sites of the Trans-Urals: artifacts and context ............................................................................................38 Moryakhina K.V. (Perm, Russian Federation) Burial Rite of the Shchukinsky Burial Ground of the Lomovatovo Archaeological Culture........................................................51 Valeev R.M. (Kazan, Russian Federation) Trade Contacts of Volga Bulgaria with the Mordvins in the 10th – Beginning of the 15th Century .........................................................63 Central Asia Medieval archaeology Kubarev G.V. (Novosibirsk, Russian Federation), Nuskabai A. (Astana, Republic of Kazakhstan) Early Medieval Memorial Enclosures in Semirechye: new data .............................72 Seregin N.N. (Barnaul, Russian Federation), Mongush K.M. (Kyzyl, Russian Federation) Early Turkic Enclosures of the Ak-Dag Complex ...................................................87 Akymbek E.Sh, Zheleznyakov B.A. (Almaty, Republic of Kazakhstan) Bronze Jugs from Ahmad's Workshop of 11th Century from Southeast Kazakhstann ............................................................................101 Baitanaev B.A. (Almaty, Republic of Kazakhstan), Ergeshbayev A.A. (Shymkent, Republic of Kazakhstan), Shayakhmetov A.H. (Almaty, Republic of Kazakhstan) Hammam from Khankurgan ..................................................................................114 Sizdikov B.S. (Turkestan, Republic of Kazakhstan), Baitanayev B.A. (Almaty, Republic of Kazakhstan), Murgabayev S.S., Bakhtybayev M.M. (Turkestan, Republic of Kazakhstan), Arynov K.S. Финно-угорская археология Поволжья (Almaty, Republic of Kazakhstan), Gursoy M. (Turkestan, Republic of Kazakhstan), Seraliyev A.A. (Astana, Republic of Kazakhstan) Mausoleums in the Medieval City of Syganak ......................................................131 CONTENT POVOLZHSKAYA ARKHEOLOGIYA № 2 (44) 2023 Varfolomeev V.V. (Karaganda, Republic of Kazakhstan) Stone Print from the Karakengir River ..................................................................145 Medieval sites in Central Russia Tochilova N.N. (Saint Petersburg, Russian Federation) Horn Ferrules from Excavation at Novgorod the Great and Smolensk .................150 Medved A.N. (Moscow, Russian Federation) Fortiﬁ cation of the Moscow State in the 16th Century and the Wattle Structures in Central Europe .....................................................160 Kolokolov A.M. (Tula, Russian Federation) Memorial Complex of the Second Half of the 7th–8th Centuries at the Hillfort Near the Village of Gora Uslan ..................................................173 Archaeological studies of the Golden Horde sites Volkov I.V., Lopan O.V., Sitdikov A.G. (Kazan, Russian Federation) Research on the Excavation CXXIV in the South-Eastern Part of the Bolgar Fortiﬁ ed Settlement ....................................................................189 Pigarev E.M. (Yoshkar-Ola, Russian Federation), Sitdikov A.G. (Kazan, Russian Federation) Mausoleum Complex near the Village of Lapas, Astrakhan Region (from the Field Diary of V.V. Dvornichenko) ...................................................209 Babenko V.A., Kolesnikova M.E. (Stavropol, Russian Federation) Golden Horde Monuments in Stavropol Region in Scientiﬁ c Work of A.A. Spitsyn ....................................................................221 Sites of the New Time period Tataurov F.S., Tataurova L.V. (Omsk, Russian Federation) Archaeological Evidence of Russian Civilization in the Cultures of the Indigenous Peoples of Western Siberia in the 16th –18th Centuries ........236 List of Abbreviations ............................................................................................. 248 Submissions .......................................................................................................... 250 ков А.М., Умаров Моряхина К.В. УДК 902/904 https://doi.org/10.24852/pa2023.2.44.51.62 ПОГРЕБАЛЬНЫЙ ОБРЯД ЩУКИНСКОГО МОГИЛЬНИКА ЛОМОВАТОВСКОЙ АРХЕОЛОГИЧЕСКОЙ КУЛЬТУРЫ © 2023 г. К.В. Моряхина Щукинский могильник расположен в 300 м к западу от д. Щукино Афанасьевского района Кировской области на небольшом мысу высотой 14 м правого берега р. Камы. Памятник был обнаружен и исследован В.А. Кананиным в 1977 г. Всего изучено 82 погребения. Щукинский могильник представляет интерес разнообразием типов по- гребального обряда. На памятнике захоронения выполнены по обряду кремации и ингумации в могильных ямах без конструктивных особенностей и в ямах с нишами, заплечиками и уступами. Заплечики и уступы фиксируются в погребениях IX в. В не- которых из них сохранились фрагменты деревянного настила, вероятно, погребенного укладывали на настил и сверху засыпали землей. Автором статьи на основе анализа погребального инвентаря выделено пять хронологических групп. Наличие на памят- нике двух захоронений VI в. подвергается сомнению, поскольку ранние материалы представлены только поясными пряжками. Датировку могильника стоит сузить и обо- значить время его функционирования VII–IX вв. В наибольшем количестве сохрани- лись поздние погребения (памятник разрушен природными факторами). Уточнение датировки памятника и выделение хронологических групп позволило автору статьи сделать предположение, что в регионе существовали одновременно две разные куль- турные традиции: погребальный обряд Щукинского могильника схож с обрядом на Аверинском II, Агафоновском I и II, при этом по ряду признаков (конструкция ям, спо- соб захоронения, заполнения слоя ям и др.) отличается от синхронных могильников Верхнего Прикамья – Плесовского, Деменковского, Каневского, Урьинского. Ключевые слова: археология, Верхняя Кама, погребальный обряд, биобрядность, погребальные сооружения, погребальный инвентарь, Средневековье, хронология. было выделено три группы погребе- ний, которые отделены столбовыми ямками. В.А. Канинин предполагал, что группы отражают определенную социальную структуру. Анализируя погребальные сооружения, В.А. Ка- нанин обратил внимание на наличие ниш-выступов, предназначение ко- торых неясно, и на то, что покойно- го укладывали на дощатый настил, в качестве перекрытия использовали бересту или шкуру животного. На па- мятнике зафиксирована биобрядность захоронения. Как указывал автор рас- копок, погребения с разным способом захоронения не выделяются плани- графически и по вещевому набору. Кремация производилась на стороне. Статистический анализ, проведенный В.А. Кананиным, позволил выявить близость Щукинского могильника по погребальному обряду с синхронным ему памятником – Аверинским II мо- гильником (Кананин, 1983, с. 80–95; было выделено три группы погребе- ний, которые отделены столбовыми ямками. В.А. Канинин предполагал, что группы отражают определенную социальную структуру. Анализируя погребальные сооружения, В.А. Ка- нанин обратил внимание на наличие ниш-выступов, предназначение ко- торых неясно, и на то, что покойно- го укладывали на дощатый настил, в качестве перекрытия использовали бересту или шкуру животного. На па- мятнике зафиксирована биобрядность захоронения. ПОГРЕБАЛЬНЫЙ ОБРЯД ЩУКИНСКОГО МОГИЛЬНИКА ЛОМОВАТОВСКОЙ АРХЕОЛОГИЧЕСКОЙ КУЛЬТУРЫ Как указывал автор рас- копок, погребения с разным способом захоронения не выделяются плани- графически и по вещевому набору. Кремация производилась на стороне. Статистический анализ, проведенный В.А. Кананиным, позволил выявить близость Щукинского могильника по погребальному обряду с синхронным ему памятником – Аверинским II мо- гильником (Кананин, 1983, с. 80–95; Щукинский могильник располо- жен в 300 м к западу от д. Щукино Афанасьевского района Кировской области на небольшом мысу высо- той 14 м правого берега р. Камы. Па- мятник был обнаружен и исследован В.А. Кананиным в 1977 г. На площа- ди 1100 кв. м было изучено 82 погре- бения. Часть памятника разрушена в результате оползня мыса. На могиль- нике фиксируются следы разграбле- ния. Памятник считается полностью изученным, датируется VI–IX вв. (Ка- нанин, 1978, с. 2). Автором раскопок была дана ха- рактеристика погребального обряда Щукинского могильника наряду с другими памятниками в обобщаю- щих работах, посвященных изуче- нию средневековых памятников вер- ховья Камы. В.А. Кананин отмечал, что «одновременно с рядовым рас- положением могил наблюдается и размещение их по группам». Так, им 51 51 51 № 1 (39) 2022 ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 4 (42) 2022 ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 2 (44) 2023 ПОВОЛЖСКАЯ АРХЕОЛОГИЯ Рис. 1. Планы погребений Щукинского могильника. 1, 2 – погребения с обрядом ин- гумации (п. 30, 32); 3, 4 – погребения с обрядом кремации (п. 31, 82). 1 – погребения без конструктивных особенностей (п. 30, 31); 2 – погребение с уступом (п. 32); 4 – по- гребение с нишей (п. 82) (план погребений по: Голдина, Кананин, 1989, рис. 30, 34). Fig. 1. Burial plans of the Shchukin burial ground. 1, 2 – burials with inhumation rite (b. 30, 32); 3, 4 – burials with cremation rite (b. 31, 82). 1 – burials without structural features (b. 30, 31); 2 – burial with a ledge (b. 32); 4 – burial with a niche (b. 82) (plan of burials according to: Goldina, Kananin, 1989, ﬁ g. 30, 34). ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 1 (39) 2022 № 4 (42) 2022 № 2 (44) 2023 Рис. 1. Планы погребений Щукинского могильника. 1, 2 – погребения с обрядом ин- гумации (п. 30, 32); 3, 4 – погребения с обрядом кремации (п. 31, 82). 1 – погребения без конструктивных особенностей (п. 30, 31); 2 – погребение с уступом (п. 32); 4 – по- гребение с нишей (п. 82) (план погребений по: Голдина, Кананин, 1989, рис. 30, 34). Fig. 1. ПОГРЕБАЛЬНЫЙ ОБРЯД ЩУКИНСКОГО МОГИЛЬНИКА ЛОМОВАТОВСКОЙ АРХЕОЛОГИЧЕСКОЙ КУЛЬТУРЫ Burial plans of the Shchukin burial ground. 1, 2 – burials with inhumation rite (b. 30, 32); 3, 4 – burials with cremation rite (b. 31, 82). 1 – burials without structural features (b. 30, 31); 2 – burial with a ledge (b. 32); 4 – burial with a niche (b. 82) (plan of burials according to: Goldina, Kananin, 1989, ﬁ g. 30, 34). Голдина, Кананин, 1989, с. 26–43). Голдина, Кананин, 1989, с. 26–43). расположения в погребении (есть обобщенное описание материалов всех памятников верховья Камы), не были выделены комплексы с набором оружия и орудий труда. В ранее опу- бликованных материалах памятник датируется широко – VI–IX вв., при этом не выделены хронологические группы на памятнике, соответствен- но, не были сделаны выводы об из- менении со временем погребального обряда и вещевого набора. Уточнение хронологии могильника позволяет бо- лее объективно проводить сравнение с синхронными памятниками Перм- ского Предуралья. , , , ) Статический анализ, проведен- ный И.В. Бочаровым по материалам могильников Пермского Предуралья, показал на памятнике повышенную тенденцию встречаемости таких эле- ментов обряда, как кости животных, культ огня (наличие углей в засыпи ямы), наличие сосудов, бус. Предметы быта, наоборот, встречаются в незна- чительном количестве. Исследовате- лем было отмечено сходство Щукин- ского могильника с Аверинским II, Агафоновским I и II, Мелехинском не- крополями (Бочаров, 2000, с. 73–86). Несмотря на то, что Щукинский могильник неоднократно выступал в качестве объекта исследования, харак- теристика обряда дана неполно. Так, предшествующие исследователи из погребальных сооружений обратили внимание на наличие ниш-выступов, но не отметили наличие уступов и заплечиков в погребениях, хотя та- кие конструкции встречаются чаще. Отсутствует описание вещевого ма- териала конкретного памятника и их На Щукинском могильнике погре- бения расположены плотно не совсем четкими рядами, часты случаи взаи- монарушения могильных ям. Можно выделить четыре ряда, вне которых располагались пять погребений в верхней (северной) части памятника и два в юго-восточной. Последние, ве- роятно, представляют собой остатки ряда, разрушенного вследствие ополз- ня мыса. Погребения ориентированы 52 52 52 ков А.М., Умаров Моряхина К.В. на ССВ (58,5%) или на СВ (36,6%), в единичных случаях на С, ВСВ, ССЗ. Ориентировка является меридиональ- ной, погребенные уложены ногами к реке. На памятнике превалируют мо- гильные ямы среднего размера (дли- ной от 160 до 260 см и шириной от 60 до 160 см) – 64,6%, также отмечено значительное количество больших ям (их длина превышает 260 см) – 32,9%. Максимальная длина достигает 350 см. Могильные ямы заполнены темно-серым суглинком. (12%) в заполнении могильных ям встречены куски углей. В двух их них зафиксированы углистые пятна: в по- гребении 1 – размеры 68×39 см, мощ- ность слоя 4 см, в погребении 23 – 83×63 см, мощность слоя 25 см. Такие углистые пятна могли быть остатками погребальных костров. В погребениях с ингумацей также преобладает ориентировка на ССВ – 62,5%, в 28,1% – на СВ, в единичных случаях на С, ССЗ. Голдина, Кананин, 1989, с. 26–43). На 53 53 53 ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 1 (39) 2022 № 4 (42) 2022 № 2 (44) 2023 Щукинском могильнике с такой тра- дицией можно связать погребения с овальными выступами. Погребения с прямоугольными выступами имеют свои особенности. Они отличают- ся большим размером в сравнении с овальными. В одном случае размер выступа 278×60 см, на его площадке обнаружены кальцинированные ко- сти, фр. серебряной подвески и развал сосуда, в самом погребении только железный предмет. В другом случае прямоугольный выступ зафиксиро- ван у безынвентарного погребения. Возможно, прямоугольные выступы – это не специальные конструкции, как отмечено было автором раскопок, а погребения неправильной формы. За исключением безынвентарного по- гребения, захоронения были совер- шены по обряду кремации. Похожие конструкции известны по материалам других памятников Пермского Пред- уралья – Аверинского II, Агафонов- ских I и II, Запосельского, Огурдин- ского, Рождественского могильников (Моряхина, Шмуратко, 2019). роткой, на высоте от 9 до 35 см от дна. Находки располагались как выше, так и ниже уровня ступенек. Восемь из десяти погребений совершены по об- ряду кремации. Погребения с уступа- ми зафиксированы на Аверинском II, Антыбарском, Запосельском, Огур- динском, Рождественском, Телячий Брод могильниках. роткой, на высоте от 9 до 35 см от дна. Находки располагались как выше, так и ниже уровня ступенек. Восемь из десяти погребений совершены по об- ряду кремации. Погребения с уступа- ми зафиксированы на Аверинском II, Антыбарском, Запосельском, Огур- динском, Рождественском, Телячий Брод могильниках. р Н.Б. Крыласова считает, что запле- чики и уступы сооружались для того, чтобы на них установить помост, на который укладывался покойный. Сме- щение находок происходило вслед- ствие того, что прогнивал помост и они обрушивались ниже его уровня (Белавин, Крыласова, 2008, с. 96). Подтверждает это предположение наличие остатков деревянного насти- ла в одном из погребений с уступом (п. 11) на Щукинском могильнике. На рассматриваемом памятнике остатки деревянного настила зафиксированы еще в шести погребениях. Находки в этих погребениях располагались либо на одном уровне, либо выше следов настила. Соответственно, можно сде- лать вывод, что покойного (или его кремированные останки) укладывали вместе с инвентарем на деревянный настил. В четырех погребениях за- фиксированы фрагменты обугленного дерева, но не представляется возмож- ным однозначно утверждать, что это остатки настила. Заплечики (ступеньки у двух или более стенок) зафиксированы в че- тырех погребениях (4,9%). В погре- бениях ступеньки располагаются по-разному: в одном случае – у двух длинных и одной короткой стенки, в двух – у двух длинных, в одном – у одной длинной и одной короткой. Голдина, Кананин, 1989, с. 26–43). В большинстве случаев хоронили в средних (81,3%) ямах, также фиксируются захоро- нения в больших (12,5%) и малых (6,2%) ямах. Одна из малых ям, ве- роятно, детское погребение (п. 14). Могильные ямы неглубокие от 44 до 93 см. В большинстве случаев (75%) вещи располагались на дне погребе- ния, украшения в порядке ношения. В трех погребениях вещи обнаруже- ны беспорядочно на разной глубине, в пяти – выше уровня дна (в двух из них зафиксированы уступы). В трех погребениях найдены зубы человека, другие кости не сохранились, что ти- пично для могильников данного реги- она. На памятнике зафиксирована биобрядность способа захоронения (рис. 1): 61% (50 погребений) – погре- бения с кремацией (к погребениям с кремацией отнесены погребения, в ко- торых были обнаружены кальциниро- ванные кости), 39% (32 погребения) – с ингумацией. К последним отнесены безынвентарные погребения, которые составляют 4,9% (п. 17, 25, 26, 29) от общего количества. Планиграфически погребения с разным способом захо- ронения не выделяются, но наиболь- шую концентрацию погребений с кре- мацией можно выделить в западной части второго ряда. Безынвентарные погребения расположены в западной части третьего ряда. На Щукинском могильнике умер- ших чаще хоронили в простых ямах прямоугольной формы с закругленны- ми углами (рис. 1: 1, 3) – 76,8% по- гребений. В погребениях с кремацией преоб- ладает ориентировка на ССВ – 56% случаев, в 42% – на СВ, одно погре- бение ориентировано на ВСВ. Встре- чаются захоронения как в средних (54%), так и больших (46%) ямах. В основном могильные ямы неглубокие (от 38 до 97 см от поверхности) – 96%, в двух случаях (п. 22, 80) отмечена глубина 103 и 118 см, причем в этих погребениях фиксируется углубле- ние в центральной части ямы. Кости и вещи расположены беспорядочно в заполнении ямы на разной глуби- не. Вероятно, засыпались вместе с землей. Только в одном погребении (п. 79) кости и вещи были уложены на дне могильной ямы в северной и центральной части. В 6 погребениях Наличие выступов-ниш (рис. 1: 4) отмечено у семи погребений (8,5%). В шести случаях выступы располага- лись у длинной СЗ или ЮВ стенки, в одном – у короткой СВ стенки. По фор- ме выступы разделяются на овальные (п. 13, 68, 77, 81, 82) и прямоугольные (п. 9, 17). Н.Б. Крыласова по материа- лам Рождественского и Огурдинского могильников интерпретировала по- гребения с выступами как имитацию жилищ-полуземлянок со входом, что, соответственно, отражало традицию в домостроительстве средневеково- го населения Пермского Предуралья (Белавин, Крыласова, 2008, с. 96). Голдина, Кананин, 1989, с. 26–43). Ступеньки могли быть как практиче- ски на уровне дна погребения, так и на высоте 17–40 см, и в таком случае находки располагались выше и ниже их уровня. Все погребения соверше- ны по обряду кремации. Погребения с заплечиками известны по материалам Огурдинского, Рождественского, Аве- ринского II, Агафоновского I могиль- ников (Моряхина, Шмуратко, 2019). Стоит отметить, что на Щукин- ском могильнике, скорей всего, не было традиции сооружать наземные погребения. В погребениях и межмогильном пространстве зафиксированы 102 столбовые ямки. Из них 29 ямок об- наружены в 13 погребениях (15,9%), которые были в верхней, центральной или нижней части. В межмогильном пространстве ямки располагались в основном в «изголовье» или в «но- гах» погребальной ямы. Исключение составляют столбовые ямки, отделя- ющие п. 20 и 41 от основной части В погребениях и межмогильном пространстве зафиксированы 102 столбовые ямки. Из них 29 ямок об- наружены в 13 погребениях (15,9%), которые были в верхней, центральной или нижней части. В межмогильном пространстве ямки располагались в основном в «изголовье» или в «но- гах» погребальной ямы. Исключение составляют столбовые ямки, отделя- ющие п. 20 и 41 от основной части На памятнике изучено десять по- гребений (12,2%) с уступами (сту- пенька у одной из стенок). Уступы (рис. 1: 2), как правило, располагались у длинной стенки, в одном случае у ко- 54 54 54 ков А.М., Умаров Моряхина К.В. Рис. 2. Украшения Щукинского могильника. 1–4 – браслеты, 5–7, 12, 13, 25, 28–30 – подвески; 8, 9, 16, 19, 20 – пряжки; 10, 15, 17, 18, 21–23 – поясные накладки; 11, 24 – наконечники ремня; 14 – пронизка; 26–27 – височные кольца; 31 – перстень. 1 – п. 44; 2–3, 31 – п. 32; 4, 15, 17,18 – п. 14; 5, 12 – п. 40; 6 – п. 60; 7, 14 – п. 79; 8, 27, 28 – п. 30; 9 – п. 53; 10, 11 – п. 54; 13 – п. 66; 16 – п. 14; 19, 20 – п. 56; 21–23, 26 – п. 12; 24 – п. 57; 25 – п. 20; 29 – п. 16; 30 – п. 11. Fig. 2. Decorations of the Shchukin burial ground. 1–4 – bracelets; 5–7, 12, 13, 25, 28–30 – pendants; 8, 9, 16, 19, 20 – buckles; 10, 15, 17, 18, 21–23 – belt pads; 11, 24 – belt tips; 14 – thread; 26, 27 – temporal rings; 31 – ring. 1 – b. 44; 2, 3, 31 – b. Голдина, Кананин, 1989, с. 26–43). 32; 4, 15, 17, 18 – b. 14; 5, 12 – b. 40; 6 – b. 60; 7, 14 – b. 79; 8, 27, 28 – b. 30; 9 – b. 53; 10, 11 – b. 54; 13 – b. 66; 16 – b. 14; 19, 20 – b. 56; 21–23, 26 – b. 12; 24 – b. 57; 25 – b. 20; 29 – b. 16; 30 – b. 11. Рис. 2. Украшения Щукинского могильника. 1–4 – браслеты, 5–7, 12, 13, 25, 28–30 – подвески; 8, 9, 16, 19, 20 – пряжки; 10, 15, 17, 18, 21–23 – поясные накладки; 11, 24 – наконечники ремня; 14 – пронизка; 26–27 – височные кольца; 31 – перстень. 1 – п. 44; 2–3, 31 – п. 32; 4, 15, 17,18 – п. 14; 5, 12 – п. 40; 6 – п. 60; 7, 14 – п. 79; 8, 27, 28 – п. 30; 9 – п. 53; 10, 11 – п. 54; 13 – п. 66; 16 – п. 14; 19, 20 – п. 56; 21–23, 26 – п 12; 24 – п 57; 25 – п 20; 29 – п 16; 30 – п 11 ; ; ; ; Fig. 2. Decorations of the Shchukin burial ground. 1–4 – bracelets; 5–7, 12, 13, 25, 28–30 – pendants; 8, 9, 16, 19, 20 – buckles; 10, 15, 17, 18, 21–23 – belt pads; 11, 24 – belt tips; 14 – thread; 26, 27 – temporal rings; 31 – ring. 1 – b. 44; 2, 3, 31 – b. 32; 4, 15, 17, 18 – b. 14; 5, 12 – b. 40; 6 – b. 60; 7, 14 – b. 79; 8, 27, 28 – b. 30; 9 – b. 53; 10, 11 – b. 54; 13 – b. 66; 16 – b. 14; 19, 20 – b. 56; 21–23, 26 – b. 12; 24 – b. 57; 25 – b. 20; 29 – b. 16; 30 – b. 11. Fig. 2. Decorations of the Shchukin burial ground. 1–4 – bracelets; 5–7, 12, 13, 25, 28–30 – pendants; 8, 9, 16, 19, 20 – buckles; 10, 15, 17, 18, 21–23 – belt pads; 11, 24 – belt tips; 14 – thread; 26, 27 – temporal rings; 31 – ring. 1 – b. 44; 2, 3, 31 – b. 32; 4, 15, 17, 18 – b. 14; 5, 12 – b. Голдина, Кананин, 1989, с. 26–43). VIII – X вв. (Моря- хина, 2018, с. 68, 108, 111–112, 125). Височные кольца (рис. 2: 26–27) были обнаружены в восьми погребе- ниях (в 9,8% от общего количества погребений; 10 экз.), представлены двумя типами: литые проволочные без привесок и с литой бипирамидальный гроздью. Первые получили распро- странение c конца V–X вв., вторые – к первой половине IX в. (Подосенова, 2009, с. 70, 78). , , ) Подвески (рис. 2: 5–7, 12, 13, 25, 28–30) были найдены в 20 погребе- ниях (в 24,4% от общего количества погребений) в области груди и в 5 погребениях (в 6,1%) в области по- яса. К ранним вариантам украшений относятся шумящие подвески с пла- стинчатой основой (VII в.), колесо- видные ажурные подвески (VIII в.) (Голдина, Кананин, 1989, рис. 68/39, 69/16); к поздним – шумящие под- вески с арочной и стержневой осно- вой (IX в.) (Голдина, Кананин, 1989, рис. 70/16, 31), подвески-ложечки VIII–IX вв. (Крыласова, 2007, с. 59, 63). Поясная гарнитура представлена пряжками (в 20 погребениях, в 24,4% от общего количества погребений), накладками (в 17 погребениях, в 20,7%, 101 экз.), наконечниками рем- ня (в 5 погребениях, в 6,1%, 7 экз.). На памятнике обнаружено два варианта ранних пряжек VI в. (рис. 2: 19–20): трехсоставные с округлым вращаю- щимся кольцом и нависающим языч- ком и круглые рамчатые (Голдина, Кананин, 1989, рис. 67/1, 4, 8). Также были найдены пряжки, характерные для VIII в. – цельнолитые с округлой задней пластиной, крепившейся к рам- ке при помощи штифтиков (рис. 2: 16) (Голдина, Кананин, 1989, рис. 69/1) – и для IX в. – с приостренной задней пластиной, украшенной насечками, и цельнолитые восьмеркообразные (рис. 2: 8–9) (Голдина, Кананин, 1989, рис. 70/1, 8). Поясные накладки (рис. 2: 10, 15, 17, 18, 21–23) весьма разно- образны, хронологически их можно разделить на две группы: VIII в. – ка- лачевидные с разомкнутыми концами и штампованные с прорезью (встре- чаются и в более позднее время) (Гол- дина, Кананин, 1989, рис. 69/5–6, 11); IX в. – круглые с петлей на обороте, сердцевидные (Голдина, Кананин, 1989, рис. 70/26), полуовальная пла- стина с прикрепленным, свободно висящим кольцом (Белавин, Крыла- сова, 2008, с. 419), в виде полумеся- ца с двумя полушарными выступами. Встречаются пронизки, типичные для памятников Пермского Предура- лья: трубочки, спиралевидные, с взду- тиями, датирующиеся VIII–XI вв. (Бе- лавин, Крыласова, 2012, с. 143–145). Также были обнаружены две птицео- бразные пронизки (рис. 2: 14), дати- рующиеся VII в. (Голдина, Кананин, 1989, рис. 68/50). Голдина, Кананин, 1989, с. 26–43). 40; 6 – b. 60; 7, 14 – b. 79; 8, 27, 28 – b. 30; 9 – b. 53; 10, 11 – b. 54; 13 – b. 66; 16 – b. 14; 19, 20 – b. 56; 21–23, 26 – b. 12; 24 – b. 57; 25 – b. 20; 29 – b. 16; 30 – b. 11. погребений. Возможно, как это отме- чал и В.А. Кананин (Кананин, 1978, с. 40), что на могильнике могли быть какие-то надмогильные сооружения и изгороди. ки залегали на разной глубине) или размещался в верхней части засыпи могильной ямы (в погребениях с ин- гумацией сосуд находился по центру ямы). Вероятно, сосуды, обнаружен- ные в верхней части засыпи, связаны с поминальной обрядностью. В 20 погребениях (24,4%) найдены зубы лошади. В погребениях с ингу- мацией они располагались чаще в из- головье, иногда в ногах или централь- ной части. На памятнике встречаются ти- пичные для ломоватовской культу- ры украшения. Женский костюмные комплексы, по классификации Н.Б. Крыласовой, представлены шестью типами: 1а, 10а, 11а (височные кольца + нагрудные украшения + пояс), 2а, 8а (нагрудные украшения + пояс), 4а (на- грудные украшения); мужские – дву- мя типами: 9а (пояс), 13а (височные кольца + пояс) (Крыласова, 2001, с. В погребениях были обнаружены целые керамические сосуды (28,1% погребений) и фрагменты керамики (45,1% погребений). Керамические сосуды в погребениях располагались двумя способами: сосуд укладывал- ся на дно могильной ямы (в погре- бениях с кремацией при этом наход- 55 55 55 ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 1 (39) 2022 № 4 (42) 2022 № 2 (44) 2023 104–105). Классификация украшений Щукинского могильника автором ста- тьи была опубликована в отдельной работе (Моряхина, 2022). Предуралья вариантами: дротовые овальные с приостренными конца- ми, четырехгранные и шестигранные в сечении, кованые пластинчатые с уплощением на концах. Дротовые браслеты с приостренными концами в Пермском Предуралье бытовали в VIII–XI вв., остальные браслеты дати- руются IX–X вв. Перстни (рис. 2: 31) представлены одного варианта – пер- сти «салтовского» типа, которые по- лучили распространение в Пермском Предуралье в кон. VIII – X вв. (Моря- хина, 2018, с. 68, 108, 111–112, 125). Предуралья вариантами: дротовые овальные с приостренными конца- ми, четырехгранные и шестигранные в сечении, кованые пластинчатые с уплощением на концах. Дротовые браслеты с приостренными концами в Пермском Предуралье бытовали в VIII–XI вв., остальные браслеты дати- руются IX–X вв. Перстни (рис. 2: 31) представлены одного варианта – пер- сти «салтовского» типа, которые по- лучили распространение в Пермском Предуралье в кон. Голдина, Кананин, 1989, с. 26–43). В целом пронизки были обнаружены в области головы (груди) в 10 погребениях (в 12,2% от общего количества погребений), в области пояса – в 7 погребениях (в 6,1%). Всего 21 экземпляр. На памятнике обнаружены укра- шения рук: браслеты – в погребениях (в 4,9% от общего количества погре- бений; 6 экз.), перстни – в четырех (в 4,9%). Браслеты (рис. 2: 1–4) пред- ставлены типичными для Пермского 56 56 56 ков А.М., Умаров Моряхина К.В. ной формы и стременами (в 2 погребе- ниях, 2,4%). В погребении 47 обнару- жены восьмеркообразные стремена, датирующиеся IX в. (Голдина, Кана- нин, 1989, рис. 70/38), в погребении 80 сохранилась лишь нижняя часть вогнутой формы. В погребениях по обряду ингумации стремена распола- гались в ногах. ной формы и стременами (в 2 погребе- ниях, 2,4%). В погребении 47 обнару- жены восьмеркообразные стремена, датирующиеся IX в. (Голдина, Кана- нин, 1989, рис. 70/38), в погребении 80 сохранилась лишь нижняя часть вогнутой формы. В погребениях по обряду ингумации стремена распола- гались в ногах. Наконечники ремня (рис. 2: 11, 24) представлены тремя типами: в виде пластины, зажимающей ремень с двух сторон, в виде продолговатой пласти- ны, в виде мечеобразной пластины. Находки бус (в 37 погребениях, 45,1% от общего количества) пред- ставлены стеклянными и каменными изделиями. В наибольшем количестве встречаются на памятнике посере- бренные и синие зонные многочаст- ные бусы, также есть одночастные зонные, глазчатые, ребристые, сердо- ликовые бусы. На памятнике встречаются уни- версальные колонувидные топоры (в 3 погребениях, 3,7% от общего коли- чества), предназначенные для рубки леса и раскалывания бревен. Такие топоры имеют широкую датировку – V–XI вв. (Данич, 2015, с. 75–76). В погребениях по обряду ингумации они обнаружены в центральной части. В погребении 79 обнаружен пред- мет пермского звериного стиля – фи- гурка человека-лося. Личина располо- жена в центральной части плакетки. Размер изделия 3×1,7 см. Датирует- ся VIII в. (Голдина, Кананин, 1989, рис. 69/27). ру р Орудия труда на памятнике немно- гочисленны. К универсальным оруди- ям можно отнести ножи (в 30 погре- бениях, 36,6% от общего количества), шило (в 3 погребениях, 3,7%), оселок (в 1 погребении, 1,2% от общего ко- личества), пряслице (в 1 погребении, 1,2%). Примечательно, что пряслице обнаружено в погребении с вооруже- нием. В одном погребении найдено кресало в виде дрота с расплющенной рабочей частью и кремень. По мне- нию Н.Б. Крыласовой, такие кресала датируются кон. VII – VIII вв. (Крыла- сова, 2007а, рис. 1). Голдина, Кананин, 1989, с. 26–43). В погребениях по обряду ингумации ножи обнаружены в центральной части или в изголовье, шило – в ногах. Вооружение представлено же- лезными наконечниками стрел (в 11 погребениях, 13,4% от общего коли- чества) и наконечниками копий (в 7 погребениях, 8,5%), которые рас- полагались в погребениях по обряду ингумации в центральной части или в ногах. Наконечники стрел обна- ружены трех типов: листовидные с расширением в нижней части (VII– IX вв.) (Данич, 2011, с. 104), ромбиче- ские с расширением в средней части и шиловидные ромбического сечения (VII–VIII вв.) (Голдина, 1985, рис. 16). Наконечники стрел в основном встречаются в виде четырехгранного стержня (VIII–XII вв.), также в одном экземпляре обнаружены ромбовидные втульчатые (IX – нач. XI вв.) и ланце- товидные (IX–X вв.) (Данич, 2010, с. 21, 22, 24). Кольчуга (в 5 погребе- ниях, 6,1%) сохранилась в виде ма- леньких фрагментов. В одном случае кольчуга обнаружена в погребении с вооружением (наконечник стрелы). Снаряжение коня представлено под- пружными пряжками (в 11 погребе- ниях, 13,4%) овальной (VII–VIII вв.) (Голдина, 1985, рис. 16) и прямоуголь- На памятнике встречается дерево- обрабатывающий инструментарий: наструги (скорбели) П-образной фор- мы (в 2 погребениях, 2,4%) и ложкарь (в 1 погребении, 1,2%). Такие орудия имеют широкую датировку (Смертин, 2021, с. 95–96). В погребении по об- ряду ингумации скобель располагался в центральной части. Стоит отметить, что орудия труда (не считая ножей) встречаются в по- гребениях с вооружением, исключе- ние составляет погребение 70, в кото- ром были скобель, оселок, кресало и 57 57 57 ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 1 (39) 2022 № 4 (42) 2022 № 2 (44) 2023 Рис. 3. Хронология Щукинского могильника (план по: Голдина, Кананин, 1989, рис. 26). Fig. 3. Chronology of the Shchukin burial ground (plan after: Goldina, Kananin, 1989, ﬁ g. 26). Рис. 3. Хронология Щукинского могильника (план по: Голдина, Кананин, 1989, рис. 26). Рис. 3. Хронология Щукинского могильника (план по: Голдина, Кананин, 1989, рис. 26). ( р ) Fig. 3. Chronology of the Shchukin burial ground (plan after: Goldina, Kananin, 1989, ﬁ g. 26). нож. В четырех погребениях (п. 3, 1 Fig. 3. Chronology of the Shchukin burial ground (plan after: Goldina, Kananin, 1989, ﬁ g. 26). В четырех погребениях (п. 3, 14, 40, 71) были завернуты в бересту или мех украшения (пояс, браслет, подве- ска). Вероятно, эти украшения были выделены из погребального набора и представляли собой особый дар умер- шему в загробный мир. нож. В межмогильном пространстве было обнаружено еще одно орудие труда – ювелирный молоточек разме- рами 10×3 см. В погребении 42 была обнаружена глиняная фигурка в виде головы жи- вотного, возможно имевшая сакраль- ное значение. В погребении 32 погребальные вещи (украшения, оружие, нож) рас- полагались на деревянном настиле и сверху были закрыты шкурой живот- ного, что могло имитировать завора- чивание. Погребение совершено по обряду кремации. В погребении 32 погребальные вещи (украшения, оружие, нож) рас- полагались на деревянном настиле и сверху были закрыты шкурой живот- ного, что могло имитировать завора- чивание. Погребение совершено по обряду кремации. На Щукинском могильнике сохра- нились фрагменты одежды. В погре- бении 19 обнаружен небольшой кусок ткани, в погребении 67 (выполнено по обряду кремации) фиксируются на кольчуге следы истлевшей ткани. В погребениях 11, 20, 56 и 63 (выпол- нены по обряду кремации) найдены небольшие куски кожи и меха. Нали- чие ткани, кожи и меха в погребени- ях с кремацией указывает на то, что в погребения укладывались не только украшения, оружие или орудия труда, но и одежда умершего. На основе анализа погребально- го инвентаря можно выделить пять хронологических групп погребений (рис. 3): VI в. – п. 56, 80. Данная группа была выделена на основе поясных пряжек, других материалов VI в. не обнаружено. Погребения располага- ются в разных частях могильника. В 58 58 58 ков А.М., Умаров Моряхина К.В. п. 80 обнаружены фрагмент стремени, при этом на могильниках, относящих- ся к харинской культуре (IV–VI вв.), стремена не встречаются (Шмуратко, 2012, электронное приложение). Воз- можно, ранние пряжки попали в более поздние погребения, что могло быть следствием разграбления могильни- ков в Средневековье. Соответственно, можно сократить датировку памятни- ка – VII–IX вв. Преобладают захоронения по обряду кремации (75%). В одном случае в заполнении ямы обнаружены угли и кости животных. Погребальный ин- вентарь представлен керамикой, укра- шениями (пояс, подвески, пронизки, бусы, височные кольца, браслет, пер- стень), наконечником стрелы, нако- нечником копья, топором, кольчугой, ножами, ложкарем. , р IX в. – 1, 2, 3, 4, 10, 12, 13, 16, 23, 24, 30, 31, 32, 36, 38, 47, 52, 53, 54, 71. Погребения ориентированы ССВ и СВ. Захоронения сделаны в ямах среднего и большого размера. Имеют- ся конструктивные особенности ямы в виде ниши, заплечиков и уступов. В четырех погребениях сохранились остатки деревянной погребальной конструкции. Преобладают захоро- нения по обряду кремации (65%). В заполнении ямы встречаются угли и кости животных. нож. По другим могиль- никам отмечается тенденция умень- шения размеров погребальных ям в позднеломоватовское время (Голдина. 1985, рис. 16). В поздних погребени- ях (VIII–IX вв.) фиксируются такие конструктивные особенности ям, как ниши, заплечики, уступы. Чаще всего такие конструктивные особенности отмечены в погребениях с кремацией. Предположительно, количество за- хоронений по обряду кремации уве- личилось в более позднее время, что опять же идет в разрез с общей ха- рактеристикой позднеломоватовских памятников, где преобладало трупо- положение (Голдина. 1985, с. 131). Оружие и орудия труда встречаются как в ранних, так и поздних погребе- ниях. Как правило, они обнаружены в одних и тех же погребениях, за ис- ключением одного случая, что гово- рит об отсутствии разделения труда, т. е. воины занимались ремеслом. Ору- дия труда представлены в основном универсальными орудиями (ножи, шила) и деревообрабатывающим ин- струментарием. Набор украшений со временем увеличивается и становится наиболее полным и разнообразным, что заметно по материалам IX в. Ранее уже исследователи пи- сали о схожести Щукинского мо- гильника с другими памятниками в верховьях Камы – Аверинским II, Агафоновским I и II – на основе раз- меров ям, биобрядности, наличия костей животных и углей в запол- нении ям (Голдина, Кананин, 1989, с. 28–43; Бочаров, 2000, с. 106–107). При этом эти памятники существенно отличаются от других одновремен- ных, расположенных немного ниже по течению Камы – Плесинсклого, Каневского, Урьинского, Деменков- ского (примечание: к Щукинскому могильнику Плесинский расположен ближе, чем Агафоновский I и II). Раз- личия заметны не по вещевому ма- териалу, а по погребальному обряду. Для перечисленных памятников ха- рактерны ямы среднего размера пря- моугольной формы без конструктив- ных особенностей, захоронения по обряду ингумации, в заполнении ям, как правило, отсутствуют угли и ко- сти животных (Моряхина, 2020). Вы- явить отличия в погребальном обряде на позднеломоватовских памятниках (а большинство погребений Щукин- ского могильника относится к этому периоду) стало возможным благода- ря уточнению датировки памятника и выделению хронологических групп. Стоит предполагать, что различия в погребальном обряде на памятниках в верхнем течении Камы указывают на наличие двух культурных традиций (разных этносов?), существовавших одновременно. нож. Погребальный ин- вентарь представлен керамикой, укра- шениями (пояс, подвески, пронизки, бусы, височные кольца, браслеты, перстни), наконечником стрелы, на- конечниками копий, топором, кольчу- гой, универсальным и деревообраба- тывающим инструментарием. VII–VIII вв. – п. 5, 8, 40, 44, 60, 67, 70, 74. Погребения ориентированы на ССВ. Захоронения сделаны в ямах среднего и большого размера. По- гребальные ямы без конструктивных особенностей. В двух погребениях сохранились остатки деревянной по- гребальной конструкции. В равном количестве выполнены захоронения по обряду ингумации и кремации. В единичных случаях в заполнении ямы обнаружены угли и кости животных. Погребальный инвентарь представ- лен керамикой, украшениями (пояс, подвески, пронизки, бусы, браслет), наконечниками стрел, кольчугой, кре- салом, универсальным и деревообра- батывающим инструментарием. VIII в. – п. 57, 63, 79. Погребения ориентированы на ССВ. Захоронения сделаны в ямах среднего размера. По- гребальные ямы без конструктивных особенностей. Захоронения выполне- ны по обряду ингумации и кремации. В одном случае в заполнении ямы обнаружены угли и кости животных. Погребальный инвентарь представ- лен керамикой, украшениями (пояс, подвески, пронизки, бусы), наконеч- ником стрелы, наконечником копья, ножами. В межмогильном пространстве за- фиксировано 10 погребальных ям: во- семь – овальной формы (средние раз- меры 33×26) и две – подквардратной (средние размеры 52×52). В заполне- нии ям встречаются вкрапления угля и прокаленной глины. Только в одной яме обнаружены вещи – развал сосуда и железный предмет. Помимо этого, в межмогильном пространстве обнару- жены два керамических сосуда между погребениями 74 и 77, рядом с погре- бением 80 – сверток с вещами. VIII–IX вв. – п. 11, 14, 37, 42, 49, 65, 66, 68, 76. Погребения ориентиро- ваны ССВ и СВ в равном количестве. Захоронения сделаны в ямах малого и среднего размера. Имеются конструк- тивные особенности ямы в виде ниши и уступов. В трех погребениях сохра- нились остатки деревянного настила. Выводы. Датировку Щукинско- го могильника, на взгляд автора ста- тьи, стоит сузить и датировать VII– IX вв. Планиграфически (рис. 3) ран- няя часть выделяется в центральной и юго-восточной части могильника. Поздние погребениях преобладают 59 59 59 ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 1 (39) 2022 № 4 (42) 2022 № 2 (44) 2023 в северо-западной части могильни- ка, также есть в центральной и се- веро-восточной части. В поздних погребениях отмечается тенденция небольшого смещения ориентиров- ки могильных ям с ССВ на СВ. Ямы большого размера встречаются как в ранней (VII в.), так и поздней (IX в.) части могильника, при этом харак- терны больше для захоронений по обряду кремации. ЛИТЕРАТУРА 1. Белавин А.М., Крыласова Н.Б. Древняя Афкула: археологический комплекс у с. Рож- дественск. Пермь: Перм. гос. пед. ун-т., 2008. 603 с. 2. Белавин А.М., Крыласова Н.Б. Огурдинский могильник. Пермь: ПГГПУ, 2012. 259 с. 3. Бочаров И.В. Средневековый погребальный обряд Верхнего Прикамья как источник реконструкции этнической истории региона: опыт статистического анализа. Дисс. … канд. ист. наук. Уфа, 2000. 235 с. 4. Голдина Р.Д. Ломоватовская культура в Верхнем Прикамье. Иркутск: Изд-во Иркут. ун-та, 1985. 280 с. 5. Голдина Р.Д., Кананин В.А. Средневековые памятники верховьев Камы. Свердловск: Уральский гос. ун-т, 1989. 216 с. у , 5. Голдина Р.Д., Кананин В.А. Средневековые памятники верховьев Камы. Свердловск: Уральский гос. ун-т, 1989. 216 с. ЛИТЕРАТУРА Костюм средневекового населения Пермского Предуралья Пермь: ПГПУ 2001 260 с В.Е. Владыкин. Ижевск: Удмуртский университет, 1983. С. 80 95. 11. Крыласова Н.Б. История прикамского костюма. Костюм средневекового населения Пермского Предуралья. Пермь: ПГПУ, 2001. 260 с. д д ур у р , 11. Крыласова Н.Б. История прикамского костюма. Костюм средневекового населения Пермского Предуралья. Пермь: ПГПУ, 2001. 260 с. р р ур р , 12. Крыласова Н.Б. Археология повседневности: материальная культура средневеково- го Предуралья. Пермь: ПГГПУ, 2007. 352 с. р р ур р 12. Крыласова Н.Б. Археология повседневности: материальная культура средневеково- го Предуралья. Пермь: ПГГПУ, 2007. 352 с. р ур р , 13. Крыласова Н.Б. 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Моряхина К.В., Шмуратко Д.В. Погребальный обряд средневековых могильни- ков Пермского Предуралья (VII–ХV вв.). Свидетельство о регистрации базы данных RU 2019622464 от 23.12.2019. 17. Моряхина К.В., Шмуратко Д.В. Погребальный обряд средневековых могильни- ков Пермского Предуралья (VII–ХV вв.). Свидетельство о регистрации базы данных RU 2019622464 от 23.12.2019. 18. Подосенова Ю.А. Височные украшения населения Пермского Предуралья в эпоху средневековья. Дисс… канд. ист. наук. Пермь, 2009. 272 с. 18. Подосенова Ю.А. Височные украшения населения Пермского Предуралья в эпоху средневековья. Дисс… канд. ист. наук. Пермь, 2009. 272 с. 19. Смертин А.Р. Деревообрабатывающий инструментарий Пермского Предуралья в эпоху Средневековья // Труды КАЭЭ. Вып. 19 / Под ред. А.М. Белавина. Пермь: ПГГПУ, 2021. С. 92–102. 19. Смертин А.Р. Деревообрабатывающий инструментарий Пермского Предуралья в эпоху Средневековья // Труды КАЭЭ. Вып. 19 / Под ред. А.М. Белавина. Пермь: ПГГПУ, 2021. С. 92–102. Информация об авторе: Информация об авторе: Моряхина Кристина Викторовна, кандидат исторических наук, доцент кафедры отечественной и всеобщей истории, археологии, Пермский государственный гумани- тарно-педагогический университет (г. Пермь, Россия); kmoryaxina@mail.ru OF THE LOMOVATOVO ARCHAEOLOGICAL CULTURE K.V. Moryakhina ЛИТЕРАТУРА 1. Белавин А.М., Крыласова Н.Б. Древняя Афкула: археологический комплекс у с. Рож дественск. Пермь: Перм. гос. пед. ун-т., 2008. 603 с. 2. Белавин А.М., Крыласова Н.Б. Огурдинский могильник. Пермь: ПГГПУ, 2012. 259 с. 3. Бочаров И.В. Средневековый погребальный обряд Верхнего Прикамья как источник реконструкции этнической истории региона: опыт статистического анализа. Дисс. … канд. ист. наук. Уфа, 2000. 235 с. у ф , 4. Голдина Р.Д. Ломоватовская культура в Верхнем Прикамье. Иркутск: Изд-во Ирку ун-та, 1985. 280 с. 5. Голдина Р.Д., Кананин В.А. Средневековые памятники верховьев Камы. Свердловск: Уральский гос. ун-т, 1989. 216 с. 60 60 60 ков А.М., Умаров Моряхина К.В. 6. Данич А.В. Наконечники копий на территории Пермского Приуралья // Вест- ник Музея археологии и этнографии Пермского Предуралья. Вып. 3/ Отв. ред. А.М. Белавин. Пермь: ПГПУ, 2010. С. 20–43. 6. Данич А.В. Наконечники копий на территории Пермского Приуралья // Вест- ник Музея археологии и этнографии Пермского Предуралья. Вып. 3/ Отв. ред. А.М. Белавин. Пермь: ПГПУ, 2010. С. 20–43. р , 7. Данич А.В. Типология бронебойных наконечников стрел Пермского Предуралья // Труды КАЭЭ. Вып. 7 / Под ред. А.М. Белавина. Пермь: ПГГПУ, 2011. С. 98–115. р 7. Данич А.В. Типология бронебойных наконечников стрел Пермского Предуралья // Труды КАЭЭ. Вып. 7 / Под ред. А.М. Белавина. Пермь: ПГГПУ, 2011. С. 98–115. ру р р 8. Данич А.В. Классификация средневековых топоров Пермского Предуралья // Труды АЭЭ. Вып. 10 / Под ред. А.М. Белавина. Пермь: ПГГПУ, 2015 С. 71–124. КАЭЭ. Вып. 10 / Под ред. А.М. Белавина. Пермь: ПГГПУ, 2015 С. 71–124. 9. Кананин В.А. Отчет об исследованиях в Афанасьевском районе Кировской области, проведенных летом 1977 года. 1978 // Архив ИА РАН. Р-1. Д. 6650. КАЭЭ. Вып. 10 / Под ред. А.М. Белавина. Пермь: ПГГПУ, 2015 С. 71 124. 9. Кананин В.А. Отчет об исследованиях в Афанасьевском районе Кировской области, проведенных летом 1977 года. 1978 // Архив ИА РАН. Р-1. Д. 6650. б б р р Д 10. Кананин В.А. Погребальный обряд средневековых могильников верховьев р. Камы // Этнические процессы на Урале и в Сибири в первобытную эпоху / Отв. ред. В.Е. Владыкин. Ижевск: Удмуртский университет, 1983. С. 80–95. р р Д 10. Кананин В.А. Погребальный обряд средневековых могильников верховьев р. Камы // Этнические процессы на Урале и в Сибири в первобытную эпоху / Отв. ред. В.Е. Владыкин. Ижевск: Удмуртский университет, 1983. С. 80–95. В.Е. Владыкин. Ижевск: Удмуртский университет, 1983. С. 80–95. 11. Крыласова Н.Б. История прикамского костюма. REFERENCES (ed.) Trudy Kamskoi arkheologo-etnograﬁ cheskoi ekspeditsii (Proceedings of the Kama Archaeological and Ethnographical Expedition) 21. Perm: Perm State Humanitarian Pedagogical University, 69–79 (in Russian). g g y ( ) 17. Moryakhina, K. V., Shmuratko, D. V. 2019. Pogrebal'nyy obryad srednevekovykh mogil'nikov Permskogo Predural'ya (VII–XV vv.). Svidetel'stvo o registratsii bazy dannykh RU 2019622464 ot 23.12.2019 (Burial rite of medieval burial grounds of the Perm Fore-Urals (7th–15th centuries). Certiﬁ cate of registration of the database RU 2019622464 dated 23.12.2019) (in Russian). g g y ( ) 17. Moryakhina, K. V., Shmuratko, D. V. 2019. Pogrebal'nyy obryad srednevekovykh mogil'nikov Permskogo Predural'ya (VII–XV vv.). Svidetel'stvo o registratsii bazy dannykh RU 2019622464 ot 23.12.2019 (Burial rite of medieval burial grounds of the Perm Fore-Urals (7th–15th centuries). Certiﬁ cate of registration of the database RU 2019622464 dated 23.12.2019) (in Russian). ﬁ f g f ) ( ) 18. Podosenova, Yu. A. 2009. Visochnye ukrasheniia naseleniia Permskogo Predural’ia v epokhu srednevekov’ia (Temple Ornaments of the Perm Cis-Urals Population in the Middle Ages). PhD Diss. Perm (in Russian). ﬁ f g f ) ( ) 18. Podosenova, Yu. A. 2009. Visochnye ukrasheniia naseleniia Permskogo Predural’ia v epokhu srednevekov’ia (Temple Ornaments of the Perm Cis-Urals Population in the Middle Ages). PhD Diss. Perm (in Russian). ( ) 19. Smertin, A. R. 2021. In Belavin, A. M. (ed.) Trudy Kamskoi arkheologo-etnograﬁ cheskoi ekspeditsii (Proceedings of the Kama Archaeological and Ethnographical Expedition) 19. Perm: Perm State Humanitarian Pedagogical University, 92–102 (in Russian). ( ) 19. Smertin, A. R. 2021. In Belavin, A. M. (ed.) Trudy Kamskoi arkheologo-etnograﬁ cheskoi ekspeditsii (Proceedings of the Kama Archaeological and Ethnographical Expedition) 19. Perm: Perm State Humanitarian Pedagogical University, 92–102 (in Russian). ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 1 (39) 2022 № 4 (42) 2022 № 2 (44) 2023 grounds of the Upper Kama region – Pleso, Demenkovsky, Kaneva, Urya. ounds of the Upper Kama region – Pleso, Demenkovsky, Kaneva, Urya. pp g y y Keywords: archaeology, Upper Kama, burial rite, bi-rite, burial structures, grave goods, Middle Ages, chronology. Keywords: archaeology, Upper Kama, burial rite, bi-rite, burial structures, grave goods, Middle Ages, chronology. K.V. Moryakhina The Shchukino burial ground is located 300 m west of the village of Shchukino, Afanasyevsky district, Kirov region, on a small cape 14 m high on the right bank of the river. Kama. The site was discovered and studied by V.A. Kananin in 1977. A total of 82 burials were studied. The Shchukino burial ground is of interest due to the variety of types of burial rites. At the site, the burials were made according to the rite of cremation and inhumation. There are grave pits without structural features and there are pits with niches, shoulders and ledges. Shoulders and ledges are recorded in burials of the 9th century. In some of them, fragments of wooden ﬂ ooring have been preserved; probably, the buried person was laid on the ﬂ oor and covered with earth from above. Based on the analysis of grave goods, the author of the article singled out 5 chronological groups. The presence on the monument of two burials of the 6th century was refuted. Early materials are represented only by belt buckles. The dating of the burial ground should be narrowed down and the time of its functioning of the 7th –9th centuries should be indicated. Late burials survived in the greatest number (the monument was destroyed by natural factors). The author of the article made an assumption that two different cultural traditions existed in the region at the same time based on the clariﬁ cation of the dating of the monument and the allocation of chronological groups. The burial rite of the Shchukino burial ground is similar to the rite at Averiny II, Agafonova I and II. The burial rite of the Shchukino burial ground differs in a number of ways (the design of the pits, the method of burial, ﬁ lling the layer of pits, etc.) from the synchronous burial 61 61 61 REFERENCES Etnicheskie protsessy na Urale i v Sibiri v per- vobytnuiu epokhu (Ethnic Processes in the Urals and Siberia in the Prehistoric Era). Izhevsk: Udmurt State University, 80–95 (in Russian). ( ) 10. Kananin, V. A. 1983. In Vladykin, V. E. (ed.). Etnicheskie protsessy na Urale i v Sibiri v per- vobytnuiu epokhu (Ethnic Processes in the Urals and Siberia in the Prehistoric Era). Izhevsk: Udmurt State University, 80–95 (in Russian). y, ( ) 11. Krylasova, N. B. 2001. 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Trudy Kamskoi arkheologo-etnograﬁ cheskoi ekspeditsii (Proceedings of the Kama Archaeological and Ethnographical Expedition) 21. Perm: Perm State Humanitarian Pedagogical University, 69–79 (in Russian). 16. Moryakhina, K. V. 2022. In Belavin, A. M. REFERENCES 1. Belavin, A. M., Krylasova, N. B. 2008. Drevniaia Afkula: arkheologicheskii kompleks u s. Rozhdestvensk (Ancient Afkula: the Archaeological Complex near the Rozhdestvensk Village). Perm: Perm State Pedagogical University (in Russian). g g y ( ) 2. Belavin, A. M., Krylasova, N. B. 2012. Ogurdinskii mogil'nik (Ogurdino Burial Ground). Perm: Perm State Humanitarian Pedagogical University (in Russian). 3. Bocharov, I. V. 2000. Srednevekovyy pogrebal'nyy obryad Verkhnego Prikam'ya kak istochnik rekonstruktsii etnicheskoy istorii regiona: opyt statisticheskogo analiza (Medieval burial rite in the Upper Kama basin as a source of reconstruction of the ethnic history of the region: the experience of statistical analysis). PhD Diss. 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Trudy Kamskoi arkheologo-etnograﬁ cheskoi ekspeditsii (Proceedings of the Kama Archaeological and Ethnographical Expedition) 7. Perm: Perm State Humanitarian Pedagogical University, 98–115 (in Russian). g g y ( ) 8. Danich, A. V. 2015. In Belavin, A. M. (ed.) Trudy Kamskoi arkheologo-etnograﬁ cheskoi ekspeditsii (Proceedings of the Kama Archaeological and Ethnographical Expedition) 10. Perm: Perm State Humanitarian Pedagogical University, 71–124 (in Russian). 9. Kananin, V. A. 1978. Otchet ob issledovaniyakh v Afanas'evskom rayone Kirovskoy oblasti, provedennykh letom 1977 goda (Report on studies in the Afanasyevo district of the Kirov region,held in the summer of 1977). Archive of the Institute of Archaeology of the Russian Academy of Sciences, F. 1. R. 1. no. 6650 (in Russian). ( ) 10. Kananin, V. A. 1983. In Vladykin, V. E. (ed.). About the Authors: Moryakhina Kristina V. Candidate of Historical Sciences, Associated Professor. Perm State Humanitarian Pedagogical University. Sibirskaya St., 24, Perm, 614000, Russian Federation; kmoryaxina@mail.ru Moryakhina Kristina V. Candidate of Historical Sciences, Associated Professor. Perm State Humanitarian Pedagogical University. Sibirskaya St., 24, Perm, 614000, Russian Federation; kmoryaxina@mail.ru Статья принята в номер 01.06.2023 г. 62 62 62
https://openalex.org/W4388499515	https://ibn.e-journal.id/index.php/KOMPUTASI/article/download/701/515	Malay	null	Aplikasi Pengelolaan dan Pemesanan Pada Pangkalan Gas LPG 3KG Desa Purun Timur Berbasis Android	Jurnal Esensi Infokom : Jurnal Esensi Sistem Informasi dan Sistem Komputer/Jurnal Esensi Infokom	2,023	cc-by	3,238	Aplikasi Pengelolaan dan Pemesanan pada Pangkalan Gas LPG 3KG Desa Purun Timur Berbasis Android 1,3 Fakultas Ilmu Komputer, Jurusan Sistem Informasi, Universitas Prabumulih Jl. Patra No.59, Sukaraja, Kec. Prabumulih, Sumatera Selatan 31111, Indonesia. 1 anggilembayu7@gmail.com*; 2 iwanhen2@gmail.com; 3 yeniyuliana@gmail.com Intisari— Berdasarkan pengamatan yang telah dilakukan pada Pangkalan Gas LPG 3KG Desa Purun Timur ini ditemukan permasalahan mengenai proses pengelolaan dan pemesanan yang mana Pengelolaan data gas yang ada selama ini hanya menggunakan cara sederhana yaitu dengan mencatat di sebuah buku besar mengenai pemesanan gas yang dipesan oleh pengecer dan konfirmasi kembali ke pelanggan melalui telepon atau melalui whatsapp bahwa pesanan sudah dicatat. Pemesanan gas LPG 3KG pemilik pangkalan hanya menginformasikan ketersediaan stok gas di jam-jam tertentu saja sehingga pengecer tidak mengetahui ketersediaan stok gas secara berkala. Hal ini tentu kurang efektif dan efisien dilihat dari segi waktu dan tenaga. Oleh karena itu, dibutuhkan sebuah aplikasi pengelolaan dan pemesanan gas LPG 3KG online yang dapat melakukan pemesanan dan memberi informasi ketersediaan gas LPG 3KG. Metode penelitian yang digunakan adalah metode deskriptif kualitatif, sedangkan untuk metode pengembangan sistemnya menggunakan metode RAD (Rapid application development). Kata Kunci: Aplikasi, Pengelolaan, Gas, Android, LPG Abstract— Based on observations that have been made at the Purun Timur Village 3KG LPG Gas Base, problems were found regarding the management and ordering process where the management of existing gas data has only used a simple method, namely by recording in a ledger regarding gas orders ordered by those who were purchased and Confirm back to the customer by telephone or via WhatsApp that the order has been recorded. Ordering 3KG LPG gas, the owner of the base only has 9 gas stocks available at certain hours, so he doesn't know the availability of gas stocks on a regular basis. This is certainly less effective and efficient in terms of time and effort. Therefore, an online 3KG LPG gas management and ordering application is needed that can place orders and provide information on the availability of 3KG LPG gas. The research method used is a qualitative descriptive method, while the system development method uses the RAD (Rapid application development) method. Keywords: Application, Management, Gas, Android, LPG dimanfaatkan sebagai pendukung mulai dari perusahaan atau bidang bisnis kecil untuk mengembangkan usahanya (Irawan 2016). Pangkalan Gas LPG 3KG Abdul Mutolip di Desa Purun Timur adalah suatu usaha bisnis yang menjual gas LPG 3KG kepada pengecer, yang terletak di Dusun 1 Desa Purun Timur Kecamatan Penukal, Kabupaten Penukal Abab Lematang Ilir (PALI). Aplikasi Pengelolaan dan Pemesanan pada Pangkalan Gas LPG 3KG Desa Purun Timur Berbasis Android Berdasarkan hasil wawancara antara peneliti dengan pemilik pangkalan yaitu bapak Abdul Mutolip. Pengelolaan data gas yang ada selama ini hanya menggunakan cara sederhana yaitu dengan mencatat di sebuah buku besar mengenai pemesanan gas yang dipesan oleh pengecer dan konfirmasi kembali oleh pemilik pangkalan melalui telepon atau melalui whatsapp bahwa pesanan sudah dicatat. Lalu pemilik pangkalan harus mencatat kembali data stok pemesanan gas yang masih tersedia. Pemesanan gas LPG 3KG pemilik pangkalan hanya menginformasikan ketersediaan stok gas di jam-jam tertentu saja sehingga pengecer tidak mengetahui ketersediaan stok gas secara berkala. Sebab itu beberapa pengecer yang ingin melakukan permintaan pemesanan gas harus mendatangi tempat Jurnal Esensi Infokom Vol 7 No. 2 Oktober 2023 Jurnal Esensi Infokom Vol 7 No. 2 Oktober 2023 I. PENDAHULUAN Perkembangan teknologi saat ini terus mengalami kemajuan terutama teknologi mobile berbasis Android yang membuat beberapa perusahaan melakukan inovasi dalam rangka memajukan perusahaannya. Salah satunya adalah dengan membuat suatu aplikasi mobile yang dapat memberikan kemudahan pelayanan bagi pelanggannya. Android merupakan sebuah sistem operasi yang berjalan pada perangkat mobile. Android memiliki lisensi open source yang membuat teknologi ini mendapat dukungan dari berbagai teknologi lainnya. Hal ini yang membuat perangkat mobile berbasis Android diminati oleh masyarakat, karena interface yang mudah dipahami dan tersedianya berbagai aplikasi yang memudahkan kehidupan sehari-hari. Perangkat mobile ini disebut dengan smartphone (Murtiwiyati dan Sobirin : 2020). Smartphone adalah perangkat yang tidak hanya sekedar digunakan untuk melakukan sms, menerima dan menjawab panggilan saja, hadirnya pusat aplikasi pada setiap ponsel pintar, maka ponsel cerdas Smartphone kini dapat 40 Jurnal Esensi Infokom Vol 7 No. 2 Oktober 2023 Jurnal Esensi Infokom Vol 7 No. 2 Oktober 2023 pangkalan atau dengan melalui telepon atau melalui whatsapp untuk menanyakan ketersediaan stok, ini membuat pemilik pangkalan gas LPG 3KG harus menjawab kembali satu per satu mengenai ketersediaan gas LPG 3KG yang ingin dipesan oleh pengecer. Hal ini tentu kurang efektif dan efisien dilihat dari segi waktu dan tenaga. Observasi Kegiatan ini dilakukan untuk mengumpulkan data dengan cara pengamatan secara langsung pada Pangkalan Gas LPG 3KG Desa Purun Timur, dan hal-hal yang berkaitan dengan permasalahan yang diteliti. Wawancara dilakukan untuk memperoleh data yang lebih akurat, agar dapat menganalisa sistem yang sedang berjalan, dengan cara melakukan wawancara bersama dengan narasumber sekaligus pemilik Pangkalan Gas LPG 3KG Desa Purun Timur. Oleh karena itu, dibutuhkan sebuah aplikasi pengelolaan dan pemesanan gas LPG 3KG online yang dapat melakukan pemesanan dan memberi informasi ketersediaan gas LPG 3KG secara berkala kepada pengecer tanpa harus menelepon, kirim pesan, dan datang ke pangkalan gas LPG 3KG sehingga memungkinkan untuk mempermudah kinerja pangkalan dalam pengelolaan data gas LPG 3KG dan pemesanan serta memudahkan pengecer dalam mendapatkan informasi ketersediaan stok gas. Studi Pustaka Studi pustaka merupakan teknik pengumpulan data yang berasal dari buku, e-book, jurnal, serta penelitian terdahulu yang berkaitan dengan penelitian yang dilakukan, baik itu objek, maupun metode yang digunakan. Metode Pengembangan Sistem Pada metode pengembangan sistem penulis menggunakan metode Rapid Application Development (RAD). Menurut Subianto (2020:47), rapid application development (RAD) adalah proses model perangkat lunak inkremental yang menekankan siklus pengembangan yang singkat. Model RAD adalah sebuah adaptasi “kecepatan tinggi” dari model waterfall, di mana perkembangan pesat dicapai dengan menggunakan pendekatan konstruksi berbasis komponen. Pengertian Aplikasi Menurut Setyawan dan Munari yang dikutip dalam Jurnal (2020), aplikasi merupakan suatu subkelas perangkat lunak computer yang memanfaatkan kemampuan komputer langsung untuk melakukan suatu tugas yang diinginkan pengguna. Pengertian Android Menurut Lala Safitri dan Sucipto Basuki yang dikutip dalam Jurnal (2020), android adalah sebuah sistem operasi perangkat mobile berbasis linux yang mencakup sistem operasi, middleware, dan aplikasi. c. Implementation (Implementasi) Pengertian Pengelolaan Menurut Suwa yang dikutip dalam Jurnal (2021:1), pengelolaan sebagai fungsi manajemen yang meliputi perencanaaan, pengorganisasian dan pengontrolan untuk mencapai efisiensi pekerjaan. a. Requirements Planning (Perencanaan Syarat-Syarat) Dalam fase ini, pengguna dan penganalisis bertemu untuk mengidentifikasikan tujuan - tujuan aplikasi atau sistem serta untuk megidentifikasikan syarat-syarat informasi yang ditimbulkan dari tujuan - tujuan tersebut. Orientasi dalam fase ini adalah menyelesaikan masalah - masalah perusahaan. Meskipun teknologi informasi dan sistem bisa mengarahkan sebagian dari sistem yang diajukan, fokusnya akan selalu tetap pada upaya pencapaian tujuan-tujuan perusahaan. b k h ( k h ) Pengertian Pemesanan Menurut Gouzali yang dikutip oleh Susanti dan Prabowo dalam Jurnal (2020), pemesanan adalah penerimaan pesanan dari pelanggan terhadap suatu produk. Lanjutan dari pemesanan adalah pengiriman produk sampai ketangan pemesan dengan selamat. I. PENDAHULUAN Jika tiap-tiap kebutuhan dan batasan ruang lingkup projek telah diketahui dengan baik, proses RAD memungkinkan tim pengembang untuk menciptakan sebuah sistem yang berfungsi penuh dalam jangka waktu yang sangat singkat. Tahapan- tahapan dalam RAD terdiri dari 3 tahapan yaitu Requirements Planning, Design Workshop, dan Implementation. Pengertian LPG Anam dan Widodo yang dikutip dalam Jurnal (2020:1), gas LPG didefinisikan sebagai propana, butana dan campuran antara propana / butana dalam bentuk cair yang tidak menimbulkan karat, tidak beracun tapi mudah terbakar. Fase ini adalah fase untuk merancang dan memperbaiki yang bisa digambarkan sebagai workshop. Penganalisis dan pemrogram dapat bekerja membangun dan menunjukkan dan pola kerja kepada pengguna. Workshop desain ini dapat dilakukan selama beberapa hari tergantung dari ukuran aplikasi yang akan dikembangkan. Selama workshop desain RAD, pengguna merespon prototipe yang ada dan penganalisis memperbaiki modul -modul yang dirancang berdasarkan respon pengguna. Jurnal Esensi Infokom Vol 7 No. 2 Oktober 2023 Jurnal Esensi Infokom Vol 7 No. 2 Oktober 2023 mengambil metode RAD di karenakan untuk mempersingkat waktu antara perancangan dan penerapan sistem informasi. mengambil metode RAD di karenakan untuk mempersingkat waktu antara perancangan dan penerapan sistem informasi. Implementasi Tampilan Halaman Registrasi Akun Pengecer Gambar 3 Tampilan Registrasi Akun Pengecer C. Implementasi Tampilan Halaman Registrasi Akun Pengecer IV. HASIL DAN PEMBAHASAN Berdasarkan dari sistem yang telah di analisis, maka peneliti mengusulkan aplikasi yang berbasis android untuk membantu pemilik pangkalan dalam mengelola dan pemesanan yang ada di pangkalan gas LPG 3KG kepada pengecer 1. Implementasi Antar Muka Pengecer A. Tampilan Halaman Splash Screen Pengecer Gambar 1 Tampilan Halaman Splash Screen Pengecer Gambar 3 Tampilan Registrasi Akun Pengecer Tampilan ini digunakan pengecer jika ingin memiliki akun supaya pengecer dapat memesan gas yang ada pada Pangkalan Gas LPG 3KG dengan cara pilih "Daftar" dan mengisi form yang terdiri dari email, username, password, nama alamat, desa, kecamatan, nomor telepon maka akun selesai dibuat dan pengecer ada akses untuk masuk ke dalam aplikasi. Gambar 1 Tampilan Halaman Splash Screen Pengecer Gambar 1 Tampilan Halaman Splash Screen Pengecer D. Tampilan Produk Pengecer Gambar 4 Tampilan Produk Pengecer Tampilan produk pada halaman tersebut pengecer dapa melihat produk yang ada pada pangkalan tersebut D. Tampilan Produk Pengecer D. Tampilan Produk Pengecer Gambar 4 Tampilan Produk Pengecer III. METODOLOGI PENELITIAN Pada fase implementasi ini, penganalisis bekerja dengan para pengguna secara intens selama workshop dan merancang aspek - aspek bisnis dan nonteknis perusahaan. Segera setelah aspek - aspek ini disetujui dan sistem - sistem dibangun dan di saring, sistem - sistem baru atau bagian dari sistem di uji coba dan kemudian di perkenalkan kepada organisasi. Berdasarkan pengertian di atas peneliti Metode penelitian digunakan untuk membahas dan menjelaskan data yang diperoleh agar dapat disimpulkan jawaban yang tepat dari rumusan masalah. Pada metode penelitian ini penulis menggunakan metode penelitian deskriptif dengan metode pendekatan kualitatif. Teknik Pengumpulan Data Teknik yang digunakan dalam mengumpulkan data pada penelitian ini, yaitu sebagai berikut: 41 D. Tampilan Produk Pengecer Pada gambar 1 ini merupakan tampilan layar awal yang muncul saat pertama kali membuka sebuah aplikasi. Gambar 4 Tampilan Produk Pengecer B. Implementasi Tampilan Halaman Login Pengecer B. Implementasi Tampilan Halaman Login Pengecer B. Implementasi Tampilan Halaman Login Pengecer Gambar 2 Tampilan Halaman Login Pengecer Tampilan login ini dibuat untuk masuk ke halaman menu utama aplikasi, dengan cara memasukkan username dan password lalu klik login, apabila belum memiliki akun maka pengecer harus mendaftarkan diri terlebih dahulu. Gambar 2 Tampilan Halaman Login Pengecer Gambar 2 Tampilan Halaman Login Pengecer Gambar 4 Tampilan Produk Pengecer Tampilan produk pada halaman tersebut pengecer dapat melihat produk yang ada pada pangkalan tersebut Gambar 2 Tampilan Halaman Login Pengecer Gambar 2 Tampilan Halaman Login Pengecer Tampilan login ini dibuat untuk masuk ke halaman menu utama aplikasi, dengan cara memasukkan username dan password lalu klik login, apabila belum memiliki akun maka pengecer harus mendaftarkan diri terlebih dahulu. Tampilan login ini dibuat untuk masuk ke halaman menu utama aplikasi, dengan cara memasukkan username dan password lalu klik login, apabila belum memiliki akun maka pengecer harus mendaftarkan diri terlebih dahulu. 42 Jurnal Esensi Infokom Vol 7 No. 2 Oktober 2023 F. Tampilan Pemesanan Pengecer Jurnal Esensi Infokom Vol 7 No. 2 Oktober 2023 G. Tampilan Transaksi Pengecer Gambar 7 Tampilan Transaksi Pengecer G. Tampilan Transaksi Pengecer G. Tampilan Transaksi Pengecer E. Tampilan Keranjang Pengecer j g g Gambar 5 Tampilan Keranjang Pengecer Gambar 5 Tampilan Keranjang Pengecer Gambar 7 Tampilan Transaksi Pengecer Gambar 5 Tampilan Keranjang Pengecer Gambar 5 Tampilan Keranjang Pengecer Pada halaman ini terdapat tabel data transaksi yang telah dibuat oleh pengecer. Pada proses tersebut, admin bertugas dan mengkonfimasi pesanan yang dipesan oleh pengecer. Halaman keranjang berfungsi sebagai tempat produk yang telah dipilih oleh pengecer sebelum produk di pesan. F. Tampilan Pemesanan Pengecer F. Tampilan Pemesanan Pengecer Gambar 6 Tampilan Pemesanan Pengecer Pada halaman ini, setelah pengecer melakukan pemesanan maka pengecer harus membayar pesanan yang telah dipesan terlebih dahulu dengan metode pembayaran transfer bank atau bayar langsung. g p y g p p g H. Implementasi Tampilan Profil Pengecer Gambar 8 Tampilan Profil Pengecer Halaman profil merupakan halaman yang berfungsi untuk mengetahui informasi dari akun pengguna. F. Tampilan Pemesanan Pengecer F. Tampilan Pemesanan Pengecer Gambar 6 Tampilan Pemesanan Pengecer Pada halaman ini, setelah pengecer melakukan pemesanan maka pengecer harus membayar pesanan yang telah dipesan terlebih dahulu dengan metode pembayaran transfer bank atau bayar langsung. esanan Pengecer Gambar 6 Tampilan Pemesanan Pengecer Tampilan Pemesanan Pengecer Gambar 6 Tampilan Pemesanan Pengecer H. Implementasi Tampilan Profil Pengecer H. Implementasi Tampilan Profil Pengecer Gambar 8 Tampilan Profil Pengecer Halaman profil merupakan halaman yang berfungsi untuk mengetahui informasi dari akun pengguna. Gambar 8 Tampilan Profil Pengecer Gambar 6 Tampilan Pemesanan Pengecer Gambar 8 Tampilan Profil Pengecer Gambar 8 Tampilan Profil Pengecer Pada halaman ini, setelah pengecer melakukan pemesanan maka pengecer harus membayar pesanan yang telah dipesan terlebih dahulu dengan metode pembayaran transfer bank atau bayar langsung. Halaman profil merupakan halaman yang berfungsi untuk mengetahui informasi dari akun pengguna. Halaman profil merupakan halaman yang berfungsi untuk mengetahui informasi dari akun pengguna. Halaman profil merupakan halaman yang berfungsi untuk mengetahui informasi dari akun pengguna. 43 Jurnal Esensi Infokom Vol 7 No. 2 Oktober 2023 K. Implementasi Tampilan Bank Pengecer oleh Admin Gambar 11 Tampilan Bank Pengecer oleh Admin 2. Implementasi Antar Muka Pengecer 2. Implementasi Antar Muka Pengecer K. Implementasi Tampilan Bank Pengecer oleh Admin K. Implementasi Tampilan Bank Pengecer oleh Admin K. Implementasi Tampilan Bank Pengecer oleh Admin Gambar 11 Tampilan Bank Pengecer oleh Admin p g I. Implementasi Tampilan Login Admin p g I. Jurnal Esensi Infokom Vol 7 No. 2 Oktober 2023 Implementasi Tampilan Login Admin Gambar 9 Implementasi Tampilan Login Admin Gambar 11 Tampilan Bank Pengecer oleh Admin Gambar 9 Implementasi Tampilan Login Admin Pada tampilan ini terdapat tabel data bank yang telah bayar oleh pengecer melalui transfer bank dan bukti pembayaran dikirim lewat whatsapp. Pada proses tersebut, admin bertugas mengkonfimasi pembayaran yang dipesan oleh pengecer. Pada tampilan login ini, admin harus memasukkan username dan password yang telah didaftarkan sebelumnya apabila ingin masuk ke sistem agar dapat mengelola proses transaksi. Admin mempunyai banyak tugas penting, seperti input gas, konfirmasi pesanan, edit data produk, menyimpan data produk, dan lain sebagainya karena admin adalah orang yang bertanggung jawab penuh atas proses transaksi yang berlangsung Pada tampilan login ini, admin harus memasukkan username dan password yang telah didaftarkan sebelumnya apabila ingin masuk ke sistem agar dapat mengelola proses transaksi. L. Implementasi Tampilan Pengecer oleh Admin L. Implementasi Tampilan Pengecer oleh Admin L. Implementasi Tampilan Pengecer oleh Admin Gambar 12 Implementasi Tampilan Pengecer oleh Admin Halaman data pengecer merupakan halaman yang dikonfirmasi oleh admin untuk mengetahui informasi dari akun pengguna/pengecer yang telah terdaftar. p p g Gambar 12 Implementasi Tampilan Pengecer oleh Admin dan lain sebagainya karena admin adalah orang yang bertanggung jawab penuh atas proses transaksi yang berlangsung J. Implementasi Tampilan Home Admin Gambar 10 Implementasi Tampilan Home Admin Fungsi tampilan Home memudahkan pengguna dalam mengakses halaman mana yang akan kita dituju. Gambar 12 Implementasi Tampilan Pengecer oleh Admin Halaman data pengecer merupakan halaman yang dikonfirmasi oleh admin untuk mengetahui informasi dari akun pengguna/pengecer yang telah terdaftar. J. Implementasi Tampilan Home Admin J. Implementasi Tampilan Home Admin J. Implementasi Tampilan Home Admin J. Implementasi Tampilan Home Admin Gambar 10 Implementasi Tampilan Home Admin Gambar 12 Implementasi Tampilan Pengecer oleh Admin Halaman data pengecer merupakan halaman yang dikonfirmasi oleh admin untuk mengetahui informasi dari akun pengguna/pengecer yang telah terdaftar. Halaman data pengecer merupakan halaman yang dikonfirmasi oleh admin untuk mengetahui informasi dari akun pengguna/pengecer yang telah terdaftar. Gambar 10 Implementasi Tampilan Home Admin Fungsi tampilan Home memudahkan pengguna dalam mengakses halaman mana yang akan kita dituju. Fungsi tampilan Home memudahkan pengguna dalam mengakses halaman mana yang akan kita dituju. 44 Jurnal Esensi Infokom Vol 7 No. 2 Oktober 2023 M. Implementasi Tampilan Produk oleh Admin Gambar 13 Implementasi Tampilan Produk oleh Admin O. Implementasi Tampilan Laporan Notifikasi oleh Admin Gambar 15 Implementasi Tampilan Laporan Notifikasi oleh Admin Jurnal Esensi Infokom Vol 7 No. 2 Oktober 2023 M. Implementasi Tampilan Produk oleh Admin O. Implementasi Tampilan Laporan Notifikasi oleh Admin O. Implementasi Tampilan Laporan Notifikasi oleh Admin O. Implementasi Tampilan Laporan Notifikasi oleh Admin Gambar 15 Implementasi Tampilan Laporan Notifikasi oleh Admin Gambar 13 Implementasi Tampilan Produk oleh Admin Gambar 15 Implementasi Tampilan Laporan Notifikasi oleh Admin Gambar 13 Implementasi Tampilan Produk oleh Admin Pada tampilan ini terdapat form yang dapat digunakan oleh admin untuk menambah produk yang akan dijual. Form yang harus diisi adalah nama produk, harga, stok, dan gambar produk. Pada tampilan ini terdapat form yang dapat digunakan oleh admin untuk menambah produk yang akan dijual. Form yang harus diisi adalah nama produk, harga, stok, dan gambar produk. Notifikasi berfungsi untuk pemberitahuan barang pesanan yang terdiri di pending, di kemas, sedang dikirim, diterima dan batal V. KESIMPULAN N. Implementasi Tampilan Laporan Transaksi oleh Admin Gambar 14 Implementasi Tampilan Laporan Transaksi oleh Admin N. Implementasi Tampilan Laporan Transaksi oleh Admin Berdasarkan penelitian yang telah dilakukan, dapat ditarik beberapa kesimpulan yaitu: Terwujudnya aplikasi pengelolaan dan pemesanan secara online di Pangkalan Gas LPG 3KG Desa Purun Timur berbasis android yang dibangun menggunakan Dart, Flutter, PHP, beserta database MYSQL. Dengan adanya aplikasi pengelolaan dan pemesanan pada Pangkalan Gas LPG 3KG Desa Purun Timur yang dibuat diharapkan dapat mempermudah pengecer dalam pemesanan gas LPG 3KG, dan bagi pemilik untuk mempermudah memproses pemesanan dari pengecer. Aplikasi ini memperluas dan meningkatkan kemajuan Pangkalan sehingga bisa dijangkau oleh masyarakat. Dengan adanya aplikasi yang dibuat pengecer atau konsumen tidak harus datang langsung ke pangkalan, cukup dengan melakukan pemesanan melalui aplikasi yang sudah dibuat. Terwujudnya aplikasi pengelolaan dan pemesanan secara online di Pangkalan Gas LPG 3KG Desa Purun Timur berbasis android yang dibangun menggunakan Dart, Flutter, PHP, beserta database MYSQL. Dengan adanya aplikasi pengelolaan dan pemesanan pada Pangkalan Gas LPG 3KG Desa Purun Timur yang dibuat diharapkan dapat mempermudah pengecer dalam pemesanan gas LPG 3KG, dan bagi pemilik untuk mempermudah memproses pemesanan dari pengecer. Aplikasi ini memperluas dan meningkatkan kemajuan Pangkalan sehingga bisa dijangkau oleh masyarakat. Dengan adanya aplikasi yang dibuat pengecer atau konsumen tidak harus datang langsung ke pangkalan, cukup dengan melakukan pemesanan melalui aplikasi yang sudah dibuat. Gambar 14 Implementasi Tampilan Laporan Transaksi oleh Admin REFRENSI Laporan transaksi dikelolah oleh admin, didalam laporan bisa melihat hasil pesanan dan laporan transaksi yang telah terselesaikan, laporan transaksi dilakukan setiap pembelian. Laporan transaksi dikelolah oleh admin, didalam laporan bisa melihat hasil pesanan dan laporan transaksi yang telah terselesaikan, laporan transaksi dilakukan setiap pembelian. [1] Murtiwiyati, Muhammad Sobirin (2020). Aplikasi Pemesanan Gas LPG Berbasis Android Menggunakan Android Studio Dan Firebase. Fakultas ilmu computer, Universitas Gunadarma Jl. Margonda Rayab100 Depok 16424. [1] Murtiwiyati, Muhammad Sobirin (2020). Aplikasi Pemesanan Gas LPG Berbasis Android Menggunakan Android Studio Dan Firebase. Fakultas ilmu computer, Universitas Gunadarma Jl. Margonda Rayab100 Depok 16424. [2] Irawan, Muhammad Indra (2016). Aplikasi Sistem Pendukung Keputusan Pemilihan Smartphone 2016 Dengan Metode Weight Product (WP). Undergraduate thesis, Universitas Muhammadiyah Gersik. [3] Setyawan dan Munari (2020). Panduan Lengkap Membangun Sistem Monitoring Kinerja Mahasiswa Intership Berbasis Web dan Global Positioning System. Bandung: Kreatid Industri Nusantara. [4] Suwa (2021:1) Manajemen Pengelolaan Dana Revitalisasi Danau Tondano Oleh Pemerintah Kabupaten 45 Jurnal Esensi Infokom Vol 7 No. 2 Oktober 2023 Mianahasa (Studi Kasus Di Balai Wilayah Sungai Sulawesi). Vol: 1. No.2. [5] Apif Susanti, Dwi Wayu Prabowo (2020) Perdagangan elektronik pada Toke -ku Digital. Diakses (tanggal 26 Agustus 2017) [6] Anam Dan Widodo (2021:3). Upaya Percepatan Proses Bongkar Muat Propylane Dikapal LPGC NO.5 SJ GAS. Vol. 3 No. 1. September 2021. [7] Subianto (2020:47). Penerapan Metode Rapid Application Development dalam Perancangan Sistem Informasi Pendataan. Jurnal Unfokam. Vol. XVI. No 1. Maret 2020. [8] Lala Safitri Dan Sucipto Basuki (2020) Analisa dan Perancangan Sistem Informasi Text Chatting Berbasis Android Wev View. Vol 8, No 2 (2020) IPSIKOM. 46
https://openalex.org/W4394923729	https://ejns.springeropen.com/counter/pdf/10.1186/s41984-024-00285-6	English	null	Frontal ghost tumour: a case report	The Egyptian Journal of Neurosurgery : the official publication of the Egyptian Society of Neurological Surgeons/Egyptian journal of neurosurgery	2,024	cc-by	2,641	© Crown 2024. 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 mate- rial. 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/. Case report Our patient was a 71 year-old right-handed female who was referred to our neurosurgical outpatient depart- ment and presented with frontal swelling and recurrent frontal headache of 3 years duration. The frontal swell- ing was initially right-sided but increased in size to cross the midline in the last 3 months prior to presentation. No swellings in other parts of the body. She reported no antecedent trauma to the head, no recurrent para- nasal infections, and no fever. At about the same time, she noticed recurrent frontal headaches which did not interfere with her daily activities of living initially but worsened within the last 1 month prior to presenta- tion requiring analgesics. No vomiting, seizures, or limb weakness. She was however noticed to have some per- sonality changes characterized by increased aggression, anhedonia, and refusal of feeds. No history suggestive of extra-cranial malignancies. She had no relevant medical co-morbidity of note. Other aspects of history were not contributory. Two weeks prior to presentation, she devel- oped facial pain with occasional blurring of vision which *Correspondence: Mesi Mathew mesi.mathew@hotmail.com 1 Division of Neurosurgery, Ahmadu Bello University Teaching Hospital, Zaria, Nigeria 2 National Hospital, Abuja, Nigeria 3 Department of Neurosurgery, Hull University Teaching Hospitals NHS Trust, Hull HU3 2JZ, United Kingdom Abstract Background Ghost tumors spontaneously disappear or decrease to less than 70% before definitive diagnosis and treatment (other than steroid treatment). We report our experience with a patient who had not received steroids, and the challenges of managing a ghost tumor from a developing country. Case presentation A 71 year old female with frontal mass, right proptosis, and frontal headache. Mass was con- firmed by cranial CT scan but entirely resolved while the patient was awaiting surgery. Further follow-up at 6 months revealed clinical and MRI evidence of recurrence. Ghost tumors are no myths and can recur! Conclusion It is imperative to closely follow up with patients who have complete resolution of brain tumors prior to definitive treatment. Keywords Ghost tumor, Cranium, Vanishing, Case report We report a case of a vanishing tumour and the key points we learnt which we believe will be useful in man- aging these cases, particularly in low-income countries. Introductionh The clinical diagnosis of a brain tumour comes with many challenges to the patient and even the managing Neuro- surgeon as it marks the onset of probably a life-long jour- ney of health care. This becomes more baffling to both parties when the lesion suddenly ‘disappears’ and can- not be found again. Cases of such experiences have been reported in the literature with various descriptions. Okita et al. in 2012 defined vanishing tumors as tumors exhib- iting the radiological feature of spontaneous disappear- ance or reduction to less than 70% of the initial tumour volume on MRI with Gd-DTPA enhancement before definitive diagnosis and treatment with drugs other than steroids [1]. Numerous causes, notably Central Nervous System (CNS) lymphoma, have been documented [2, 3]. Egyptian Journal of Neurosurgery Egyptian Journal of Neurosurgery Mathew et al. Egyptian Journal of Neurosurgery (2024) 39:25 https://doi.org/10.1186/s41984-024-00285-6 Mathew et al. Egyptian Journal of Neurosurgery (2024) 39:25 https://doi.org/10.1186/s41984-024-00285-6 Open Access Mathew et al. Egyptian Journal of Neurosurgery (2024) 39:25 Page 2 of 5 Cranial CT scan showed a hyperdense soft tissue mass with intracranial extension compressing the frontal lobes (Fig. 1a). The lesion showed some enhancement with contrast, with minimal peri-lesional edema. There was sclerosis of the right frontal bone with a moth-eaten appearance and associated spiculations. No paranasal or intracranial collections. Her Fasting Blood Glucose was 4.7 mmol/L. The full Blood Count result was within normal limits. Retroviral screening was negative. Thy- roid and abdominopelvic ultrasound scans revealed nor- mal findings. necessitated an Ophthalmology review where she also had a cranial CT scan and was referred for neurosurgical care based on CT findings. i At presentation, she was found to be in some painful distress with an otherwise normal general physical find- ing. Vital signs were within normal limits (T = 36.3, Pulse rate = 90 beats/min, Blood Pressure = 120/80 mmHg). She had a GCS of 15 with preserved higher cerebral func- tions. Cranial nerves functions were preserved. Pupils were 3 mm and briskly reactive to light (direct and con- sensual) bilaterally. She had normal muscle bulk, tone, power, and deep tendon reflexes across the limbs. Exami- nation of the Head revealed an irregular mass extending from the right frontal area, across the midline extending from the right supra-orbital margin to the bregma with normal overlying skin. No differential warmth and the mass was non-tender. It measured 18 × 14 cm (cranio- caudal and lateral dimensions). It was firm, non-mobile, and non-pulsatile. She had no other scalp masses. Exami- nations of the anterior neck, breast, abdomen, and other systems were clinically normal. i A diagnosis of Frontal Meningioma to rule out soft tissue tumour (sarcoma) was made. She was placed on analgesics and requested to do cranial MRI and other pre-op investigations. On her pre-surgical assessment visit 4 weeks later, she was unable to do the requested MRI on financial grounds. She however reported resolu- tion of headache and the frontal mass. Clinical evaluation showed minimal right supraorbital swelling. She was able to do a Brain CT scan 3 months later which revealed a normal study (Fig. 1b). The residual right supraorbital Fig. 1 a and b Cranial CT scan before and after the disappearance of tumour Fig. 1 a and b Cranial CT scan before and after the disappearance of tumour Fig. 1 a and b Cranial CT scan before and after the disappearance of tumour Page 3 of 5 Mathew et al. Egyptian Journal of Neurosurgery (2024) 39:25 Page 3 of 5 to be aware of this oncologic phenomenon. In our situa- tion, we did not anticipate it, so we had to discuss it with the patient and her family convincing them that this is a documented phenomenon in clinical practice with a possibility of recurrence. The phenomenon could also come with the tendency for patients to fail to adhere to outlined management protocols. In the typical context of a developing country, the physician might end up being caught in the web of the clinical-supernatural debate. Our patient believed a miracle had happened! Rather than raising objections against the patient’s beliefs, we opined that repeat imaging at intervals would go further in ascertaining the miraculous. This was a more noble approach to take than an outright jettison of the patient’s faith. In the setting of ghost tumors, this complementary approach will be better suited than a conflicting one.h mass also resolved completely. However, at 6 months fol- low up she noticed right supraorbital swelling and mild headaches. A contrasted cranial MRI revealed evidence of recurrence (Fig. 2). Discussion Frassanito et al. [3] proposed a classification of Ghost tumors into Vanishing tumors (GhT1), Tumour-like lesions (GhT2), and False tumors (GhT3). According to their classification, a Vanishing tumour describes a space-occupying lesion that shows the radiologic features consistent with the diagnosis of a tumour but unexpect- edly disappears or shrinks in size on follow-up radiologic studies, behaving like a ghost! Furthermore, the term spontaneous remission or spontaneous regression (SR) of cancer translates into the recovery of a patient from cancer in the absence of a disease-specific treatment or in the presence of inadequate therapy [4]. While this phe- nomenon may be the norm in many common ailments, it remains an exception in malignancies hence the concerns when a ghost tumour is encountered in clinical practice. l So why do some tumors vanish? The honest answer is still up for research! Spontaneous tumour regression has been associated with apoptosis, immune system changes, micro-environment, and more or less with DNA onco- genic suppression [7]. Factors that stimulate the immune system have also been postulated and are numer- ous including bacterial products, enzymes, infections, and hormones [8]. The infection-induced remission of tumors has been proposed. Acute infections are believed to trigger the immune system to eliminate tumors. This has been independently demonstrated with Streptococ- cus pyogenes [4]. Various brain conditions have been reported to exhibit this phenomenon. These include, but are not limited to, primary central nervous system lymphoma (PCNSL), gliomas, tumefactive multiple sclerosis, granulomas or tuberculomas, and clival chordoma [5, 6]. PCNSL accounts for up to 50% of cases, but the phenomenon has also been reported with oligoastrocytoma, colonic tumors, renal tumors, etc. [2, 3] When this occurs, the implications could be far-reaching and need to be contained professionally. Patients could understand- ably question the credibility of the entire clinical process. Therefore, it is important for clinicians managing cancers As with our experience, there is a possibility of not having a histological diagnosis in these cases, at least initially. In this patient, the lesion disappeared com- pletely while the patient was awaiting tumour resection as seen in the repeat cranial CT scan (compare Fig. 1a Fig. 2 a and b MRI Scan at 6 months showing recurrence Fig. 2 a and b MRI Scan at 6 months showing recurrence Mathew et al. Egyptian Journal of Neurosurgery (2024) 39:25 Page 4 of 5 Deoxyribonucleic acid Deoxyribonucleic acid DNA and b). Discussion Our patient had no prior steroid therapy or any form of intervention specifically directed at the tumour apart from oral analgesics (Paracetamol and Codeine). Such resolution of tumors without intervention has also been reported by Kheiri et al. [9] The use of ster- oids prior to biopsy has been associated with the reso- lution of lymphomas in the literature. In a study of 162 PCNSL over a 20-year period in Tokyo, Yim et al. [10] reported a 14% increase in steroid-associated vanish- ing tumour per 100 mg of dexamethasone equivalent. Sometimes, the resolution of brain lesions can only be picked by imaging. It has been reported that approxi- mately 1% of patients with brain tumors will have dis- appearing lesions at the time of surgery and because this possibility exists, updated imaging before surgery is recommended [11]. Availability of data and materials l bl Availability of data and materials Not applicable. References k 1. Okita Y, Narita Y, Miyakita Y, Ohno M, Fukushima S, Maeshima A, et al. Long-term follow-up of vanishing tumors in the brain: How should a lesion mimicking primary CNS lymphoma be managed? Clin Neurol Neurosurg. 2012;114:1217–21. 2. Bromberg J, Siemers M, Taphoorn M. Is a “Vanishing tumor” always a lymphoma? Neurology. 2002;59:762–4. 3. Frassanito P, Tamburrini G, Massimi L, Caldarelli M, Di Rocco C. Ghost tumors of the central nervous system: definition, clinical implications, and proposal of classification. World Neurosurg. 2015;84:663–70. Consent for publication Consent for this publication was obtained from the patient for the publication of this case report. Ethical approval and consent to participate Ethical approval was obtained from the Health Research Ethics Committee of the Ahmadu Bello University Teaching Hospital, Zaria with the number ABUTHZ/HREC/F43/2023 for this study. Consent to participate was also obtained. Acknowledgements I k l d h I acknowledge the supportive role played by the son of this patient who was the next of Kin in making this experience to be published. Author contributions MM Conceptualization, Design, Drafting, Literature search, and Review of the Manuscript. IG Initial Drafting, Literature search, and Review of the Manuscript. AAA Manuscript Review. SIG Manuscript Review. MRM Review of the final Manuscript. AOJ Manuscript Review. Received: 19 August 2023 Accepted: 22 November 2023 Received: 19 August 2023 Accepted: 22 November 2023 Received: 19 August 2023 Accepted: 22 November 2023 Declarations On follow-up, our patient had a recurrence of the right proptosis 6 months after the initial complete disappearance of the tumour, and a follow-up MRI of the brain also showed evidence of intracranial tumour recurrence (Fig. 2). Gupta et al. [12] in their experi- ence, reported a tumour recurrence time of 5 months following the disappearance of a cerebellar cystic medulloblastoma in India. Despite our professional counsel, it has proved difficult for the patient and the family to come to terms and consent to neurosurgical intervention. In a long-term follow-up study of van- ishing tumors by Okita et al. [1] in which 10 patients were studied, 5 had a median recurrence duration of 7 months, and they recommended MRI follow-up for all patients with vanishing tumors for up to 5 years. This phenomenon underscores the concepts of individ- ualization of patients and the need for interest in “outli- ers” whose pathology does not behave as the majority [13]. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Received: 19 August 2023 Accepted: 22 November 2023 Conclusions h Ghost tumors are no myths, they do occur! As we go about the business of caring for the sick, it is important that clinicians are aware of this phenomenon. It is also important to follow up on patients whose tumors have vanished using clinical and radiological tools to detect recurrence. As clinicians, our posture must not lean towards antagonizing what has happened, but we should pragmatically walk the patients through the known phases of this poorly understood phenomenon. p pi g 4. Radha G, Lopus M. The spontaneous remission of cancer: current insights and therapeutic significance. Transl Oncol. 2021. https://doi. org/10.1016/j.tranon.2021.101166. 5. Bander ED, Kocharian G, Liechty B, Tsiouris AJ, Schwartz TH. Spontane- ous regression of a clival chordoma. Case report Acta Neurochir (Wien). 2020;162:433–6. 6. Hamed SA, Mekkawy MA, Abozaid H. Differential diagnosis of a vanishing brain space occupying lesion in a child. World J Clin Cases. 2015;3:956–64. 7. Salman T. Spontaneous tumor regression. J Oncol Sci. 2016. https://doi. org/10.1016/j.jons.2016.04.008. 8. Cole WH. Efforts to explain spontaneous regression of cancer. J Surg Oncol. 1981;17:201–9. Abbreviations MRI Magnetic resonance imaging CT Computerized tomography CNS Central nervous system GhT Ghost tumour SR Spontaneous regression PCNSL Primary CNS lymphoma Competing interests The authors have no competing interests to declare. Abbreviations 9. Kheiri G, Habibi Z, Nejat F. Spontaneous regression of congenital brain tumors: a report of two cases. Child’s Nerv Syst. 2021;37:3901–5. 10. Yim E, Umemura Y, Sun Y, Junck L. The vanishing tumour phenomenon in the diagnosis of primary CNS lymphoma. Neuro Oncol. 2020;22(Supple- ment 2):ii84. 10. Yim E, Umemura Y, Sun Y, Junck L. The vanishing tumour phenomenon in the diagnosis of primary CNS lymphoma. Neuro Oncol. 2020;22(Supple- ment 2):ii84. Page 5 of 5 Mathew et al. Egyptian Journal of Neurosurgery (2024) 39:25 Mathew et al. Egyptian Journal of Neurosurgery (2024) 39:25 11. Sutherland CS, Kelly JJP, Morrish W, Sutherland GR. Identification of disap- pearing brain lesions with intraoperative magnetic resonance imaging prevents surgery. Neurosurgery. 2010;67:1061–5. 12. Gupta V, Kumar S, Tatke M, Singh A, Sinha S, Singh D. Disappearing cystic cerebellar medulloblastoma: the ghost tumour. Neurol India. 2001;49:291–4. 13. Eldridge L. Spontaneous Remission of Cancer Incidence and Causes [Internet]. 2021 [cited 2023 Oct 10]. Available from: https://www.veryw ellhealth.com/spontaneous-remission-of-lung-cancer-a-rare-miracle- 3971875 Mathew et al. Egyptian Journal of Neurosurgery (2024) 39:25 Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Publisher’s Note S i N i Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations.
https://openalex.org/W2019418883	https://jm.copernicus.org/articles/5/4/1986/jm-5-4-1986.pdf	English	null	Unusually large Late Campanian – Early Maastrichtian foraminifera from the Southern North Sea Basin	Journal of micropalaeontology	1,986	cc-by	3,336	I NTRO DlJCTION fractions in a nest of sieves (SOOpm, 250-500pm, 180'- 250pm, 12.5-1 80pm). The foraminifera of Chalk sequences from three boreholes drilled by Shell (U K) Exploration and Production Limited in the Southern North Sea Basin during 1965-1971 have been investigated in order to establish a local biostratigraphical zonation for the Upper Cretaceous. The boreholes are Shell 44i2.1 (5C5.2'38" N., 02'23'35"E.) on the southern edge of the Mid North Sea High, and Shell 49119.1 (50"23'48"N., 02"45'21"E.) and Shell 49/20.2 (53"27'26.056"N., 02" 58' 15.124" E.) in the Indefatigable Gas Field (Fig. 1). Geophysical logs are a major tool for correlation and analysis of such offshore sequences but where a full suite of these is not available, or where a monotonous response is produced such as that through the Chalk Group, detaikd subdivision must rely heavily on micro- palaeontolalgical criteria. The 500pm fraction of the samples from 1500ft.- 2000ft. in Borehole 49/19.1 and 287Oft.-3800ft. in Borehole 49120.2 yielded extraordinarily large specimens (see P1. 1, figs. Sa, b) of a low diversity fauna of agglu- tinated and calcareous benthic foraminifera (see below). Only rare planktonic forms were recorded. This unusual assemblage was not recovered from Borehole 44/2.1. The 500pm fraction of the samples from 1500ft.- 2000ft. in Borehole 49/19.1 and 287Oft.-3800ft. in Borehole 49120.2 yielded extraordinarily large specimens (see P1. 1, figs. Sa, b) of a low diversity fauna of agglu- tinated and calcareous benthic foraminifera (see below). Only rare planktonic forms were recorded. This unusual assemblage was not recovered from Borehole 44/2.1. SY ST E MAT IC PA LA E 0 NT 0 L 0 GY Arenobulirnina courta (Marie) (Pl. 1, fig. 1) (Pl. 1, fig. 1) Remarks. This species was described originally from the Campanian (rnucronata Zone) of the Paris Basin (Marie, 1941). The test is free and globular; the chambers are arranged trochospirally in three to four whorls and are inflated to very inflated in the last whorl which makes up over half of the test. The sutures are distinct and de- pressed, and the aperture is a single slit or semi-circular opening midway along the internal margin of the last chamber at the junction of the four sutures of the final whorl. The wall is thick and the surface rugose. The species has been compared with Bulirnina obesa Reuss, first described from the Upper Cretaceous of Poland, but the relationship between the two species remains unclear. J.rnicropa/aeonto/., 5(1): 11-17, April 1986 J.rnicropa/aeonto/., 5(1): 11-17, April 1986 Unusually large Late Campanian - Early Maastrichtian foraminifera from the Southern North Sea Basin KIM C. BALL Paleoservices Ltd., Unit 15, Paramount Industrial Estate. Sandown Road, Watford WD2 4X ABSTRACT-A low diversity fauna of unusually large (individuals of two to three times the normal size) agglutinated and calcareous benthic foraminifera is recorded from the Late Campanian - Early Maastrichtian Chalk sequences of two boreholes (Shell 49119.1 and Shell 49120.2) in the Southern North Sea Basin. Associated planktonic species are extremely rare. This distinctive assemblage provides a useful local stratigraphical marker. I NTRO DlJCTION p g Most borehole samples from offshore are generally available only as "ditch cuttings" which are tiny chips of rock cut by the drill bit. In the present study, these were sampled at 30ft. intervals. The cuttings are brought to the surface by drilling mud that is circulated to lubricate the drill bit ;and to prevent the well caving in. One of the several problems encountered when ditch cutting are used in biostratigraphical studies is that of "cavings" which is material introduced into the mud system from stratigraphical levels higher up the well. To compensate for this, biostratigraphical and chronostratigraphical boundaries are drawn at extinctions (i.e. first down-hole appearances) of fossil taxa. The Upper Cretaceous stage boundaries in the three boreholes examined have been established on this basis using the known foraminifera1 ranges from onshore Britain and elsewhere in Europe. These boundaries are shown, together with the geo- physical log signatures (Gamma Ray and Formation Density) for Borehole 49120.2 in Fig. 2. Dimensions. Average Bigger forms Height: 0.63 mm Height: 1.3mm Width : 0.49 mm Width : l.Omm Arenobulimina elevata (d'orbigny) (PI. 1, fig. 2) ( (PI. 1, fig. 2) Remarks. This species was described originally from the Campanian of the Paris Basin (d'orbigny, 1840) and is very similar toA. courta (see above). However, it may be distinguished by its bigger size (four to five whorls) and high trochospire. SAMPLE PREPARATION AND EXAMINATION Bigger forms Height: 2.0mm Width : 1.7 mm Bigger forms Height: 2.0mm Width : 1.7 mm Dimensions. Average Height: 1.3mm Width : 1.1 mm Dimensions. Average Height: 1.3mm Width : 1.1 mm Dimensions. Average Height: 1.3mm Width : 1.1 mm Each sample was disaggregated in white spirit, washed through a 75pm sieve, dried and then split into four size 11 Ball KEY + Shell boreholes used in this study boundary _.-- international Scale Fig. 1 . Location of the three boreholes in the Southern North Sea Basin used in this study. Scale 1 . Location of the three boreholes in the Southern North Sea Basin used in this study Eggerellina brevis (d’orbigny) Eggerellina brevis (d’orbigny) distinctly overlapping whorls and are obscured by the strong ornamentation of randomly arranged grooves which roughen the surface and distinguish the species from other members of the genus. The grooves may be the result of dissolution of the sponge spicules that were once cemented together to form the test. The sutures are flush, and the aperture varies from a simple semi- circular interiomarginal opening on the final chamber to a narrow slit extending up the apertural face. gg ( g y) Remarks. This species was described originally from the Campanian of the Paris Basin (d’orbigny, 1840). It shows a wide range of morphological variation and well documented dimorphism (microspheric and megalo- spheric forms). However, its taxonomic history is rather confused. The type figures show clearly the character- istic dominance of the last overlapping whorl, the tri- lobed appearance and the ‘key-hole’ shaped aperture. y Two and a half chambers are usually visible from the summit. The wall is agglutinated but composed of cal- careous particles. Dimensions. Average Bigger forms Height: 0.53 mm Width : 0.49mm Height: 0.83 mm Width : 0.77 mm Atuxophrugmium vuriabile (d’orbigny) (PI. 1, figs. Sa, b) Dimensions. Average Bigger forms Height: 0.56mm Height: 1.17mm Width : 0.49mm Width : 0.96 mm Ataxophragmium rimosum (Marsson) (PI. 1, fig. 4) Dimensions. Average Bigger forms Height: 0.56mm Height: 1.17mm Width : 0.49mm Width : 0.96 mm Ataxophragmium rimosum (Marsson) (PI. 1, fig. 4) Dimensions. Average Height: 0.56mm Width : 0.49mm Remarks. This species was described originally from the Campanian of the Paris Basin (d’orbigny, 1840) and is type of the genus Ataxophragmium. It is a highly variable form with a free test which is subglobular to elongate with a variable outline but always broader than high. The chambers are arranged in a helical spire of two to three loosely to tightly coiled overlapping whorls. (PI. 1, fig. 4) Remarks. This species was described originally from the Chalk of Riigen Island, East Germany (Marsson, 1878). The test is free and subspherical; the chambers are arranged in a tightly coiled helical spire of two to three 12 Late Campanian - Early Maastrichtian foraminifera from the Southern North Sea Basin U. MAASTRICHTIAN 0 G . Eggerellina brevis (d’orbigny) q 1.0 WE R M A ASTR I C H T I AN UPPER CAMPANIAN MIDDLE CAMPANIAN LOWER CAMPANIAN - - SA NTO N I AN /CON I AC I A N TUR ON IAN -- PLENUS MARLS CENOMANIAN .100 2 F.D.C. 3 BOREHOLE SHELL 49120.2 range of occurrence of unusually large foraminifera KEY recorded flint bands C.D.C. Formation Density Log ( g b m 3 ) 5.R.L. Gamma Ray Log (API units) depths in feet -~ ~ Fig. 2. Stratigraphical interpretation of the Chalk sequence in Shel! 493/20.2. BOREHOLE SHELL 49120.2 M A ASTR I C H T I AN UPPER CAMPANIAN MIDDLE CAMPANIAN LOWER CAMPANIAN SA NTO N I AN /CON I AC I A N Fig. 2. Stratigraphical interpretation of the Chalk sequence in Shel! 493/20.2. 13 Ball The sutures are flush to slightly depressed. The aperture is usually a simple semi-circular opening at the inner margin of the last chamber, but it may vary in form and be a narrow, elongate slit that extends up the apertural face which is always flattened. The wall is thick and the surface smooth or slightly rugose. The considerable morphological variation shown by this species has resulted in much taxonomic confusion and in the past, certain variants have been accommodated in other, sometimes new, genera (e.g. Orbignyna, Ataxogyroidina) and species (e.g. concava Marie, cylindrica Marie, gibbosa Marie, ovoidea Marie). species and the present form cannot be accommodated with globulosa and a new specific name is required. Sweicicki (unpublished thesis, 1980) created the new species mariae for it, but until this taxon is published formally, the identificationAtaxophragmium sp. nov. A. is favoured. Dimensions. Average Bigger forms Height: 0.72 mm Width : 0.64mm Height: 1.2 mm Width : 1.05mm Orbignyna ovata von Hagenow (PI. 1, fig. 7) Remarks. This species was described originally from the Chalk of Rugen Island, East Germany (von Hagenow, 1842). The test is free, elongate and compressed. The chambers are indistinct and uninflated; the first five or six are arranged in a broadly rounded planispiral coil and are strongly overlapping on the inner coiled margin biit later, they expand moderately and uniformly and the coil becomes evolute with the last chamber or two being partially uncoiled. The sutures are initially indistinct but later distinct, depressed and curved. Eggerellina brevis (d’orbigny) The apertural face is weakly inflated with a distinct depression about the aperture which is a simple, terminal circular to elliptical pit. The wall is coarsely agglutinated and the surface is roughened by many longitudinal grooves as in Ataxo- phragmium rimosum (see above). Dimensions. Average Bigger forms Height: 0.34 mm Width : 0.31 mm Height: 0.91 mm Width : 0.86 mm Ataxophragmium sp. nov. A (PI. 1, fig. 6) Remarks. This species has a free, asymmetrical test which is sometimes subspherical tending to plano- convex. The chambers are indistinct, slightly inflated and higher than broad, and are arranged trochospirally usually in two (but sometimes in up to three) over- lapping evolute whorls. There are six chambers per whorl and the last chamber of each tends to overlap the previous whorl. The sutures are distinct and flush. The aperture is a simple, sub-rectangular, deep slit extending along the interiomarginal suture of the last chamber; the apertural face is flattened. The wall is thick and finely agglutinated with much calcareous cement; the surface is smooth. Dimensions. Average Bigger forms Height: 0.88 mm Width : 0.66mm Height: 1.4 mm Width : 1.2 mm Orbignyna sherlocki Barnard (PI. 1, fig. 8) Marie (1941) referred an identical form from the Campanian (mucronata Zone) of the Paris Basin to Nonionina globulosu von Hagenow, 1842. However, several authors (e.g. Marsson, 1878; Cushman, 1931 ; Visser, 1951) have concluded that von Hagenow’s un- figured species was based on a calcareous perforate form, not an agglutinated form. Therefore, Marie’s Remarks. This species was described originally from the Campanian (mucronata Zone) of Council’s Pit, New- market Road, Norwich, Norfolk (Barnard, 1953). It is distinguished from 0. ovata (see above) by its conical form, circular and weakly inflated apertural face, and moderately rugose surface. g p , , ; , Fig. 11. Lenticulina sp., side view, MPK 4099; Shell 49120.2, 3590ft. Explanation of Plate 1 All figures are x 35. Specimen numbers refer to the coilections of the British Geological Survey (Keyworth). All figures are x 35. Specimen numbers refer to the coilections of the British Geological Survey (Keyworth). Fig. 1. Arenobulimina courtu (Marie), apertural view, MPK 4088; Shell 49120.2, 3650 ft. Fig. 1. Arenobulimina courtu (Marie), apertural view, MPK 4088; Shell 49120.2, 3650 ft. Fig. 1. Arenobulimina courtu (Marie), apertural view, MPK 4088; Shell 49120.2, 3650 ft. . 1. Arenobulimina courtu (Marie), apertural view, MPK 4088; Shell 49120.2, 3650 ft 2 Arenobulimina elevuta (d’orbigny) apertural view MPK 4089; Shell 49120 2 35 Fig. 1. Arenobulimina courtu (Marie), apertural view, MPK 4088; Shell 49120.2 . 2. Arenobulimina elevuta (d orbigny), apertural view, MPK 4089; Shell 49120.2, 35 . 3. Eggerellina hrevis (d’orbigny), apertural view, MPK 4090; Shell 49120.2, 3020ft. g ( g y), p , ; , Fig. 3. Eggerellina hrevis (d’orbigny), apertural view, MPK 4090; Shell 49120.2, 3020ft. g ( g y), p , ; , Fig. 3. Eggerellina hrevis (d’orbigny), apertural view, MPK 4090; Shell 49120.2, 3020ft. ( g y), p , ; , ophragmium rirnosurn (Marsson), apertural view, MPK 4091 ; Shell 49/20.2, 3020 ft. g gg ( g y) p Fig. 4. Ataxophragmium rirnosurn (Marsson), apertural view, MPK 4091 ; Shell 49/20. Fig. 4. Ataxophragmium rirnosurn (Marsson), apertural view, MPK 4091 ; Shell 49/20.2, 3020 ft. Fig. 5.Ataxophragmium variabile (d’orbigny): (a) apertural view, MPK 4092; Shell 49/20.2, 3620ft.; (b) apertura view, MPK 4093; Shell 44/2.1, 3360ft., ‘normal’ size. Fig. 5.Ataxophragmium variabile (d’orbigny): (a) apertural v view, MPK 4093; Shell 44/2.1, 3360ft., ‘normal’ size. Fig. 5.Ataxophragmium variabile (d’orbigny): (a) apertural view, MPK 4092; Shell 4 view, MPK 4093; Shell 44/2.1, 3360ft., ‘normal’ size. Fig. 6. Atuxophragmium sp. nov. A, apertural view, MPK 4094; Shell 49120.2, 323 , ; , , g p g p , p , ; , Fig. 7. Orbignyna ovata von Hagenow, side view, MPK 4095; Shell 49i20.2, 3200f g g y g Fig. 8. Orbignyna sherlocki Barnard, side view, MPK 4096; Shell 49120.2, 3620ft. g g y Fig. 9. Cibicidoides (?) voltziana (d’orbigny), spiral view, M PK 4097; Shell 49/20.2, 3200 ft. Fig. 9. Cibicidoides (?) voltziana (d’orbigny), spiral view, M PK 4097; g p Fig. 11. Lenticulina sp., side view, MPK 4099; Shell 49120.2, 3590ft. Explanation of Plate 1 14 Campanian - Early Maastrichtian foraminifera from the Southern North Sea Basin Late Campanian - Early Maastrichtian foraminifera from the Southern North Sea Basin 15 15 Ball 0. sherlocki Barnard and Praebulimina obtusa (d’orbigny), followed by the Late Campanian forms Gavelinella monterelensis (Marie), Globorotalites hiltermanni Kaever, Globorotalites micheliniana (d’orbigny), Osangularia cordieriana (d’orbigny) and rarely the planktonic Archaeoglobigerina cretacea (d’orbigny). Dimensions. Average Bigger forms Height: 0.62 mm Width : 0.44mm Height: 1.1 mm Width : 1.02 mm Cibicidoides (?) voltziana (d’ Orbigny) (Pl. 1, fig. 9) ( , g ) Remarks. This species was described originally from the Upper Campanian of the Paris Basin and England (d’orbigny, 1840). The characteristic large plano- convex test with prominent calcite bosses make this species distinctive although its generic position is unclear. It has been referred to a number of genera in- cluding Cibicides, Cibicidoides, Gavelinella and Gavelinopsis, all of which display similarities in basic morphology but differ in their test wall structure. Most foraminifera1 tests from the Upper Chalk have under- gone fine-scale recrystallisation resulting in a secondary heterogeneous granular wall structure (Reiss, 1959 ; Schlanger & Douglas, 1974) and therefore the true generic position of this species cannot be established; study of unrecrystallised material may clarify the position. ACKNOWLEDGEMENTS This work was carried out during the tenure of a CASE award with the Natural Environment Research Council and Shell (U.K.) Exploration and Production Ltd. at Plymouth Polytechnic under the supervision of Professor Malcolm B. Hart (1980-1982). This support is gratefully acknowledge. g y g I wish to thank Shell (U.K.) Exploration and Produc- tion Ltd. and Esso Exploration and Production (U.K.) Ltd. for providing the material which appears here with their permission. p I am grateful to Dr. Haydon W. Bailey (Paleoservices Ltd.) and Professor Malcom B. Hart for helpful dis- cussion and to Mr. Stephen Crittenden (Paleoservices Ltd.) for critically reading and commenting on an early draft of this paper. p Dimensions. Average Bigger forms Diameter: 0.51 mm Diameter: 1.02 mm Height : 0.22 mm Height : 0.45 mm Lenticulina spp. (Pl. 1, figs. 10, 11) Bigger forms Diameter: 1.02 mm Height : 0.45 mm p Dimensions. Average Bigger forms Diameter: 0.51 mm Diameter: 1.02 mm Height : 0.22 mm Height : 0.45 mm Lenticulina spp. (Pl. 1, figs. 10, 11) Dimensions. Average Dimensions. Average Diameter: 0.51 mm p p Finally, I am indebted to Dr. Beris M. Cox (B.G.S., Keyworth) for her invaluable help and enthusiasm during the preparation of this paper. pp (Pl. 1, figs. 10, 11) ( , g , ) The unornamented Lenticulina are in most cases nearly impossible to speciate. They have long stratigraphical ranges which reduces their potential as useful bio- stratigraphic indicators and in the present study, no effort was made to determine them beyond generic level. Manuscript received April 1984 Revised manuscript accepted March 1985 y g Dimensions. Average Bigger forms Diameter 1 : 0.59mm Diameter 2: 0.51 mm Diameter 1 : 1.77 mm Diameter 2 : 1.54 mm Dimensions. Average Bigger forms Diameter 1 : 0.59mm Diameter 2: 0.51 mm Diameter 1 : 1.77 mm Diameter 2 : 1.54 mm Dimensions. Average Bigger forms Diameter 2: 0.51 mm Diameter 2 : 1.54 mm Manuscript received April 1984 Revised manuscript accepted March 1985 CONCLUSIONS The low diversity but dominant fauna of unusually large benthic foraminifera described above has been recorded from the same stratigraphical interval in the two boreholes studied from Block 49 - Shell 49/19.1 and Shell 49120.2. It is suggested that the occurrence of this distinctive assemblage provides a useful local stratigraphical marker. g p All chronostratigraphical and biostratigraphical inter- pretations were based on down-hole appearances which have been related to known forminiferal ranges from onshore Britain and elsewhere in Europe (e.g. Koch, 1977; Sweicicki, 1980 (unpubl.); Hart et al., 1981). The assemblage is dated as Early Maastrichtian - Late Campanian because of the down-hole appearances of the Maastrichtian forms Osangularia navarroana (Cushman), Praebulimina laevis (Beissel) and planktonic Rugoglobigerina rugosa (Plummer) together with Areno- bulimina elevata (d’orbigny), Bolivinoides laevigatus Marie, B. miliaris Hiltermann & Koch, Cibicidoides (?) voltziana (d’orbigny), Orbignyna ovum von Hagenow, 16 Late Carnpanian - Early Maastrichtian foraminifera from the Southern North Sea Basin REFERENCES Barnard, T. & Banner, F. T. 1953. Arenaceous Foraminifera from the Upper Cretaceous of England. Q. Jl Geol. SOC. Lond., 109, 173-216, pls. 7-9. p Cushman, J. A. 1931. A preliminary report on the foraminifera of Tennessee. Bull. Tenn. Div. Geol., Nashville, 41, 1-62, pk. 21-23. Hagenow, F. von. 1842. Monographie der Rugenschen Kreide. Versteinerungen pt. Mollusken E. Cephalopoda Fora- minifera. Neues Jb. Miner. Geog. Geol., 9, 568-575. p Hart, M. B., Bailey, H. W., Fletcher, B. N., Price, R. J. & Sweicick.i, A. 1981. Cretaceous. In Jenkins, D. G. &. Murray, J. W. (Eds.), Stratigraphical Atlas of Fossil Foraminifera, 14!)-227, pls. 7.1-7.25, text figs. 7.1-7.16. Ellis Horwood Lttl., Chichester for British Micropalaeontological Society. p g y Koch, W. 1977. Biostratigraphie in der Oberkreide und Taxonomie von Foraminiferen. Geol. Jb ., Hannover, Reihe A 38, 128 pp., 19 pls. pp p Marie, P. 1941. Les Foraminiferes de la Craie a Belemnitella niucronata du Bassin de Paris. Mern. Mus. natn. Hist. nut., Paris, n.s. 12, 1-296, pls. 1-37. , , p Marsson, T. F. 1878. Die Foraminiferen der weissen Schreib- kreide der Insle Riigen. Mitt. neturw. Ver. Neu-Vorpomm., Berlin, 10, 115-196, pls. 1-5. , , p Orbigny, A. d’ 1840. Memoire sur les Foraminiferes de la craie blarrche du Bassin de Paris. Mern. SOC. Geol. Fr., Paris, 4 (l), 1-51, PIS. 1-4. Reiss, 2. 1059. The wall structure of Cibicides, Planulina, Gyroidinoides and Globorotalites. Micropaleontology , New York, 5, 355-357, pl. 1. ( ) , , , p Schlanger, S. 0. & Douglas, R. G. 1974. Pelagic ooze-chalk- limestone transition and its implications for marine strati- graphy. In Hsu, K. J. & Jenkyns, H. C. (Eds.), Pelagic Sediments: On Land and Under the Sea. Int. Ass. Sediment. Spec. Publ’. , 1, 117-148. p Sweicicki, A. 1980. A foraminiferat biostratigraphy of the Cam- panian and Maastrichtian Chalks of the United Kingdom. Unpub. Ph.D. Thesis (CNAA), Plymouth Polytechnic. p ( ), y y Visser, A. M. 1951. Monograph of Foraminifera from the type locality of the Maastrichtian (S. Limburg, Netherlands). Leidse Geol. Med., Leiden, 16, 197-360, 16 pls. 17 17
https://openalex.org/W3011947568	https://hal.archives-ouvertes.fr/hal-02525149/document	English	null	A Rao-Blackwellized Particle Filter With Variational Inference for State Estimation With Measurement Model Uncertainties	IEEE access	2,020	cc-by	11,974	A Rao-Blackwellized particle filter with variational inference for state estimation with measurement model uncertainties Cheng Cheng Jean Yves Tourneret Xiaodong Lu A Rao-Blackwellized particle filter with variational inference for state estimation with measurement model uncertainties Ch Ch J Y T t Xi d L Cheng Cheng, Jean-Yves Tourneret, Xiaodong Lu To cite this version: Cheng Cheng, Jean-Yves Tourneret, Xiaodong Lu. A Rao-Blackwellized particle filter with variational inference for state estimation with measurement model uncertainties. IEEE Access, 2020, 8, pp.55665- 55675. 10.1109/ACCESS.2020.2981948. hal-02525149 Open Archive Toulouse Archive Ouverte Open Archive Toulouse Archive Ouverte OATAO is an open access repository that collects the work of Toulouse researchers and makes it freely available over the web where possible This is an author’s version published in: https://oatao.univ-toulouse.fr/25810 HAL Id: hal-02525149 https://hal.science/hal-02525149v1 Submitted on 30 Mar 2020 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. Open Archive Toulouse Archive Ouverte Open Archive Toulouse Archive Ouverte A Rao-Blackwellized Particle Filter With Variational Inference for State Estimation With Measurement Model Uncertainties CHENG CHENG 1, JEAN-YVES TOURNERET 2, (Fellow, IEEE), AND XIAODONG LU1 1School of Astronautics, Northwestern Polytechnical University, Xi’an 710072, China 2ENSEEIHT-IRIT-TéSA, University of Toulouse, 31071 Toulouse, France Corresponding author: Cheng Cheng (cheng.cheng@nwpu.edu.cn) This work was supported by the National Natural Science Foundation of China under Grant 61901380 and Grant 61761136001. ABSTRACT This paper develops a Rao-Blackwellized particle ﬁlter with variational inference for jointly estimating state and time-varying parameters in non-linear state-space models (SSM) with non-Gaussian measurement noise. Depending on the availability of the conjugate prior for the unknown parameters, the joint posterior distribution of the state and unknown parameters is approximated by using an auxiliary particle ﬁlter with a probabilistic changepoint model. The distribution of the SSM parameters conditionally on each particle is then updated by using variational Bayesian inference. Experiments are ﬁrst conducted on a modiﬁed nonlinear benchmark model to compare the performance of the proposed approach with other state-of-the-art approaches. Finally, in the context of GNSS multipath mitigation, the proposed approach is evaluated based on data obtained from a measurement campaign conducted in a street urban canyon. INDEX TERMS Joint state and parameter estimation, Rao-blackwellized particle ﬁlter, state-space models, variational inference. To cite this version: Cheng, Cheng and Tourneret, Jean-Yves and Lu, Xiaodong A Rao-Blackwellized particle filter with variational inference for state estimation with measurement model uncertainties. (2020) IEEE Access, 8. 55665-55675. ISSN 2169-3536 Any correspondence concerning this service should be sent to the repository administrator: tech-oatao@listes-diff.inp-toulouse.fr Received March 3, 2020, accepted March 16, 2020, date of publication March 19, 2020, date of current version March 30, 2020. Received March 3, 2020, accepted March 16, 2020, date of publication March 19, 2020, date of current version March 30, 2020. Received March 3, 2020, accepted March 16, 2020, date of publication March 19, 2020, date of current version March 30, 2020. Digital Object Identifier 10.1109/ACCESS.2020.2981948 The associate editor coordinating the review of this manuscript and approving it for publication was Yingsong Li . is work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ I. INTRODUCTION and parameter estimation is to assign a prior distribution to the model parameters and augment the state vector by including these unknown parameters. When it cannot be computed in closed form, the joint posterior distribution of all variables can be approximated by using sequential Monte Carlo (SMC) techniques [4]–[6]. Another possibility is to exploit the expectation-maximization (EM) algorithm, i.e., the posterior distribution of the state is obtained in the expectation step and then the maximum likelihood estimator of the unknown parameters is updated in the maximization step [7]–[10]. Recently, variational inference-based approaches in SSMs have been extensively studied for state estimation in the presence of model uncertainty [11]. In the case of linear SSMs, approximate separable distributions for state and noise parameters can be updated by iteratively solv- ing the coupled equations by using variational Bayesian (VB) inference [12]–[14]. A VB-based Kalman ﬁlter was also proposed for linear SSMs with non-stationary heavy-tailed noises [15], [16]. In the case of nonlinear SSMs, the noise parameters are usually marginalized out and the posterior distribution of the state is approximated by using an appro- priate particle ﬁlter (PF). Then the distribution of the noise State-space models (SSMs), composed of dynamic and mea- surement equations, are applied to a wide variety of signal processing problems, especially in positioning, tracking and navigation [1], [2]. A central problem when using these models is to recursively infer the state based on a sequence of measurements. In general, sensors delivering measure- ments are assumed to be in their nominal state of work, i.e., the parameters in the measurement equation have to be exactly speciﬁed a priori. However, in realistic contexts, these parameters can be time-varying due to abruptly changing environment, leading to the problem of state estimation in the presence of model uncertainty. Early attempts to solve this problem are based on Gaussian mixture approximations, such as the interacting multiple model (IMM) algorithm [3]. Since the reliability of the IMM is dependent on the number and choice of models, some approaches for jointly estimating state and parameter for SSMs have been proposed. Another solution for joint state The associate editor coordinating the review of this manuscript and approving it for publication was Yingsong Li . For more information, see https://creativecommons.org/licenses/by/4.0/ 55665 C. I. INTRODUCTION Cheng et al.: Rao-Blackwellized PF With Variational Inference for State Estimation With Measurement Model Uncertainties parameters conditionnally on each state particle can be cal- culated by using VB inference [17], [18]. The paper is organized as follows: The problem of jointly estimating state and time-varying parameters in non-linear SSMs with non-Gaussian measurement noise is presented in Section II. Section III studies the proposed new RBPF based on VB inference. The performance of this ﬁlter is evaluated in Section IV, ﬁrst using simulated data based on a modiﬁed nonlinear benchmark model, and then using experimental data in the context of GNSS multipath mitigation in urban canyons. Conclusions are ﬁnally reported in Section V. Considering that state and measurement equations depend linearly on a subset of the state vector, a Rao-Blackwellized particle ﬁlter (RBPF) (also known as marginalized particle ﬁlter) was proposed in [19], [20]. The ﬁlter was introduced for state and measurement equations depending linearly on a subset of the state vector and non-linearly on the other state variables. The idea of the Rao-Blackwellization is to marginalize the distribution of interested with respect to the state variables appearing linearly in the state and measure- ment equations, allowing these linear components to be pro- cessed using analytical methods (such as the Kalman ﬁlter) and the non-linear components by SMC techniques [21]. Rao-Blackwelization has been used successfully in many applications including navigation using an inertial navigation system and a terrain-aided positioning [22], multiple target tracking [23] and simultaneous localization and mapping (SLAM) [24]. Some theoretical works derived the asymptotic variance of the Rao-Blackwell particle ﬁlter in order to deter- mine in which cases it is interesting to use an RBPF in place of a standard PF with an increased number of particles [25]. When there is no linear sub-structure in the state vector, which is the case in this work, the standard Rao-Blackwellization cannot be applied directly. II. PROBLEM FORMULATION In this paper, we consider the following nonlinear discrete- time SSM related to a hidden state vector xk ∈Rnx and the measurement vector yk ∈Rny xk = f (xk−1) + ωk (1) yk = hθk (xk) + vk (2) (1) (2) (1) (2) where k = 1, . . . , K denotes the kth time instant, f (·) is the state transition function, ωk denotes the process noise with a zero mean Gaussian distribution of covariance matrix Qk, hθk (·) is the measurement function depending on a vector of parameters θk ∈Rnθ and vk denotes the measurement noise. In practice, the unknown parameter vector θk can be time- varying due to abruptly changing environments leading to different sensor functioning conditions. In addition, the mea- surement noise vk can be non-Gaussian due to abruptly changing environments leading to outliers corrupting the measurements. Since the Student-t distribution is heavy- tailed compared to the Gaussian distribution and is robust to outliers, this work assumes that the measurement noise has a Student-t distribution. This distribution is obtained by integrating an inﬁnite mixture of Gaussians with the same mean and scaled precision matrices [32], i.e., Several approaches can be found in the literature for jointly estimating state and static parameter in the frame of the RBPF. The common way is to replace analytical methods with different types of SMC samplers for implementing the parameter estimation, such as the particle Markov chain Monte Carlo [26], the twisted particle ﬁlter [27] and the nested particle ﬁlter [28], [29]. However, these approaches cannot be easily applied to cases where the parameters appearing in the measurement equation are time-varying. To address this problem, an RBPF based on a Dirichlet pro- cess mixture (DPM) was studied in [30]. In addition, the adap- tive parameter ﬁlter was proposed in [31] to implement a probabilistic changepoint model in the frame of the RBPF for estimating jointly the state vector and the time-varying unknown parameters. St (vk\|3k, νk) = Z ∞ 0 N vk\|0, (κk3k)−1 ×γ (κk\|νk/2, νk/2) dκk (3) (3) where St (·) denotes the student-t distribution and N (·) is the multivariate Gaussian probability density function (pdf) (here with zero mean and precision matrix κk3k), κk is an auxiliary precision scalar, γ (·) is the gamma distribution with shape and inverse scale parameters both equal to νk 2 and νk adjusts the thickness of the distribution tail. II. PROBLEM FORMULATION Independent conjugate gamma distributions are assigned a priori to 3k, κk and νk, i.e., the joint prior of (3k, κk, νk) is deﬁned as where St (·) denotes the student-t distribution and N (·) is the multivariate Gaussian probability density function (pdf) (here with zero mean and precision matrix κk3k), κk is an auxiliary precision scalar, γ (·) is the gamma distribution with shape and inverse scale parameters both equal to νk 2 and νk adjusts the thickness of the distribution tail. Independent conjugate gamma distributions are assigned a priori to 3k, κk and νk, i.e., the joint prior of (3k, κk, νk) is deﬁned as This paper studies an RBPF with variational inference for jointly estimating state and time-varying parameters in non- linear state-space models with measurement noise distributed according to a Student-t distribution. The interest of using this Student-t distribution is to obtain a robust estimator of the state vector. The idea of the resulting RBPF ﬁlter is to marginalize the joint distribution of interest with respect to the parameters of the Student-t distribution, to sample the remaining state and unknown parameters by using an auxiliary particle ﬁlter (APF) with a probabilistic change- point model, and to sample the Student-t parameters by using VB inference. The proposed approach combines the advantages of the adaptive parameter estimation (APE) ﬁlter introduced in [31] and the robust PF studied in [18] to build a joint state and time-varying parameter estimator in the presence of non-Gaussian measurement noise. q (3k, κk, νk) = Nd Y s=1 γ λs,k\|αs,k, βs,k ×γ κk\|ν1 k , ν2 k γ (νk\|ak, bk) (4) ×γ κk\|ν1 k , ν2 k γ (νk\|ak, bk) (4) (4) where 3k = diag λ1,k, · · · , λNd,k (a diagonal matrix with diagonal elements λ1,k, · · · , λNd,k), Nd is the dimension of 3k, αk, βk, ν1 k , ν2 k , ak, bk are the hyperparameters of the prior distributions at the kth time instant. 55666 VOLUME 8, 2020 C. Cheng et al.: Rao-Blackwellized PF With Variational Inference for State Estimation With Measurement Model Uncertainties A. UPDATING xk, θk SAMPLES BASED ON THE AUXILIARY PARTICLE FILTER Considering that both the parameter vector θk and the noise statistical parameters ξk = {3k, κk, νk} are time-varying in abruptly changing environments, the aim of this paper is to infer the states xk and the time-varying parameters θk, ξk given measurements y1:k = y1, · · · , yk . Assuming that the propagation models for xk and θk are independent, the posterior distribution p xk, θk\|y1:k can be recursively updated according to Bayes’ rule III. A RAO-BLACKWELLIZED PARTICLE FILTER WITH VARIATIONAL INFERENCE (10) Since the joint posterior distribution of all unknown vari- ables p xk, θk, ξk\|y1:k has a complex expression and cannot be calculated in closed form, we propose to study a PF approximating this posterior by using sequential importance sampling. The joint posterior distribution of the state and unknown parameters can be factorized as follows where the predictive distribution of the state p xk\|y1:k−1 can be obtained by using the Chapman-Kolmogorov equa- tion. This paper assumes that the predictive distribution p θk\|y1:k−1 can depend on θk−1 or on a non-informative prior distribution (i.e., can change to a value independent of θk−1). More precisely, the probabilistic model for the changepoint locations proposed in [34] is used for adap- tively estimating the time-varying parameter vector θk when a changepoint has occurred. We also assume that there is a probability η (ﬁxed to an a priori value for a given applica- tion) of a changepoint at each time instant. As a consequence, the predictive distribution of θk can be deﬁned as follows p xk, θk, ξk\|y1:k = p ξk\|xk, θk, y1:k p xk, θk\|y1:k (5) where the noise statistical parameters ξk have been marginal- ized out in the second term of the right hand side. We propose to approximate p xk, θk\|y1:k by using an empirical density following the principle of PFs p θk\|y1:k−1 = ( φθk−1 (θk) with probability 1 −η ψ (θk) with probability η (11) p θk\|y1:k−1 = ( φθk−1 (θk) with probability 1 −η ψ (θk) with probability η (11) (11) p xk, θk\|y1:k ≈ N X i=1 ωi kδ (xk, θk) − xi k, θi k (6) (6) where φθk−1 (·) denotes a prior distribution for θk given θk−1 (corresponding to the absence of changepoint) and ψ (·) denotes a non-informative prior distribution (corresponding to the presence of changepoint). Note that, as explained in [31], a large value for change probability η will intro- duce excess parameters from the diffuse prior ψ (θ) when no changepoint has occurred, whereas too small value for η will lead to difﬁculties in handling time-varying parameters. III. A RAO-BLACKWELLIZED PARTICLE FILTER WITH VARIATIONAL INFERENCE Replacing (6) in (5) leads to 3) Generate two sets of weights for presence and absence of changepoint at time k, i.e., p xk, θk, ξk\|y1:k ≈ N X i=1 ωi kp ξk\|xi k, θi k, y1:k ×δ (xk, θk) − xi k, θi k (9) ωi k,1 ∝p yk\|xi k, θi k,1, ξi,− k with θi k,1 ∼φθk−1 (θ) (12) ωi k,2 ∝p yk\|xi k, θi k,2, ξi,− k with θi k,2 ∼ψ (θ) (13) where i = 1, . . . , N. Thus N equally-weighted parti- cles xi θi N at the (k 1)th time instant lead ωi k,1 ∝p yk\|xi k, θi k,1, ξi,− k with θi k,1 ∼φθk−1 (θ) (12) ωi k,2 ∝p yk\|xi k, θi k,2, ξi,− k with θi k,2 ∼ψ (θ) (13) ωi k,1 ∝p yk\|xi k, θi k,1, ξi,− k with θi k,1 ∼φθk−1 (θ) (12) ωi k,1 ∝p yk\|xi k, θi k,1, ξi,− k with θi k,1 ∼φθk−1 (θ) (9) (12) ωi k,2 ∝p yk\|xi k, θi k,2, ξi,− k with θi k,2 ∼ψ (θ) (13) where the conditional distribution p ξk\|xi k, θi k, y1:k is the distribution of the noise parameter vector conditionally on the ith particle xi k, θi k . In this paper, we propose to approximate the distribution p ξk\|xi k, θi k, y1:k using VB inference. As a consequence, samples xi k, θi k are generated using an APF, and then the posterior distribution of ξk conditionally on xi k, θi k is calculated according to VB inference and replaced in (9), allowing the joint distribution of the state, parameters and hyperparameters to be determined. where the conditional distribution p ξk\|xi k, θi k, y1:k is the distribution of the noise parameter vector conditionally on the ith particle xi k, θi k . In this paper, we propose to approximate the distribution p ξk\|xi k, θi k, y1:k using VB inference. As a consequence, samples xi k, θi k are generated using an APF, and then the posterior distribution of ξk conditionally on xi k, θi k is calculated according to VB inference and replaced in (9), allowing the joint distribution of the state, parameters and hyperparameters to be determined. III. A RAO-BLACKWELLIZED PARTICLE FILTER WITH VARIATIONAL INFERENCE Based on the previous assumptions, we propose to sample (xk, θk) based on the APF by using the following steps: where φθk−1 (·) denotes a prior distribution for θk given θk−1 (corresponding to the absence of changepoint) and ψ (·) denotes a non-informative prior distribution (corresponding to the presence of changepoint). Note that, as explained in [31], a large value for change probability η will intro- duce excess parameters from the diffuse prior ψ (θ) when no changepoint has occurred, whereas too small value for η will lead to difﬁculties in handling time-varying parameters. Based on the previous assumptions, we propose to sample (xk, θk) based on the APF by using the following steps: where N is the number of particles, δ (·) is the Dirac delta function, xi k, θi k is the ith particle and ωi k is the correspond- ing weight at the kth time instant, which can be updated as follows [33] ωi k ∝p yk, xk, θk\|y1:k−1 ωi k−1. (7) (7) The conditional density p yk, xk, θk\|y1:k−1 can be obtained by the following marginalization 1) Sample xi k according to a proposal distribution q xk\|xi k−1, y1:k for i = 1, . . . , N where q (·) is the optimal importance distribution introduced in [20]. i 1) Sample xi k according to a proposal distribution q xk\|xi k−1, y1:k for i = 1, . . . , N where q (·) is the optimal importance distribution introduced in [20]. i p yk, xk, θk\|y1:k−1 = Z p yk, xk, θk\|ξk, y1:k−1 ×p ξk\|y1:k−1 dξk (8) (8) 2) Propagate the previous particles ξi k−1 by using the evo- lution equation of the noise parameters to create new particles ξi,− k , where the superscript ‘‘-’’ means that the quantity is computed a priori. where p ξk\|y1:k−1 is the predictive distribution of the noise statistical parameters. B. CALCULATING p ξk\|xi k, θi k, y1:k BASED ON VARIATIONAL BAYESIAN INFERENCE (21) The posterior distribution p ξk\|xi k, θi k, y1:k conditionally on the ith particle xi k, θi k cannot be calculated in closed form. According to the VB inference, this posterior can be approximated by another distribution q ξk , which is factorized into single-variable factors based on the mean- ﬁeld theory [35], i.e., q ξk = q (3k) q (κk) q (νk). Accord- ing to the VB approximation, the log marginal likelihood ln p yk\|xi k, θi k, ξk can be deﬁned by using the following identity where s = 1, . . . , Ns and 9 (·) denotes the digamma func- tion. According to (18)-(20), the hyperparameters of the pos- terior distribution conditionally on the ith particle xi k, θi k can be updated as follows [13] αi,+ s,k = αi,− s,k + 1 2 (22) βi,+ s,k = βi,− s,k + 1 2E h κi k i ys,k −hs,θi k xi k 2 (23) ν1,i,+ k = E νi k + 1 2 (24) ν2,i,+ k = E νi k 2 (22) (23) ln p yk\|xi k, θi k, ξk = L + KL (q\|\|p) (15) h L = Z q ξk ln p yk, ξk\|xi k, θi k, y1:k−1 q ξk dξk (16) ln p yk\|xi k, θi k, ξk = L + KL (q\|\|p) (15) (15) (24) with with L = Z q ξk ln p yk, ξk\|xi k, θi k, y1:k−1 q ξk dξk (16) (16) k 2 + yk −hθi k xi k T E 3i k yk −hθi k xi k 2 (25) ai,+ k = ai,− k + 1 2 (26) bi,+ k = bi,− k + E κi k 2 −E ln κi k 2 −1 2 (27) 2 + yk −hθi k xi k T E 3i k yk −hθi k xi k 2 (25) and (25) KL (q\|\|p) = Z q ξk ln q ξk p ξk\|xi k, θi k, y1:k dξk (17) (17) (26) where L is a variational lower bound for the log-marginal likelihood, KL (q\|\|p) is the Kullback-Leibler (KL) diver- gence between the true posterior and its approximation. B. CALCULATING p ξk\|xi k, θi k, y1:k BASED ON VARIATIONAL BAYESIAN INFERENCE Con- sidering that the KL divergence is non-negative, minimiz- ing the KL divergence can be achieved by maximizing the lower bound L, which results in computing expectations with respect to q (3k), q (κk) and q (νk) in turn. Straightforward computations lead to (27) where s = 1, · · · , Nd, i = 1, · · · , N. Note that the super- script ‘‘+’’ in the above equation means that the quantity is computed a posteriori. In order to maintain the conjugacy for the distribution of the noise statistical parameters, αs,k, βs,k, ak, bk are propagated as follows [12] αi,− s,k = ραi,+ s,k−1 βi,− s,k = ρβi,+ s,k−1 ai,− k = ρai,+ k−1 bi,− k = ρbi,+ k−1 (28) ln q (3k) ∝Eκk,νk h ln p yk\|xi k, θi k, 3k, κk + ln p (3k) i = Ns X s=1 αs,k −1 2 ln λs,k − βs,k + 1 2E [κk] ys,k −hs,θi k xi k 2 λs,k (18) ln q (κk) ∝E3k,νk h ln p yk\|xi k, θi k, 3k, κk + ln p (κk\|νk) i = E [νk] −1 2 ln κk −E νi k 2 κk ln q (3k) ∝Eκk,νk h ln p yk\|xi k, θi k, 3k, κk + ln p (3k) i = Ns X s=1 αs,k −1 2 ln λs,k − βs,k + 1 2E [κk] ys,k −hs,θi k xi k 2 λs,k (18 (28) where ρ is a forgetting factor, which can be considered as a weight balancing the historical estimates and the current information. Accordingly, a small forgetting factor can track faster changes in the noise statistical parameters whereas a larger value of this factor induces a slower response [17]. i (18) ln q (κk) ∝E3k,νk h ln p yk\|xi k, θi k, 3k, κk + ln p (κk\|νk) i = E [νk] −1 2 ln κk −E νi k 2 κk ln q (κk) ∝E3k,νk h ln p yk\|xi k, θi k, 3k, κk + ln p (κk\|νk) i = E [νk] −1 2 ln κk −E νi k 2 κk Thus p ξk\|xi k, θi k, y1:k can be approximated by iteratively calculating (22)-(27) until an iteration stopping rule is satis- ﬁed. III. A RAO-BLACKWELLIZED PARTICLE FILTER WITH VARIATIONAL INFERENCE Cheng et al.: Rao-Blackwellized PF With Variational Inference for State Estimation With Measurement Model Uncertainties 5) The weights for the selected N particles are updated according to the APF, i.e.,  ji ji ji − yk −hθi k xi k T E [3k] yk −hθi k xi k 2 κk (19) 5) The weights for the selected N particles are updated according to the APF, i.e., − yk −hθi k xi k T E [3k] yk −hθi k xi k 2 κk 5) The weights for the selected N particles are updated according to the APF, i.e., − yk −hθi k xi k T E [3k] yk −hθi k xi k 2 κk 5) The weights for the selected N particles are updated according to the APF, i.e., − yk −hθi k xi k T E [3k] yk −hθi k xi k 2 κk (19) ωi k =    p yk\|xji k, θji k,1, ξji,− k ωji k,1 when ji ∈{1, · · · , N} p yk\|xji k, θji k,2, ξji,− k ωji k,2 when ji ∈{N + 1, · · · , 2N} (14) ωi k =    p yk\|xji k, θji k,1, ξji,− k ωji k,1 when ji ∈{1, · · · , N} p yk\|xji k, θji k,2, ξji,− k ωji k,2 when ji ∈{N + 1, · · · , 2N} (14) 2 ln q (νk) ∝Eκk [ln p (κk\|νk) + ln p (νk)] = ak −1 2 ln νk − bk + E [κk] 2 −E [ln κk] 2 −1 2 where i = 1, · · · , Ns, ys,k and hs,θi k (·) de   p yk\|xji k, θji k,1, ξji,− k ωji k,1 when ji ∈{1 N} 2 ln q (νk) ∝Eκk [ln p (κk\|νk) + ln p (νk)] = ak −1 ln νk ln q (νk) ∝Eκk [ln p (κk\|νk) + ln p (νk)] = ak −1 2 ln νk − bk + E [κk] 2 −E [ln κk] 2 −1 2 νk (20) ωi k =   , when ji ∈{1, · · · , N} p yk\|xji k, θji k,2, ξji,− k ωji k,2 when ji ∈{N + 1, · · · , 2N} (14) (14) (20) where i = 1, · · · , Ns, ys,k and hs,θi k (·) denote the sth measurement and the corresponding nonlinear function. III. A RAO-BLACKWELLIZED PARTICLE FILTER WITH VARIATIONAL INFERENCE The expectations in the above equations can be expressed as follows where i = 1, · · · , Ns, ys,k and hs,θi k (·) denote the sth measurement and the corresponding nonlinear function. The expectations in the above equations can be expressed as follows where i = 1, · · · , N and ji denotes the index of the ith selected particle. E λs,k = αs,k βs,k E [κk] = ν1 k ν2 k E [ln κk] = 9 ν1 k −ln ν2 k E [νk] = ak bk (21) III. A RAO-BLACKWELLIZED PARTICLE FILTER WITH VARIATIONAL INFERENCE where i = 1, . . . , N. Thus N equally-weighted parti- cles xi k−1, θi k−1 N i=1 at the (k −1)th time instant lead to 2N samples at the kth time instant. where i = 1, . . . , N. Thus N equally-weighted parti- cles xi k−1, θi k−1 N i=1 at the (k −1)th time instant lead to 2N samples at the kth time instant. p 4) In order to estimate θk, N particles are selected from the 2N samples with probabilities proportional to n (1 −η) ω1 k,1, · · · , (1 −η) ωN k,1 o and n ηω1 k,2, · · · , ηωN k,2 o . 4) In order to estimate θk, N particles are selected from the 2N samples with probabilities proportional to n (1 −η) ω1 k,1, · · · , (1 −η) ωN k,1 o and n ηω1 k,2, · · · , ηωN k,2 o . 55667 VOLUME 8, 2020 C. A. ILLUSTRATIVE EXAMPLE i i i 10: Initialize αi,+ s,k (0) = αji,− s,k , βi,+ s,k (0) = βji,− s,k , ai,+ k (0) = aji,− k and i 10: Initialize αi,+ s,k (0) = αji,− s,k , βi,+ s,k (0) = βji,− s,k , ai,+ k (0) = aji,− k and i 10: Initialize αs,k (0) = αs,k , βs,k (0) = βs,k , ak (0) = ak and bi,+ k (0) = bji,− k where s = 1, · · · , Nd and i = 1, . . . , N. 11: for i = 1, . . . , N do 12: for r = 1, . . . , rmax do 13: Compute ν1,i,+ k (r) , ν2,i,+ k (r) according to (24) and (25); 14: Compute n αi,+ s,k (r) , βi,+ s,k (r) oNd s=1, ai+ k (r) and bi+ k (r) according to (22), (23), (26) and (27); 15: if the parameters change by less than 0.001 then 16: stop the iteration; 17: else 18: set r = r + 1; 19: end if 20: end for 21: end for 22: Normalize ωi k = ˜ωi k/ PN i=1 ˜ωi k and perform particle resam- pling. % Step 3: (Recursion) k = k + 1. 23: Go to Step 1. bi,+ k (0) = bji,− k where s = 1, · · · , Nd and i = 1, . . . , N. 11: for i = 1, . . . , N do 12: for r = 1, . . . , rmax do 13: Compute ν1,i,+ k (r) , ν2,i,+ k (r) according to (24) and (25); • APE ﬁlter with known variance [31]: The APE ﬁlter is combined to the APF with a changepoint model and the measurement noise model is assumed to be distributed according to a N (0, 1) distribution. • APE ﬁlter with unknown variance [31]: In the presence of measurement outliers, the estimation accuracy of the APE ﬁlter can be improved by considering that the measurement noise is a zero-mean Gaussian distribution with unknown variance, which is estimated with other parameters, i.e., the APE ﬁlter with unknown variance is combined to the APF with a changepoint model and the conjugate prior for the variance of Gaussian distribution is updated by using the state particles and measurements. Algorithm 1 Proposed RBPF With Variational Inference Algorithm 1 Proposed RBPF With Variational Inference Inputs: xi k−1, θi k−1, n αi s,k−1, βi s,k−1 oNd s=1 , ai k−1, bi k−1 N i=1 Outputs: xi k, θi k, n αi s,k, βi s,k oNd s=1 , ν1,i k , ν2,i k , ai k, bi k N i=1 % Step 1: Generate samples of (xk, θk) by using the APF. and bk in (23) and (27) require to know the values of ν1 k and ν2 k . Therefore, the estimates of ν1 k and ν2 k in (24) and (25) at the (r + 1)th iteration are calculated by using the estimates of αk, βk, ak and bk at the rth iteration. Finally, note that the estimates of βk and bk at the (r + 1)th iteration can be implemented based on the last estimates of ν1 k and ν2 k . The proposed RBPF with VB inference for joint state and time- varying parameter estimation is summarized in Alg. 1. Inputs: xi k−1, θi k−1, n αi s,k−1, βi s,k−1 oNd s=1 , ai k−1, bi k−1 N i=1 Outputs: xi k, θi k, n αi s,k, βi s,k oNd s=1 , ν1,i k , ν2,i k , ai k, bi k N i=1 n o s 1 i=1 % Step 1: Generate samples of (xk, θk) by using the APF 1: Generate xi k ∼ p xk\|xi k−1 by using (1), and then com- pute n αi,− s,k , βj,− s,k oNd s=1 , ai,− k , bi,− k according to (28) and generate νi,− k ∼γ νk\|ai,− k , bi,− k where i = 1, . . . , N. A. ILLUSTRATIVE EXAMPLE More details about these algorithms are provided below , , 5: Sample index ji in {1, · · · , 2N} with probabilities n (1 −η) ωi k,1 oN i=1 and n ηωi k,2 o2N i=N+1; 6: Propagate xi k ∼p xk\|xji k−1 ; , , 5: Sample index ji in {1, · · · , 2N} with probabilities n (1 −η) ωi k,1 oN i=1 and n ηωi k,2 o2N i=N+1; 5: Sample index ji in {1, · · · , 2N} with probabilities n (1 −η) ωi k,1 oN i=1 and n ηωi k,2 o2N i=N+1; 6: Propagate xi k ∼p xk\|xji k−1 ; 6: Propagate xi k ∼p xk\|xji k−1 ; 7: If ji ∈{1, · · · , N}, update θi k ∼φ θji k,1 (θ) and compute ˜ωi k ∝ ! 7: If ji ∈{1, · · · , N}, update θi k ∼φ θji k,1 (θ) and compute ˜ωi k ∝ p yk\|xji k ,θji k,1, κji k 3ji k −1! ωji k,1 ; p yk\|xji k ,θji k,1, κji k 3ji k −1! ωji k,1 ; 8: If ji ∈{N + 1, · · · , 2N}, set θi k = θji k,2 and compute ˜ωi k ∝ p yk\|xji k ,θji k,2, κji k 3ji k −1! ωji k,2 ; 9 d f • IMM Algorithm [1]: The models in the IMM differ by the choice of the parameter a where 10 equally spaced values of a are sampled in the range [−18, 18] and the measurement noise associated with each model is assumed to be distributed according to a N (0, 1) distribution. Moreover, the unscented Kalman ﬁlter is embedded in the IMM in order to estimate the state for the nonlinear SSM. k,2 9: end for % Step 2: Calculating q (λk, κk, vk) by using VB inference. i i i k,2 9: end for % Step 2: Calculating q (λk, κk, vk) by using VB inference. B. CALCULATING p ξk\|xi k, θi k, y1:k BASED ON VARIATIONAL BAYESIAN INFERENCE Note that the updates for ν1 k and ν2 k in (24) and (25) are functions of αk, βk, ak and bk, whereas the updates for βk 55668 VOLUME 8, 2020 C. Cheng et al.: Rao-Blackwellized PF With Variational Inference for State Estimation With Measurement Model Uncertainties A. ILLUSTRATIVE EXAMPLE 2: Generate n λi s,k ∼γ λk\|αi,− s,k , βi,− s,k oNd s=1 and κi k ∼ γ κk\| νi,− k 2 , νi,− k 2 where i = 1, . . . , N. In order to evaluate the proposed RBPF with variational infer- ence, we use the following modiﬁed benchmark model [6] for the illustrations 3: Generate θi k,1 ∼ φθk−1 (θ) and θi k,2 ∼ ψ (θ), and then compute ωi k,1 ∝ p yk\|xi k, θi k,1, κi k3i k −1 and ωi k,2 ∝ p yk\|xi k, θi k,2, κi k3i k −1 where 3i di h λi λi i d i 1 N 3: Generate θi k,1 ∼ φθk−1 (θ) and θi k,2 ∼ ψ (θ), and then compute ωi k,1 ∝ p yk\|xi k, θi k,1, κi k3i k −1 and ωi k,2 ∝ p yk\|xi k, θi k,2, κi k3i k −1 where 3i k = diag h λi 1,k, · · · , λi Nd,k i and i = 1, · · · , N. 4: for i = 1, . . . , N do xk = 0.5 xk−1 + 25 xk−1/ 1 + x2 k−1 + 8 cos (1.2 k) + ωk yk = x2 k /a + vk xk = 0.5 xk−1 + 25 xk−1/ 1 + x2 k−1 + 8 cos (1.2 k) + ωk yk = x2 k /a + vk where ωk ∼ N (0, 0.5), a is a time-varying param- eter taking values {5, −6, 7, 14, 5} with changes occur- ing at time instants {0, 40, 80, 120, 160}. The noise vk is the non-Gaussian measurement noise whose distri- bution is (1 −ε) N (0, 1) + εD where ε denotes the outlier probability, D denotes the outlier distribution, which was set to U (−20, 20) where U (·) is the uniform distribution [13], [36]. We compare the proposed approach with the interacting multiple model (IMM) algorithm [1] and the APE ﬁlter [31]. A. ILLUSTRATIVE EXAMPLE In the changepoint model applied to the APE ﬁlter and pro- posed strategy, the predictive distribution of a conditionally upon ak−1 is assumed to be a mixture of multivariate Gaus- sian distributions based on the kernel smoothing method [4], i.e., φak−1 (a) ≈ PN i=1 ωi k−1N a\|ζ i k−1, h2 Vk−1 where ζ i k−1 = τai k−1 + (1 −τ) a, Vk−1 = PN i=1 ωi k−1 ai k−1 −a 2, a = PN i=1 ωi k−1ai k−1, h2 is the kernel smoothing parameter, τ = √ 1 −h2 is the shrinkage parameter of the kernel mean, i 1 k 1 k 1 τ = √ 1 −h2 is the shrinkage parameter of the kernel mean, VOLUME 8, 2020 C. Cheng et al.: Rao-Blackwellized PF With Variational Inference for State Estimation With Measurement Model Uncertainties TABLE 1. Parameters used for the simulations on synthetic data. FIGURE 1. Mean of the estimates ba for 100 MC runs in the absence of outliers. FIGURE 2. Mean of the estimates ba for two different outlier probabilities (100 MC runs). TABLE 1. Parameters used for the simulations on synthetic data. FIGURE 1. Mean of the estimates ba for 100 MC runs in the absence of outliers. TABLE 1. Parameters used for the simulations on synthetic data. FIGURE 1. Mean of the estimates ba for 100 MC runs in the absence of outliers. while the non-informative prior pdf ψ (a) was assumed to be a uniform distribution. The related parameters used in all test scenarios are provided in Table 1. Nm = 100 Monte Carlo simulations have been run for any approach to compute the average root mean square errors (ARMSEs) of the estimates deﬁned by q (KNm)−1 PK k=1 PNm m=1 (bxk (m) −xk)2, where bxk (m) is the mth state estimate and K is the number of time instants. All algorithms have been coded using MATLAB and run on a laptop with Intel i-5 and 8 GB RAM. 1 FIGURE 2. Mean of the estimates ba for two different outlier probabilities (100 MC runs). outliers, whereas the estimation performance of the APE ﬁlter is robust to outliers when the variance of measurement noise is assumed to be time-varying and jointly estimated with the state. 1The estimator of the parameter a is denoted asba A. ILLUSTRATIVE EXAMPLE ARMSEs of state estimates for different outlier probabilities. collected from Nd in-view satellites in the presence of MP can be deﬁned using the following compact form yk = hθk (xk) + vk (29) (29) with hθk (xk) = h1,θ1,k (xk) , . . . , hNd,θNd,k (xk) T (30) (30) and hs,θs,k (xk) =∥ps,k −pk ∥+bk + θs,k (31) (31) TABLE 3. Execution times for different numbers of particles. TABLE 3. Execution times for different numbers of particles. where s = 1, . . . , Nd, xk is state vector for describing the dynamic of the vehicle in the earth-centered earth-ﬁxed T the dynamic of the vehicle in the earth-centered earth-ﬁxed (ECEF) frame at the kth time instant, yk = y1,k, . . . , yNd,k T is the pseudo-range measurement vector composed of all in-view satellites, ps,k = xs,k, ys,k, zs,k T and pk = (xk, yk, zk)T are the sth satellite and vehicle positions in the ECEF frame, bk is the GNSS receiver clock offset, ∥· ∥and (·)T denote the Euclidean norm and the transpose of a vector, respectively. In the presence of MP, θk = θ1,k, · · · , θNd,k T denotes the mean value jumps appearing on the pseudo-range measurement of all in-view satellites (θs,k = 0 in the absence of MP) and vk is the measurement noise distributed according to the Student-t distribution (as in (3)). In practice, an impor- tant property of MP signals is that they are not only depend on the relative position of the receiver and GNSS satellites, but also on the environment where the receiver is located, especially in urban canyons. Thus the mean value jump θk and the measurement noise parameters ξk = {3k, κk, vk} can be considered as unknown time-varying model parameters, which need to be estimated jointly with the vehicle state xk. In addition, the state vector xk considered in this paper is deﬁned as follows [42], [43] VB iterations is embedded in the APF before performing the update of the unknown parameter distribution, which leads to a higher computation load for APF, as reported in Table 3. Note that the computational cost of the proposed approach was evaluated in the following two situations: (a) the algo- rithm is stopped when the stopping rule in Line 15 of Alg. A. ILLUSTRATIVE EXAMPLE 1 is satisﬁed, (b) the number of variational iterations is set to its maximum value rmax. It is clear that the corresponding com- putational cost can be efﬁciently reduced by introducing the stopping rule of Line 15 of Alg. 1 in the proposed approach. A. ILLUSTRATIVE EXAMPLE However, the performance of the proposed approach is less affected by the presence of outliers due to the Student-t distribution, which is more robust to outliers than the Gaussian distribution. The ARMSEs of the state estimates obtained with the three approaches for different outlier prob- abilities are reported in Table 2. In presence or absence of outliers, the ARMSEs obtained with the proposed approach and the APE ﬁlter are obviously smaller than the one obtained with the IMM. Moreover, the state estimation accuracy of the proposed approach is better than that of the APE ﬁlter in the absence of outliers. Finally, thanks to the good robustness of the Student-t distribution to outliers, the proposed approach provides more accurate state estimates for any outlier proba- bility. Fig. 1 displays the means and standard deviations of ba 1 computed using 100 MC simulations for the IMM, the APE ﬁlter with known variance and the proposed approach in the absence of outliers (i.e., ε = 0). Considering that the changepoint model for parameter a is taken into account both in the proposed approach and in the APE ﬁlter, the esti- mation accuracies for parameter a obtained with these two approaches are obviously better than the one obtained with the IMM. Since the Student-t distribution provides almost the same solution as the Gaussian distribution in the absence of outliers, similar mean and standard deviation for the estimator ba should be obtained with the proposed approach and the APE ﬁlter. The means and standard deviations of ba computed using 100 MC simulations for different outlier probabilities are depicted in Fig. 2. It is clear that the estimation perfor- mance of the APE ﬁlter with known variance for param- eter a severely degrades in the presence of measurement Table 3 shows the execution times for 100 MC runs by using different numbers of particles for the proposed approach and the APE ﬁlter with unknown variance. The main difference between the proposed approach and the APE ﬁlter with unknown variance is that the computation of the 55670 VOLUME 8, 2020 VOLUME 8, 2020 h Variational Inference for State Estimation With Measurement Model Uncertainties C. Cheng et al.: Rao-Blackwellized PF With Variational Inference for State Estimation With Measurement Model Uncertainties d PF With Variational Inference for State Estimation With Measurement Model Uncertainties TABLE 2. ARMSEs of state estimates for different outlier probabilities. TABLE 2. B. GNSS MULTIPATH MITIGATION IN URBAN CANYONS This section validates the proposed RBPF with VB inference in the context of multipath (MP) interference mitigation for GNSS receivers in urban canyons. MP signals are mainly due to the fact that a signal transmitted by a navigation satellite is very likely to be reﬂected or diffracted and can follow different paths before arriving at the GNSS receiver [37]. Depending on the availability of the direct signal, reﬂected MP signals affecting the received direct GNSS signal can be divided into two situations: (a) MP interfereces resulting from the sum of the direct signal and of delayed reﬂections handled by the GNSS receiver; (b) non-line-of-sight (NLOS) signals resulting from a unique reﬂected signal received and tracked by the GNSS receiver [38], [39]. Accordingly, the pseudo- range measurement noise error in the MP interference sit- uation is considered as a non-Gaussian stochastic process, while a mean value jump is present on the pseudo-range measurement in the NLOS signal situation [40]–[42]. Since our aim is to estimate the pseudo-range measurement error resulting from MP rather than to identify the type of MP, the impact of MP signals on the pseudo-range measurement in this work is formulated as an unknown mean value jump and a non-Gaussian measurement noise distributed accord- ing to the Student-t distribution in this work. Assuming the atmospheric propagation errors can be compensated within the GNSS receiver, the pseudo-range measurement model xk = (xk, ˙xk, yk, ˙yk, zk, ˙zk, bk, dk)T (32) (32) where k = 1, . . . , K denotes the kth sampling time instant, ˙pk = (˙xk, ˙yk, ˙zk)T is the vehicle velocity in the ECEF frame, where k = 1, . . . , K denotes the kth sampling time instant, ˙pk = (˙xk, ˙yk, ˙zk)T is the vehicle velocity in the ECEF frame, dk is the GNSS receiver clock drift. The velocity can be reasonably modelled as a random walk, e.g., ¨x = ex where ex is a zero mean Gaussian noise of variance σ 2 a . For short- term applications in which the periodical clock resets of the GNSS receiver are not taken into account, the GNSS receiver clock offset bk and its drift dk can also be modelled as random walks, i.e., ˙bk = dk + eb and ˙dk = ed where eb and ed are zero-mean Gaussian white noises of variance σ 2 b and σ 2 d . 2The sign of the mean value jump depends on the value of the MP signal carrier phase relative to the direct signal. When the relative carrier phase belongs to −90◦, 90◦ , the sign of the mean value jump is positive. When the relative carrier phase belongs to −180◦, −90◦ or 90◦, 180◦ , the sign of the mean value jump is negative. B. GNSS MULTIPATH MITIGATION IN URBAN CANYONS The standard deviations of the process noise, the clock offset and drift noises were set to σa = 1 m/s2, σb = 3c×10−10 m and σd = 2πc×10−10 m/s, respectively, where c = 3 × 108 m/s denotes the velocity of light. The nominal standard deviation of the pseudo-range measurement noise used for the standard APF was adjusted by cross-validation and was set to 4 m, and the number of par- ticles for all three approaches is 5000. The other parameters used in the proposed approach are reported in Table 1. FIGURE 4. Distance to the origin versus time. used to evaluate the method proposed in [42]. A synchro- nized integrated navigation system composed of a Novatel receiver coupled to a tactical grade IMAR IMU was used to provide a reference trajectory. Taking advantage of a ground reference station, differential corrections were performed to obtain position accuracy close to 1 m for the reference trajec- tory, which is considered as the ground truth. For assessing the algorithm performance, the vehicle was equipped with a UBLOX 6T receiver. This receiver delivered not only the position, velocity and time solution, but also, for each satel- lite, the raw pseudo-range and Doppler frequency measure- ments, as well as the navigation message. It allowed us to compute satellite locations, and to perform timing and prop- agation correction on the measured pseudo-range. Data were collected in street urban canyons during which the receiver was strongly affected by MP signals, and post-processed using Matlab. used to evaluate the method proposed in [42]. A synchro- nized integrated navigation system composed of a Novatel receiver coupled to a tactical grade IMAR IMU was used to provide a reference trajectory. Taking advantage of a ground reference station, differential corrections were performed to obtain position accuracy close to 1 m for the reference trajec- tory, which is considered as the ground truth. For assessing the algorithm performance, the vehicle was equipped with a UBLOX 6T receiver. This receiver delivered not only the position, velocity and time solution, but also, for each satel- lite, the raw pseudo-range and Doppler frequency measure- ments, as well as the navigation message. It allowed us to compute satellite locations, and to perform timing and prop- agation correction on the measured pseudo-range. Data were collected in street urban canyons during which the receiver was strongly affected by MP signals, and post-processed using Matlab. Fig. B. GNSS MULTIPATH MITIGATION IN URBAN CANYONS In order to evaluate the proposed algorithm, we propose to compare its positioning estimation accuracy with that obtained using the standard APF and the Dirichlet process mixture (DPM)-based RBPF studied in [30]. In [30], the pseudo-range measurement noise in the absence of MP is assumed to have a zero mean Gaussian FIGURE 3. Urban canyon trajectory used in the proposed experiments (obtained with Google Earth). FIGURE 4. Distance to the origin versus time. FIGURE 5. Typical example of estimated mean value jumps. FIGURE 5. Typical example of estimated mean value jumps. FIGURE 3. Urban canyon trajectory used in the proposed experiments (obtained with Google Earth). FIGURE 3. Urban canyon trajectory used in the proposed experiments (obtained with Google Earth). FIGURE 3. Urban canyon trajectory used in the proposed experiments (obtained with Google Earth). FIGURE 5. Typical example of estimated mean value jumps. FIGURE 4. Distance to the origin versus time. not change during the time interval (78 s, 159 s), as the vehicle is stopped in the middle of two buildings. As it appears at the LOS frequency (the Doppler frequency related to the vehicle velocity is zero), pseudo-range measurements are severely impacted by mean value jump during this period. The in-view satellites observed during the experience are satellites #3, #6, #19, #26, and #27 (i.e., Nd = 5). The non- informative prior used for the mean value jump is a uni- form distribution deﬁned by ψ θs,k = U (−20 m, 20 m),2 where s = 1, · · · , Nd. In order to evaluate the proposed algorithm, we propose to compare its positioning estimation accuracy with that obtained using the standard APF and the Dirichlet process mixture (DPM)-based RBPF studied in [30]. In [30], the pseudo-range measurement noise in the absence of MP is assumed to have a zero mean Gaussian distribution with a nominal standard deviation, whereas the mean and standard deviation of the measurement noise can change abruptly or evolve slowly during a long period of time in the presence of MP, leading to time-varying measurement noise parameters. The DPM-approach of [30] was considered in this work since it provided interesting results for state- space models with time-varying parameters, in particular for MP mitigation in urban canyons. B. GNSS MULTIPATH MITIGATION IN URBAN CANYONS Based on the above assumptions, the discrete-time state model which describes the propagation of the vehicle state xk can be formulated as where k = 1, . . . , K denotes the kth sampling time instant, ˙pk = (˙xk, ˙yk, ˙zk)T is the vehicle velocity in the ECEF frame, dk is the GNSS receiver clock drift. The velocity can be reasonably modelled as a random walk, e.g., ¨x = ex where ex is a zero mean Gaussian noise of variance σ 2 a . For short- term applications in which the periodical clock resets of the GNSS receiver are not taken into account, the GNSS receiver clock offset bk and its drift dk can also be modelled as random walks, i.e., ˙bk = dk + eb and ˙dk = ed where eb and ed are zero-mean Gaussian white noises of variance σ 2 b and σ 2 d . Based on the above assumptions, the discrete-time state model which describes the propagation of the vehicle state xk can be formulated as xk = Fk\|k−1xk−1 + ek (33) (33) where k = 1, . . . , K denotes the kth sampling time instant, ek = ex, ey, ez, eb, ed T is a zero mean Gaussian noise vec- tor of covariance matrix Qk. Details on the matrices Fk\|k−1 and Qk can be found in [44]. The experimental data was collected during a measurement campaign carried out in Toulouse center (France) and was VOLUME 8, 2020 55671 C. Cheng et al.: Rao-Blackwellized PF With Variational Inference for State Estimation With Measurement Model Uncertainties FIGURE 3. Urban canyon trajectory used in the proposed experiments (obtained with Google Earth). FIGURE 4. Distance to the origin versus time. FIGURE 5. Typical example of estimated mean value jumps. not change during the time interval (78 s, 159 s), as the vehicle is stopped in the middle of two buildings. As it appears at the LOS frequency (the Doppler frequency related to the vehicle velocity is zero), pseudo-range measurements are severely impacted by mean value jump during this period. The in-view satellites observed during the experience are satellites #3, #6, #19, #26, and #27 (i.e., Nd = 5). The non- informative prior used for the mean value jump is a uni- form distribution deﬁned by ψ θs,k = U (−20 m, 20 m),2 where s = 1, · · · , Nd. B. GNSS MULTIPATH MITIGATION IN URBAN CANYONS 5 displays typical estimates for the mean value jumps bθk impacting the pseudo-range measurements of all in-view satellites during the measurement campaign. According to Fig. 3 shows the trajectory considered in our measurement campaign (lasting 240 s). Fig. 4 displays the evolution of the distance to the starting point of the trajectory (considered in our experiment) versus time, where the original point is deﬁned as the initial position on the trajectory and the trip distance represents the horizontal distance travelled from the initial position. It is clear that the distance to the origin does 55672 VOLUME 8, 2020 VOLUME 8, 2020 VOLUME 8, 2020 C. Cheng et al.: Rao-Blackwellized PF With Variational Inference for State Estimation With Measurement Model Uncertainties TABLE 4. Elevation angles for in-view satellites. FIGURE 6. Positioning errors versus time. TABLE 4. Elevation angles for in-view satellites. TABLE 4. Elevation angles for in-view satellites. solution if these interferences are not processed within the receiver. Conversely, the positioning errors obtained with the DPM-based RBPF and the proposed approach remains lower than 20 m, conﬁrming that the MP signals affecting the pseudo-range measurements have been mitigated. We think that the slightly better positioning accuracy obtained with the proposed approach with respect to the DPM-based RBPF is due to a good estimation of the locations of the mean value jumps, which is possible thanks to the use of the change-point model deﬁned in (11). Note that the MP amplitude, delay and phase are constant with respect to those of the direct signal when the vehicle stops in the middle of two buildings (leading to a constant jump). As a consequence, the impact result- ing from the MP signal is the maximum during this period (i.e., 78 s-159 s). V. CONCLUSION This paper proposed a Rao-Blackwellized particle ﬁlter with variational inference for jointly estimating state and time- varying parameters in non-linear state-space models with non-Gaussian measurement noise. The proposed approach embedded the variational Bayesian inference in an auxiliary particle ﬁlter with a probabilistic changepoint model to build a joint state and time-varying parameter estimator in the pres- ence of non-Gaussian measurement noise. A simulation study was conducted by using a modiﬁed nonlinear benchmark model in order to compare the performance of the proposed approach with the interacting multiple model and the adaptive parameter estimation ﬁlter. Since the Student-t distribution considered in the proposed approach is more robust to out- liers, the proposed approach provided better estimate accu- racy for the state and unknown parameters when compared to the other strategies. Finally, the proposed approach was validated by processing GNSS receiver data collected from a measurement campaign carried out in an urban environment and proved its efﬁciency for MP mitigation, resulting in improved positioning accuracy. FIGURE 6. Positioning errors versus time. One of the drawbacks of the proposed approach is the requirement that a conjugate prior of the unknown parame- ters needs to be available for optimizing the objective func- tion in the proposed variational Bayesian inference. Recent work on stochastic variational inference might solve this problem by applying stochastic optimization to the objective function [45]. Applying stochastic variational inference for estimating the state and parameters of the changepoint model investigated in this work is currently under investigation. Validating the proposed method using data from other mea- surement campaigns and high-dimensional state-space mod- els is also an interesting prospect. FIGURE 6. Positioning errors versus time. the results obtained with the proposed approach, the pseudo- range measurement of satellites #3, #6 and #27 are less affected by MP than the other received signals. 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The elevation angles of all in-view satellites during the measurement cam- paign are reported in Table 4. Note that the elevation angles for satellites #3, #6 and #27 are larger than 75◦, whereas the elevation angles for satellites #16 and #19 are less than or equal to 60◦. In general, the signals acquired with low elevation angles (for satellites #16 and #19) are more likely to be affected by MP, which seems to be the case here. ACKNOWLEDGMENT The authors would like to thank Audrey Giremus from the University of Bordeaux for helpful discussions on Dirichlet process mixtures. Fig. 6 displays the positioning errors (horizontal and vertical errors versus time) for the different approaches. It is clear that MP signals severely impair the positioning 55673 55673 VOLUME 8, 2020 C. Cheng et al.: Rao-Blackwellized PF With Variational Inference for State Estimation With Measurement Model Uncertainties C. Cheng et al.: Rao-Blackwellized PF With Variational Inference for State Estimation With Measurement Model Uncertaint REFERENCES He was the General Chair of the CIMI workshop on optimization and statistics in image processing hole in Toulouse, in 2013 (with F. Malgouyres and D. Kouamé) and of the Inter- national Workshop on Computational Advances in Multi-Sensor Adaptive Processing CAMSAP, in 2015 (with P. Djuric) and in 2019 (with D. Brie). He was a member of different technical committees, including the Signal Processing Theory and Methods (SPTM) Committee of the IEEE Signal Pro- cessing Society, from 2001 to 2007 and 2010 to 2015, and the EURASIP SAT committee on Theoretical and Methodological Trends in Signal Processing, from 2015 to 2019. In 2019, he joined the board of directors of EURASIP. He has been serving as an Associate Editor for the IEEE TRANSACTIONS ON SIGNAL PROCESSING, from 2008 to 2011 and 2015 to 2019, and the EURASIP Journal on Signal Processing, from 2013 to 2019. CHENG CHENG was born in Xi’an, China, in 1986. He received the B.S. and M.S. degrees from Northwestern Polytechnical University, Xi’an, in 2008 and 2011, respectively, and the Ph.D. degree from the Institut Supérieur de l’Aéronautique et de l’Espace, Toulouse, France, in 2015. He is currently an Assistant Professor with the School of Astronautics, Northwestern Polytechnical University. His research interests include Bayesian ﬁltering for various navigation systems and aerospace engineering. systems and aerospace engineering. JEAN-YVES TOURNERET (Fellow, IEEE) received the Ingénieur degree in electrical engi- neering from the Ecole Nationale Supérieure d’Electronique, d’Electrotechnique, d’Infor- matique, d’Hydraulique et des Télécommunica- tions (ENSEEIHT) de Toulouse, in 1989, and the Ph.D. degree from the National Polytechnic Insti- tute, Toulouse, in 1992. He is currently a Professor with the University of Toulouse (ENSEEIHT) and a member of the IRIT Laboratory (UMR 5505 of the CNRS). His research activities are centered around statistical signal and image processing with a particular interest to Bayesian and Markov chain Monte Carlo (MCMC) methods. He has been involved in the organiza- tion of several conferences, including the European Conference on Signal Processing EUSIPCO’02 (a Program Chair), the International Conference ICASSP’06 (plenaries), the Statistical Signal Processing Workshop SSP’12 XIAODONG LU was born in Xi’an, China, in 1979. He received the B.S., M.S., and Ph.D. degrees from Northwestern Polytechnical Univer- sity, Xi’an, in 2000, 2003, and 2006, respectively. He is currently an Associate Professor with the School of Astronautics, Northwestern Polytech- nical University. REFERENCES 899–924, May 2007. [44] A. Giremus, J.-Y. Tourneret, and V. Calmettes, ‘‘A particle ﬁltering approach for joint Detection/Estimation of multipath effects on GPS mea- surements,’’ IEEE Trans. Signal Process., vol. 55, no. 4, pp. 1275–1285, Apr. 2007. [22] T. Schon, F. Gustafsson, and P.-J. Nordlund, ‘‘Marginalized particle ﬁlters for mixed linear/nonlinear state-space models,’’ IEEE Trans. Signal Pro- cess., vol. 53, no. 7, pp. 2279–2289, Jul. 2005. [23] S. Särkkä, A. Vehtari, and J. Lampinen, ‘‘Rao-Blackwellized particle ﬁlter for multiple target tracking,’’ Inf. Fusion, vol. 8, no. 1, pp. 2–15, Jan. 2007. [45] C. Zhang, J. Bütepage, H. Kjellström, and S. Mandt, ‘‘Advances in varia- tional inference,’’ IEEE Trans. Pattern Anal. Mach. Intell., vol. 41, no. 8, pp. 2008–2026, Aug. 2019. [24] S. Thrun, ‘‘Particle ﬁlters in robotics,’’ in Proc. Uncertain. Artif. Intell., Alberta, WC, Canada, 2002, pp. 511–518. 55674 VOLUME 8, 2020 C. Cheng et al.: Rao-Blackwellized PF With Variational Inference for State Estimation With Measurement Model Uncertainties C. Cheng et al.: Rao-Blackwellized PF With Variational Inference for State Estimation With Measurement Model Uncertaint (international liaisons), the International Workshop on Computational Advances in Multi-Sensor Adaptive Processing CAMSAP 2013 (local arrangements), the Statistical Signal Processing Workshop SSP’2014 (special sessions), the Workshop On Machine Learning For Signal Pro- cessing MLSP’2014 (special sessions). He was the General Chair of the CIMI workshop on optimization and statistics in image processing hole in Toulouse, in 2013 (with F. Malgouyres and D. Kouamé) and of the Inter- national Workshop on Computational Advances in Multi-Sensor Adaptive Processing CAMSAP, in 2015 (with P. Djuric) and in 2019 (with D. Brie). He was a member of different technical committees, including the Signal Processing Theory and Methods (SPTM) Committee of the IEEE Signal Pro- cessing Society, from 2001 to 2007 and 2010 to 2015, and the EURASIP SAT committee on Theoretical and Methodological Trends in Signal Processing, from 2015 to 2019. In 2019, he joined the board of directors of EURASIP. He has been serving as an Associate Editor for the IEEE TRANSACTIONS ON SIGNAL PROCESSING, from 2008 to 2011 and 2015 to 2019, and the EURASIP Journal on Signal Processing, from 2013 to 2019. (international liaisons), the International Workshop on Computational Advances in Multi-Sensor Adaptive Processing CAMSAP 2013 (local arrangements), the Statistical Signal Processing Workshop SSP’2014 (special sessions), the Workshop On Machine Learning For Signal Pro- cessing MLSP’2014 (special sessions). REFERENCES His research interests include sensor data fusion, integrated navigation, and multitarget cooperative detection systems. 55675 VOLUME 8, 2020
https://openalex.org/W3150584435	https://www.frontiersin.org/articles/10.3389/fcimb.2021.620010/pdf	English	null	Draft Genome of Proteus mirabilis Serogroup O18 Elaborating Phosphocholine-Decorated O Antigen	Frontiers in cellular and infection microbiology	2,021	cc-by	7,360	ORIGINAL RESEARCH published: 25 March 2021 doi: 10.3389/fcimb.2021.620010 Draft Genome of Proteus mirabilis Serogroup O18 Elaborating Phosphocholine-Decorated O Antigen Proteus mirabilis is a pathogenic, Gram-negative, rod-shaped bacterium that causes ascending urinary tract infections. Swarming motility, urease production, bioﬁlm formation, and the properties of its lipopolysaccharide (LPS) are all factors that contribute to the virulence of this bacterium. Uniquely, members of the O18 serogroup elaborate LPS molecules capped with O antigen polymers built of pentasaccharide repeats; these repeats are modiﬁed with a phosphocholine (ChoP) moiety attached to the proximal sugar of each O unit. Decoration of the LPS with ChoP is an important surface modiﬁcation of many pathogenic and commensal bacteria. The presence of ChoP on the bacterial envelope is correlated with pathogenicity, as decoration with ChoP plays a role in bacterial adhesion to mucosal surfaces, resistance to antimicrobial peptides and sensitivity to complement-mediated killing in several species. The genome of P. mirabilis O18 is 3.98 Mb in size, containing 3,762 protein-coding sequences and an overall GC content of 38.7%. Annotation performed using the RAST Annotation Server revealed genes associated with choline phosphorylation, uptake and transfer. Moreover, amino acid sequence alignment of the translated licC gene revealed it to be homologous to LicC from Streptococcus pneumoniae encoding CTP:phosphocholine cytidylyltransferase. Recognized homologs are located in the O antigen gene clusters of Proteus species, near the wzx gene encoding the O antigen ﬂippase, which translocates lipid-linked O units across the inner membrane. This study reveals the genes potentially engaged in LPS decoration with ChoP in P. mirabilis O18. Edited by: Andres Felipe Opazo-Capurro, University of Concepcion, Chile Reviewed by: Michael Marceau, Universite´ Lille Nord de France, France Salim T. Islam, Universite´ du Que´ bec, Canada *Correspondence: Grzegorz Czerwonka gczerwonka@ujk.edu.pl Specialty section: This article was submitted to Clinical Microbiology, a section of the journal Frontiers in Cellular and Infection Microbiology Received: 21 October 2020 Accepted: 02 March 2021 Published: 25 March 2021 Keywords: Proteus mirabilis, phosphocholine, lipopolysaccharide, urinary tract infection, genome Edited by: Andres Felipe Opazo-Capurro, University of Concepcion, Chile Citation: Proteus mirabilis is an opportunistic Gram-negative bacterial pathogen that swarms across solid surfaces, which often leads to catheter-associated urinary tract infections. The hindered eradication of P. mirabilis results in recurrent urinary tract infections (Schaffer and Pearson, 2015). Flagella- based swarming motility and bioﬁlm formation, as well as the production of urease, hemolysin, and lipopolysaccharide (LPS) endotoxin, all contribute to the virulence of P. mirabilis. The presence of a long-chain O antigen is important for resistance to normal serum, and is involved in swarming Czerwonka G, Gmiter D and Durlik-Popin´ ska K (2021) Draft Genome of Proteus mirabilis Serogroup O18 Elaborating Phosphocholine-Decorated O Antigen. Front. Cell. Infect. Microbiol. 11:620010. doi: 10.3389/fcimb.2021.620010 March 2021 \| Volume 11 \| Article 620010 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Genome of Proteus mirabilis O18 Czerwonka et al. motility as well as the formation of the cell-surface glycocalyx (Knirel et al., 2011). The O antigen consists of repeating polysaccharide units (O units) that typically contain two to eight sugar residues that deﬁne the serological speciﬁcity of Gram- negative bacteria (Liu et al., 2014). Due to the high structural diversity of P. mirabilis O antigens, infections by different serotypes may activate different host immune responses. To date, 83 serogroups within the genus Proteus have been identiﬁed (Siwińska et al., 2020). An interesting and unique modiﬁcation of the P. mirabilis O antigen is its decoration with phosphocholine (ChoP), which occurs in strains belonging to the O18 serogroup (Fudala et al., 2003). Decoration of glycans with ChoP protects bacteria from innate and adaptive immune system responses, and modiﬁes interactions with host proteins engaged in bacterial pathogenesis (Young et al., 2013). conservation between wzy genes of different serotypes enables the use of this gene as a molecular marker for molecular serotyping (Wang et al., 2017). The ﬁrst complete genome of P. mirabilis was characterised for the widely-studied strain HI4320 (serogroup O28) isolated from the urine of a patient with a long-term indwelling urinary catheter. Genome annotation revealed the coding sequences and genomic locations of previously-characterized virulence determinants. From this annotation, genes predicted to be involved in LPS biosynthesis were identiﬁed (Pearson et al., 2008). Subsequent studies have reported that Proteus serogroups can be genetically distinguished based on the sequences of their respective O antigen biosynthesis clusters; however, information about the O18 serogroup was incomplete (Yu et al., 2017). Here we report the draft genome sequence of P. Citation: mirabilis strain PrK 34/57 belonging to the O18 serogroup, including the fully-sequenced and annotated O antigen biosynthesis gene cluster. Biosynthesis of ChoP and decoration of LPS involves four enzymes: choline kinase (LicA), choline transmembrane transporter (LicB), CTP:phosphocholine cytidylyltransferase (LicC) and lipopolysaccharide cholinephosphotransferase (LicD). The ﬁrst steps in this process is choline uptake from the cell surroundings, involving choline permease LicB and phosphorylation of free choline to ChoP by choline kinase LicA (Young et al., 2013). Choline uptake is carried by both LicB and BetT (a high-afﬁnity choline transporter). The BetT permease contributes to the utilisation of choline in the osmoprotection system, in which choline is oxidized to form glycine betaine that acts as an osmoprotectant, as demonstrated for Escherichia coli (Fan et al., 2003). In the LPS decoration pathway, phosphorylated choline is modiﬁed via the CDP- choline pathway to CDP-ChoP by LicC. This enzyme catalyzes the transfer of a cytidine monophosphate from CTP to phosphocholine to form CDP-choline (Kwak et al., 2002). The ﬁnal event in the decoration process is CDP-ChoP transfer to a target glycan, mediated by LicD (Young et al., 2013). This enzyme decorates cell surface structures such as LPS in Gram- negative species, e.g. H. inﬂuenzae and commensal Neisseria, the pili of Neisseria meningitidis, and lipoteichoic acids and teichoic acids of S. pneumoniae (Geiger et al., 2013). Importantly, only in P. mirabilis and Morganella morganii, the O antigens are decorated with ChoP (Clark and Weiser, 2013). Sequencing Libraries were prepared using the Nextera XT kit (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s protocol. Libraries were sequenced on the NextSeq machine (Illumina) with 2 × 150 bp paired-end reads with a depth of 200-fold coverage. Over 91.00% of bases of sequencing reads had quality scores ≥Q30. Our studies revealed the homologs of licABCD genes presumably responsible for LPS decoration with ChoP. The biosynthesis of O antigen is regulated by genes which are generally organized in an O antigen gene cluster (Yu et al., 2017). Certain O antigens may contain unique modiﬁcations, and genes engaged in their processing are also located within this cluster (Yu et al., 2017). In the Wzx/Wzy-dependent assembly pathway the O antigen repeating units are linked to a lipid carrier (undecaprenyl pyrophosphate) and engage a transmembrane ﬂippase (Wzx) that ﬂips O units from the cytoplasm to the periplasm, where O antigen subunits are polymerized by Wzy. The length of the O antigen chain is regulated by the action of Wzz, a polysaccharide copolymerase (PCP) (Islam and Lam, 2014). The conservation of the O antigen gene cluster among bacteria is low, and heterogeneity of this region results in the synthesis of different O antigens (Islam and Lam, 2014). The low Bacterial Strains and DNA Extraction Bacterial Strains and DNA Extraction P. mirabilis strain PrK 34/57 (O18) was obtained from the Czech National Collection of Type Cultures in Prague, Czech Republic. Cells were grown overnight in 50 ml of LB (Biocorp, Warsaw, Poland), diluted in ratio 1:100 with fresh LB medium and cultivated for following tests at 37°C with shaking (160 rpm) for 12–18 h in an Ecotron incubator (Infors HT, Basel, Switzerland). Genomic DNA was isolated with a GenElute™ Bacterial Genomic DNA Kit (Sigma-Aldrich, Saint Louis, MO, USA) according to manufacturer’s protocol from 1.5 ml of overnight culture. Final elution was performed with 100 ml of nuclease-free water. DNA quality was assessed using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientiﬁc, Waltham, MA, USA). ac e a S a s a d ac o P. mirabilis strain PrK 34/57 (O18) was obtained from the Czech National Collection of Type Cultures in Prague, Czech Republic. Cells were grown overnight in 50 ml of LB (Biocorp, Warsaw, Poland), diluted in ratio 1:100 with fresh LB medium and cultivated for following tests at 37°C with shaking (160 rpm) for 12–18 h in an Ecotron incubator (Infors HT, Basel, Switzerland). Genomic DNA was isolated with a GenElute™ Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org The Virulome Investigation The presence of virulence driving genes was investigated using a local data base created with the makeblastdb option of BLAST+ (Camacho et al., 2009). Genes for data base construction were selected based on previously-described genome of P. mirabilis HI4320 strain. The database included genes responsible for ureolitic, proteolitic and hemolytic activity, motility (ﬂagellum synthesis and chemotaxis), and ﬁmbriae synthesis. Resistance Gene Identiﬁer The resistome of P. mirabilis PrK 34/57 was predicted from nucleotide data based on homology and SNP models using the Resistance Gene Identiﬁer (RGI) based on The Comprehensive Antibiotic Resistance Database (CARD; https://card.mcmaster.ca) (Alcock et al., 2020). The DNA was used as a data type, selection criteria was selected as perfect and strict hits only, the nudges above 95% was excluded, and the sequence quality was selected as a high coverage. Genome ‘De Novo’ Assembly The trimmed Illumina reads were assembled using Unicycler, an assembly pipeline for bacterial genomes (Wick et al., 2017). The Unicycler Version 0.4.8.0 was available online on the Galaxy web Server (https://usegalaxy.org/). The Unicycler pipeline functions as a SPAdes-optimizer. For the assembly, the default options of SPADes were selected, that includes turned on error correction, the k-mer in a range of 0.2 to 0.95 (expressed as a fraction of the read length). Contigs with a fraction of the chromosomal depth lower than 0.25 were ﬁltered out. In terms of Unicycler options, March 2021 \| Volume 11 \| Article 620010 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Genome of Proteus mirabilis O18 Czerwonka et al. output of Mauve, was used to visualize the genomes comparison with the R package genoPlotR (Guy et al., 2011). the Normal Bridge mode (moderate contigs size and moderate misassembly rate) was selected. Contigs shorter than 500 bp were excluded from the ﬁnal assembly. Genome Accession Number This Whole Genome Shotgun Project of P. mirabilis PrK 34/57 has been deposited at GenBank (http://www.ncbi.nlm.nih.gov) under the accession JAAMPE000000000. The version described in this paper is version JAAMPE010000000. Variant Calling Analysis For variant calling analysis of PrK 34/57, the Illumina reads were tested using Snippy (Galaxy Version 4.4.5+galaxy2) with the default parameters and HI4320 as a reference genome (https:// github.com/tseemann/snippy) (Bush et al., 2020). Genome Annotation Analysis of KEGG pathways in PrK 34/57 was conducted by GhostKOALA, an automated metagenome annotation server that characterizes gene functions and pathways based on KEGG Orthology sequence assignments (Kanehisa et al., 2016). As an input ﬁle, the Amino-Acid FASTA ﬁle generated by RAST was used. The obtained contigs were reordered relative to the P. mirabilis HI4320 reference genome using Mauve Contig Mover (MCM) of Mauve 2.4.0 software to facilitate the study (Darling et al., 2004; Rissman et al., 2009). The default parameters were used. Genome sequence of PrK 34/57 was primarily functionally annotated by Rapid Annotation Subsystems Technology (RAST) server using the ClassicRAST annotation scheme, FIGfams version 70, automatic error correction, and automatic frame shift correction (Aziz et al., 2008; Gmiter et al., 2019). Further, during WGS (Whole Genome Shotgun) submission to NCBI, it was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al., 2016) to ensure better insight into the genomic features. Phylogenetic Analysis y g y In order to perform the P. mirabilis PrK 34/57 comparative study, selected P. mirabilis, both complete and WGS type, genomes were downloaded from NCBI and presented in Table 1. For WGS genomes, the reordering of contigs was performed using MCM as above. The comparative genomics include the phylogenomic analysis based on average nucleotide identities (ANI) using FastANI algorithm (Jain et al., 2018) and single nucleotide polymorphisms (SNPs) using CSI Phylogeny webserver (Kaas et al., 2014). The CSI Phylogeny webserver was used with the default options, including minimum depth at SNP positions = 10×, minimum relative depth at SNP positions = 10%, minimum distance between SNPs = 10 bp, minimum SNP quality = 30, minimum read mapping quality = 25 and minimum Z-score = 1.96. The obtained phylogeny tree includes the P. mirabilis HI4320 as a reference genome. The tree resulted from the CSI Phylogeny webserver was visualized using Interactive Tree Of Life (iTOL) version 5 (Letunic and Bork, 2019). To determine global rearrangement structure between P. mirabilis PrK 34/57 and other genomes, the progressiveMauve option of Mauve was used (Darling et al., 2004; Darling et al., 2010). The backbone ﬁle, an Genome Overview TABLE 2 \| Proteus mirabilis PrK 34/57 genome assembly statistics based on RAST annotation server. Attribute Value Genome size 3,970,593 %GC 38.7 N50 (bp) 203,813 L50 (bp) 8 Number of contigs (with PEGs) 60 Number of subsystems 497 Number of coding sequences 3,620 Number of RNAs 70 which are important for the ability of P. mirabilis strains to swarm over solid surfaces. We also identiﬁed genes encoding urease subunits (nitrogen metabolism category), an important enzyme for P. mirabilis survival in the urinary tract. Furthermore, genes involved in choline transport and metabolism were identiﬁed (miscellaneous category). The detailed subsystems present in PrK 34/57 are summarised in Table 3. Variant calling is a useful comparative genomic method that provides insight into organismal differences at the nucleotide level (Fariq et al., 2019). A set of variants are identiﬁed from tested and reference genomes, including small polymorphisms, speciﬁcally single-nucleotide polymorphisms (SNPs), insertions and deletions (indels), multi- nucleotide polymorphisms (MNPs) and complex events (composite insertion and substitution events) smaller than the length of short-read sequencing alignment. During our current analysis, 18,240 variants were identiﬁed between PrK 34/57 and HI4320. For all variants, their FILTERs status was PASS, indicating that variants in the raw data are true calls and not false positives resulting from low coverage depth. Observed variants were divided into ﬁve categories, and numbers and frequencies are summarized in Table 4. Using GhostKOALA annotation server totally 2342 entries (64.7%) was annotated in the genome of PrK 34/57. The distribution and frequency of KEGG pathways was presented on Figure 1. evident, with only a few genome rearrangement events. These occasional events are illustrated by the crossing lines that link Locally Collinear Blocks (LCBs). LCBs were calculated by Mauve to identify conserved segments that appear to be internally free from genome rearrangements. However, the PrK 34/57 genome displays blocks below the genome’s center line, which suggests the presence of inverted regions in PrK 34/57 (regions that align in the reverse complement (inverse) orientation) compared with other strains (Darling et al., 2004). Genome Overview The genome of P. mirabilis strain PrK 34/57 was determined to be 3,970,593 bp in length, with a GC content of 38.7%. It was assembled into 60 contigs (>500 bp). Based on PGAP annotation, there are 3,722 genes, of which 3,594 (96.6%) are protein-coding genes, 56 (1.5%) are pseudogenes and the remaining 72 are predicted RNA-coding genes, including 66 tRNAs, 2 rRNAs and 4 ncRNAs. The general features of P. mirabilis PrK 34/57 assembly identiﬁed by RAST are presented in Table 2. In contrast to PGAP, 3,620 genes were annotated using RAST, which were assigned to 497 subsystems. Importantly, among virulence, disease and defence subsystem categories, we identiﬁed genes involved in bacteriocin tolerance and production, as well as tolerance to antibiotics and heavy metals (including efﬂux pumps). Moreover, PrK 34/57 possesses 56 genes related to iron acquisition and metabolism. Additionally, there is a set of 57 genes related to motility and chemotaxis, both of TABLE 1 \| Proteus mirabilis genomes used in the study. Strain Accession number Genome size (bp) Reference HI4320 AM942759 4,063,606 Pearson et al. (2008) BB2000 CP004022 3,846,754 Sullivan et al. (2013) GN2 CP026581 4,012,640 Li et al. (2018) BC11-24 CP026571 4,021,165 Lei et al. (2018) K1609 CP028522 3,817,795 Gmiter et al. (2019) K670 CP028356 3,935,626 Gmiter et al. (2019) Pr2921 LGTA00000000 3,924,499 Giorello et al. (2016) 1230_SSON NZ_JVXV01000000 3,923,692 Roach et al. (2015) PM_125 NZ_LWUL00000000 3,955,474 Yu et al. (2016) TABLE 1 \| Proteus mirabilis genomes used in the study. March 2021 \| Volume 11 \| Article 620010 3 Genome of Proteus mirabilis O18 Czerwonka et al. TABLE 3 \| Subsystem distribution of Proteus mirabilis PrK 34/57 via RAST server-based annotation. Subsystems Counts Cofactors, Vitamins, Prosthetic groups, Pigments 249 Cell wall and Capsule 161 Virulence, Disease and Defense 73 Potassium metabolism 25 Photosynthesis 0 Miscellaneous 45 Phages, Prophages, Transposable elements, Plasmids 62 Membrane transport 173 Iron acquisition and metabolism 56 RNA metabolism 223 Nucleosides and Nucleotides 100 Protein metabolism 273 Cell division and Cell cycle 38 Motility and Chemotaxis 57 Regulation and Cell signalling 99 Secondary metabolism 4 DNA metabolism 105 Fatty acids, Lipids, and Isoprenoids 110 Nitrogen metabolism 26 Dormancy and Sporulation 6 Respiration 151 Stress response 132 Metabolism of Aromatic compounds 3 Amino acids and Derivatives 367 Sulfur metabolism 37 Sulfur metabolism 35 Carbohydrates 327 TABLE 3 \| Subsystem distribution of Proteus mirabilis PrK 34/57 via RAST server-based annotation. March 2021 \| Volume 11 \| Article 620010 The Virulome Investigation 1230_SSON BB2000 BC11-24 GN2 HI4320 K670 K1609 PM_125 Pr2921 PrK 34/57 1230_SSON 100 98.7973 98.6261 98.8935 99.1878 99.0408 98.7498 99.0670 98.9698 99.0348 BB2000 98.7973 100 99.0143 98.8578 98.8301 98.8168 99.3476 98.8259 98.7436 98.7991 BC11-24 98.6261 99.0143 100 98.6209 98.6625 98.6047 98.8936 98.5968 98.5448 98.6077 GN2 98.8935 98.8578 98.6209 100 98.8563 99.0234 98.7216 98.8451 98.9670 98.9807 HI4320 99.1878 98.8301 98.6625 98.8563 100 98.9659 98.7721 99.0081 98.9928 98.9781 K670 99.0408 98.8168 98.6047 99.0234 98.9659 100 98.7839 98.8571 99.0467 99.1747 K1609 98.7498 99.3476 98.8936 98.7216 98.7721 98.7839 100 98.6677 98.7019 98.7622 PM_125 99.0670 98.8259 98.5968 98.8451 99.0081 98.8571 98.6677 100 98.8820 98.9628 Pr2921 98.9698 98.7436 98.5448 98.9670 98.9928 99.0467 98.7019 98.8820 100 99.0904 PrK_34_57 99.0348 98.7991 98.6077 98.9807 98.9781 99.1747 98.7622 98.9628 99.0904 100 Values represent the average nucleotide identity in percent. The ANI values for P. mirabilis PrK 34/57 strain are indicated by bold font. FIGURE 2 \| Whole genome phylogeny of Proteus mirabilis strains based on single nucleotide polymorphisms (SNPs) using CSI Phylogeny webserver. The sequence of studied P. mirabilis PrK 34/57 strain is indicated by bold font. The obtained Newick ﬁle was visualized using Interactive Tree Of Life (iTOL) version 5. TABLE 5 \| ANI similarity matrix between studied Proteus mirabilis genomes. FIGURE 2 \| Whole genome phylogeny of Proteus mirabilis strains based on single nucleotide polymorphisms (SNPs) using CSI Phylogeny webserver. The sequence of studied P. mirabilis PrK 34/57 strain is indicated by bold font. The obtained Newick ﬁle was visualized using Interactive Tree Of Life (iTOL) version 5. FIGURE 2 \| Whole genome phylogeny of Proteus mirabilis strains based on single nucleotide polymorphisms (SNPs) using CSI Phylogeny webserver. The sequence of studied P. mirabilis PrK 34/57 strain is indicated by bold font. The obtained Newick ﬁle was visualized using Interactive Tree Of Life (iTOL) version 5. The Virulome Investigation TABLE 5 \| ANI similarity matrix between studied Proteus mirabilis genomes. 1230_SSON BB2000 BC11-24 GN2 HI4320 K670 K1609 PM_125 Pr2921 PrK 34/57 1230_SSON 100 98.7973 98.6261 98.8935 99.1878 99.0408 98.7498 99.0670 98.9698 99.0348 BB2000 98.7973 100 99.0143 98.8578 98.8301 98.8168 99.3476 98.8259 98.7436 98.7991 BC11-24 98.6261 99.0143 100 98.6209 98.6625 98.6047 98.8936 98.5968 98.5448 98.6077 GN2 98.8935 98.8578 98.6209 100 98.8563 99.0234 98.7216 98.8451 98.9670 98.9807 HI4320 99.1878 98.8301 98.6625 98.8563 100 98.9659 98.7721 99.0081 98.9928 98.9781 K670 99.0408 98.8168 98.6047 99.0234 98.9659 100 98.7839 98.8571 99.0467 99.1747 K1609 98.7498 99.3476 98.8936 98.7216 98.7721 98.7839 100 98.6677 98.7019 98.7622 PM_125 99.0670 98.8259 98.5968 98.8451 99.0081 98.8571 98.6677 100 98.8820 98.9628 Pr2921 98.9698 98.7436 98.5448 98.9670 98.9928 99.0467 98.7019 98.8820 100 99.0904 PrK_34_57 99.0348 98.7991 98.6077 98.9807 98.9781 99.1747 98.7622 98.9628 99.0904 100 Values represent the average nucleotide identity in percent. The ANI values for P. mirabilis PrK 34/57 strain are indicated by bold font. TABLE 5 \| ANI similarity matrix between studied Proteus mirabilis genomes. 1230_SSON BB2000 BC11-24 GN2 HI4320 K670 K1609 PM_125 Pr2921 PrK 34/57 1230_SSON 100 98.7973 98.6261 98.8935 99.1878 99.0408 98.7498 99.0670 98.9698 99.0348 BB2000 98.7973 100 99.0143 98.8578 98.8301 98.8168 99.3476 98.8259 98.7436 98.7991 BC11-24 98.6261 99.0143 100 98.6209 98.6625 98.6047 98.8936 98.5968 98.5448 98.6077 GN2 98.8935 98.8578 98.6209 100 98.8563 99.0234 98.7216 98.8451 98.9670 98.9807 HI4320 99.1878 98.8301 98.6625 98.8563 100 98.9659 98.7721 99.0081 98.9928 98.9781 K670 99.0408 98.8168 98.6047 99.0234 98.9659 100 98.7839 98.8571 99.0467 99.1747 K1609 98.7498 99.3476 98.8936 98.7216 98.7721 98.7839 100 98.6677 98.7019 98.7622 PM_125 99.0670 98.8259 98.5968 98.8451 99.0081 98.8571 98.6677 100 98.8820 98.9628 Pr2921 98.9698 98.7436 98.5448 98.9670 98.9928 99.0467 98.7019 98.8820 100 99.0904 PrK_34_57 99.0348 98.7991 98.6077 98.9807 98.9781 99.1747 98.7622 98.9628 99.0904 100 Values represent the average nucleotide identity in percent. The ANI values for P. mirabilis PrK 34/57 strain are indicated by bold font. FIGURE 2 \| Whole genome phylogeny of Proteus mirabilis strains based on single nucleotide polymorphisms (SNPs) using CSI Phylogeny webserver. The sequence of studied P. mirabilis PrK 34/57 strain is indicated by bold font. The obtained Newick ﬁle was visualized using Interactive Tree Of Life (iTOL) version 5. TABLE 5 \| ANI similarity matrix between studied Proteus mirabilis genomes. The Virulome Investigation The fastANI analysis of the P. mirabilis PrK 34/57 genome revealed high similarity with other genomes. ANI values were 98.6077 to 99.1747% compared with BC11-24 and K670 genomes, respectively. These values far exceed the generally accepted 95% cut-off level for taxonomy afﬁliation of newly sequenced genomes (Figueras et al., 2014). The results of detailed comparison of the genomes are presented in Table 5. To better explore virulence potential of P. mirabilis strain PrK 34/57 the presence of genes involved in expression of most important virulence factors was tested using local virulence factor databases. From this analysis, genes associated with all major virulence factors were detected in PrK 34/57. The sequence similarity with HI4320 was up to 100% depending on gene class. Interestingly, though previously detected in strain HI4320, genes encoding ﬁmbriae 3 (ﬁm3A) were not detected in PrK 34/57 (Table 6). A phylogenetic tree based on SNPs identiﬁed from whole genome comparisons (Figure 2) resulted in three major clades: PrK 34/57; the HI4320 reference genome; and BB2000, another frequently studied P. mirabilis strain (Sullivan et al., 2013). The PrK 34/57 genome is most similar to the previously reported genome of K670 (Gmiter et al., 2019), and shares greater similarity with HI43200 than BB2000. These observations correspond to the results of ANI-based analysis. Similar results were obtained following analysis of whole genome phylogeny of P. mirabilis strains based on the Mauve comparison (data not shown). The global rearrangement structure of the studied P. mirabilis genomes is visualized in Figure 3. Signiﬁcant similarity between genomes is TABLE 4 \| Summary of variants in Proteus mirabilis PrK 34/57 genome against P. mirabilis HI4320 reference strain. Total number of variations Categories SNPs MNPs Insertions Deletions Complex Count 18,240 16,489 245 106 117 1283 Frequency (%) 100 90.40 1.34 0.58 0.64 7.03 March 2021 \| Volume 11 \| Article 620010 TABLE 4 \| Summary of variants in Proteus mirabilis PrK 34/57 genome against P. mirabilis HI4320 reference strain. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Czerwonka et al. Genome of Proteus mirabilis O18 FIGURE 1 \| KEGG pathway analysis of Proteus mirabilis PrK 34/57 strain. Proteins were identiﬁed and categorized using the GhostKOALA tool against the Amino-Acid ﬁle generated by RAST server. FIGURE 1 \| KEGG pathway analysis of Proteus mirabilis PrK 34/57 strain. Proteins were identiﬁed and categorized using the GhostKOALA tool against the Amino-Acid ﬁle generated by RAST server. Identiﬁcation of Putative Antibiotic Resistance Genes (amikacin, tobramycin) and beta-lactams (ampicillin, piperacillin, amoxicillin), and the results are presented in Table 7. Antimicrobial peptides resistance in this strain is presumably supported by homologs of Klebsiella pneumoniae KpnFH efﬂux pumps (Poirel et al., 2017; Aghapour et al., 2019). Based on the Comprehensive Antibiotic Research Database (CARD), the Resistance Gene Identiﬁer (RGI) tool identiﬁed homologs of genes responsible for resistance to aminoglycosides March 2021 \| Volume 11 \| Article 620010 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Genome of Proteus mirabilis O18 Czerwonka et al. FIGURE 3 \| Whole genome comparison of Proteus mirabilis genomes using progressiveMauve option of Mauve software. Genomes are represented in blue, and blocks with borders of different colors are homologous between genomes The backbone ﬁle was visualized using the R package genoPlotR. FIGURE 3 \| Whole genome comparison of Proteus mirabilis genomes using progressiveMauve option of Mauve software. Genomes are represented in blue, and blocks with borders of different colors are homologous between genomes The backbone ﬁle was visualized using the R package genoPlotR. FIGURE 3 \| Whole genome comparison of Proteus mirabilis genomes using progressiveMauve option of Mauve software. Genomes are represented in blue, and blocks with borders of different colors are homologous between genomes The backbone ﬁle was visualized using the R package genoPlotR. TABLE 6 \| Presence of genes related to Proteus mirabilis virulence in PrK 34/57. Virulence factor Presence/absence Enzymatic activity Ureolytic activity + Proteolytic activity + Haemolytic activity + Motility and chemotaxis Flagellum + Chemotaxis + Fimbriae and pili: MR/P Fimbriae + mrp' + Fimbriae 3 - UCA (NAF) + Fimbriae 5 + Fimbriae 6 + Fimbriae 7 + Fimbriae 8 + PMF(MR/K) + Fimbriae 10 + PMP + Fimbriae 12 + ATF + Fimbriae 14 + Fimbriae 15 + Fimbriae 16 + Fimbriae 17 + TABLE 6 \| Presence of genes related to Proteus mirabilis virulence in PrK 34/57. chain-length regulator protein Wzz (Islam and Lam, 2014; Yu et al., 2017). The P. mirabilis O antigen gene cluster is located between cpxA and secB genes, and contains both the wzx and wzy genes (Wang et al., 2010; Yu et al., 2017). Genome annotation using the RAST server identiﬁed the genes involved in choline transport and metabolism. Identiﬁcation of Putative Antibiotic Resistance Genes Three of the genes were licA located at open reading frame (ORF) ORF04, licB located at ORF05, and licD located at ORF08 based on the cluster scheme proposed by Yu and colleagues (Yu et al., 2017). The O antigen cluster contained one additional ORF (ORF06); amino acid sequence analysis by Phyre2 software (Kelley et al., 2015) revealed 98% coverage, and a 224 amino acid polypeptide was modelled with 100.0% conﬁdence with ctp:phosphocholine cytidylytransferase (LicC) as the single highest scoring template. The licABC genes overlap with each other, suggesting they are co-transcribed during the O antigen assembly process. The sequence of the licD gene overlaps with that of the wzx gene. Both regions are divided by a short intergenic sequence. The licABC genes are localized between the wemA gene, encoding a UDP-galactofuranosyl transferase according to Phyre2 (conﬁdence 98.1%, coverage 49%), separated from licD by wzx encoding a ﬂippase. The complete O antigen biosynthesis cluster for the P. mirabilis O18 serogroup is presented on Figure 4. DISCUSSION P. mirabilis is a Gram-negative, commensal bacterium, which is frequently responsible for catheter-associated urinary tract infections. In this work, we determined the draft genome of P. mirabilis strain PrK 34/57 (O18) containing the unique ChoP modiﬁcation of O antigen. Furthermore, the obtained data allowed us to investigate the general features of the studied genome. PGAP and RAST annotation revealed the presence of important genes involved in P. mirabilis pathogenicity, including genes responsible for swarming motility, ureolytic activity and iron acquisition P. mirabilis is a Gram-negative, commensal bacterium, which is frequently responsible for catheter-associated urinary tract infections. In this work, we determined the draft genome of P. mirabilis strain PrK 34/57 (O18) containing the unique ChoP modiﬁcation of O antigen. Furthermore, the obtained data allowed us to investigate the general features of the studied genome. PGAP and RAST annotation revealed the presence of important genes involved in P. mirabilis pathogenicity, including genes responsible for swarming motility, ureolytic activity and iron acquisition Most O antigen gene clusters are located at ﬁxed positions on bacterial chromosomes, and genetic variation in these gene clusters is largely responsible for the diversity of O antigen forms (Liu et al., 2008). There are three types of molecular biosynthesis pathways for O antigens: Wzx/Wzy-dependent, synthase-dependent, and ABC transporter-dependent pathways (Bertani and Ruiz, 2018). The Wzx/Wzy-dependent pathway is the most common, and the main components are the integral inner membrane ﬂippase Wzx, the polymerase Wzy, and the March 2021 \| Volume 11 \| Article 620010 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 6 Drug Class Resistance Mechanism % Identity of Matching Region % Length of Reference Sequence otic, tetracycline antibiotic antibiotic efﬂux 42.17 99.34 antibiotic efﬂux 53.79 101.02 ﬂuoroquinolone antibiotic, penam antibiotic efﬂux 98.1 100.00 antibiotic inactivation 96.77 100.00 otic, diaminopyrimidine antibiotic, antibiotic efﬂux 92.98 101.64 ﬂuoroquinolone antibiotic, iotic, carbapenem, cephalosporin, biotic, penem antibiotic efﬂux 72.71 100.00 aminoglycoside antibiotic, ycline antibiotic, peptide antibiotic, antibiotic efﬂux 68.81 100.92 amycin, penam antibiotic target alteration 51.45 98.03 otic antibiotic target alteration 84.58 100.00 antibiotic target alteration 94.91 96.09 Czerwonka et al. Genome of Proteus mirabilis O18 FIGURE 4 \| Proteus mirabilis O18 O antigen gene cluster arrangement. Highlighted genes represent licABCD genes and its organisation in the Wzx/Wzy-dependent O antigen biosynthesis gene cluster. Scheme modiﬁed from Yu et al. (2017). FIGURE 4 \| Proteus mirabilis O18 O antigen gene cluster arrangement. Highlighted genes represent licABCD genes and its organisation in the Wzx/Wzy-dependent O antigen biosynthesis gene cluster. Scheme modiﬁed from Yu et al. (2017). (Różalski etal., 1997).Thestructure andorganisationofthePrK 34/ 57 genome was compared with other P. mirabilis strains, and the genome ofPrK34/57isgenerallysimilarintermsoffeaturessuchas genome size, GC ratio and the number of coding sequences (Pearson et al., 2008; Sullivan et al., 2013; Shi et al., 2016; Gmiter et al., 2019). In the present study, the HI4320 genome was used as a reference,sincethisstrainhasbeenstudiedextensively(Armbruster et al., 2018). Other complete and WGS genomes were also included to better understand the general position of PrK 34/57 among P. mirabilis isolates from around the world. P. mirabilis PrK 34/57 shares high similarity with strain HI4320, but it is most closely related to the previously studied K670 strain. Simple variant calling analysis was performed using HI4320 and PrK 34/57. The results indicated few P. mirabilis PrK 34/57 polymorphisms (<20,000 variants), especially considering the high level of polymorphisms revealed in previously studied Acidithiobacillus ferrooxidans strains (>70,000variants) (Fariqetal.,2019).These variantcalling analyses mayberelevantforfuturestudiesonPrK34/57.Similarconclusions about the high genetic relatedness between PrK 34/57 and other strainscanbedrawnfromtheANIresults,whichwereinagreement with previous observations (Armbruster et al., 2018). ﬁnding reveals that ChoP attachment to O antigen repeating unit in P. mirabilis is probably regulated by two independent regulatory regions, upstream the licABC and wzx/licD clusters, and it occurs as two independently controlled events. The decoration of a single O antigen unit presumably occurs before ﬂipping of the undecaprenyl phosphate-linked O units to the periplasmic face of the inner membrane by the Wzx ﬂippase. In conclusion, determination of the P. DATA AVAILABILITY STATEMENT p The obtained draft genome sequence of ChoP-containing P. mirabilis O18 strain PrK 34/57 is important in a serological context and will be beneﬁcial during subsequent studies of the pathogenicity of this bacterium. Choline uptake and LPS decoration protects bacteria from osmotic pressure, and enables avoiding the immune system recognition. Choline uptake is provided in P. mirabilis O18 by a high-afﬁnity choline transport protein BetT. Choline gained in this way is involved in the glycine betaine biosynthesis pathway, which acts as an osmoprotectant (Deole and Hoff, 2020). The other way for choline recruitment into the cell is the ChoP decoration pathway, where licABC genes products (kinase, permease, and cytidylyltransferase, respectively) incorporate, and modify choline molecule. In the next step, CDP- choline is attached to the target glycans by the transferase LicD (Young et al., 2013). In P. mirabilis O18 antigen gene cluster genes licABC overlaps, which suggest that they are organised in a separate cluster, and expressed simultaneously. The action of this cluster may result in incorporation choline into the cell and its modiﬁcation to CDP-choline. The licD encoding transferase overlaps with the sequence of the wzx ﬂippase, which suggests that both genes are co-transcribed, and expression of licD depends on expression of wzx and is regulated with the same factors. This The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ supplementary material. mirabilis O18 genome sequence revealed the presence and localisation of genes licABCD potentially involved in choline uptake, modiﬁcation and decoration of LPS. Enzymes involved in ChoP decoration, a choline kinase, choline permease, and ctp:phosphocholine cytidylytransferase are encoded by the licABC cluster. This cluster is separated from licD homolog (encoding lipopolysaccharide cholinephosphotransferase) by the wzx gene encoding a crucial LPS biosynthesis transmembrane ﬂippase. The results allowed us to present the provisional organisation of the O antigen gene cluster in the P. mirabilis O18 serogroup. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org REFERENCES doi: 10.1016/ j.jmb.2015.11.006 Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K., et al. (2009). BLAST+: Architecture and applications. BMC Bioinf. 10, 1–9. doi: 10.1186/1471-2105-10-421 Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., and Sternberg, M. J. (2015). The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858. doi: 10.1038/nprot.2015-053 Clark, S. E., and Weiser, J. N. (2013). Microbial modulation of host immunity with the small molecule phosphorylcholine. Infect. Immun. 81, 392–401. doi: 10.1128/IAI.01168-12 Darling, A. C. 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All authors provided critical feedback and helped shape the research, analysis, and manuscript. March 2021 \| Volume 11 \| Article 620010 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 8 Czerwonka et al. Genome of Proteus mirabilis O18 All authors contributed to the article and approved the submitted version. Rector’s Grant 2019 (no SIGR.RN.20.061.604). DG received the PhD scholarship from the NSC Grant No. 2019/32/T/NZ1/00515. All authors contributed to the article and approved the submitted version. FUNDING This research was supported by the Polish National Science Centre (Grant No. 2017/01/X/NZ6/01141), and Jan Kochanowski University We would like to thank Prof. Wiesław Kaca for his contribution of a discussion to our paper. REFERENCES 9, 478. doi: 10.3389/ fmicb.2018.00478 Figueras, M. J., Beaz-Hidalgo, R., Hossain, M. J., and Liles, M. R. (2014). 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Macromol. 163, 1168–1174. doi: 10.1016/j.ijbiomac.2020.07.012 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. Sullivan, N. L., Septer, A. N., Fields, A. T., Wenren, L. M., and Gibbs, K. A. (2013). The Complete Genome Sequence of Proteus mirabilis Strain BB2000 Reveals Differences from the P. mirabilis Reference Strain. Genome Announc. 1, e00024–13-e00024-13. doi: 10.1128/genomeA.00024-13 Copyright © 2021 Czerwonka, Gmiter and Durlik-Popińska. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). March 2021 \| Volume 11 \| Article 620010 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 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. Tatusova, T., Dicuccio, M., Badretdin, A., Chetvernin, V., Nawrocki, E. P., Zaslavsky, L., et al. (2016). NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res. 44, 6614–6624. doi: 10.1093/nar/gkw569 Wang, Q., Torzewska, A., Ruan, X., Wang, X., Rozalski, A., Shao, Z., et al. (2010). Molecular and genetic analyses of the putative Proteus O antigen March 2021 \| Volume 11 \| Article 620010 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 10
https://openalex.org/W4241937397	https://www.researchsquare.com/article/rs-52344/v1.pdf	English	null	Effect of the Pumping-injection flow rate on Heat Transfer Characteristic of Borehole Heat Exchangers for Coupling Ground-Source Heat Pump System	Research Square (Research Square)	2,020	cc-by	19,783	Jiuchen Ma 1,2 , Qian Jiang 1,2,* , Qiuli Zhang 1,2 , Yacheng Xie 1,2 , Yahui Wang 1,2 , Feiyu Yi 1,2 5 * Correspondence: 596797782@qq.com 1 School of Energy Safety Engineering, Tianjin Chengjian 6 University, Tianjin 300384, China. 7 Full list of author information is available at the end of the article 8 Abstract: A coupling ground source heat pump system (CGSHP) is established in areas 9 where groundwater is shallow but the seepage velocity is weak, which sets up pumping and 10 injection wells on both sides of borehole heat exchangers (BHEs). A convection-dispersion 11 analytical model of excess temperature in aquifer that considers groundwater forced seepage 12 and axial effects and thermal dispersion effects is proposed. A controllable forced seepage 13 sandbox is built by equation analysis method and similarity criteria. Through indoor test and 14 the proposed analytical model, the correctness and accuracy of the numerical simulation 15 software FEFLOW7.1 is verified. The influence of different pumping-injection flow rate on 16 the heat transfer characteristic of BHEs is studied by numerical simulation. The results show 17 that the average heat efficiency coefficient of BHEs increases and the heat influence range of 18 downstream BHEs expands with the increasing of pumping-injection flow rate. The relation 19 curve between Pe and the increment of heat transfer rate per unit depth of BHEs (Δq) is 20 distributed as Gaussian function. The pumping-injection flow rate that makes Darcy velocity 21 reaches 0.6×10-6~1.4×10-6 m∙s-1 in the aquifer is the best reference range for CGSHP system，so 22 400~600 m3∙d-1 is taken as the best pumping-injection flow rate in this paper. 23 Keywords: coupling ground source heat pump system; groundwater forced seepage; heat transfer; laboratory experiment; numerical simulation; analytical model Page 1 of 22 Page 1 of 22 Effect of the Pumping-injection flow rate on Heat 1 Transfer Characteristic of Borehole Heat 2 Exchangers for Coupling Ground-Source Heat 3 Pump System 4 Jiuchen Ma 1,2 , Qian Jiang 1,2,* , Qiuli Zhang 1,2 , Yacheng Xie 1,2 , Yahui Wang 1,2 , Feiyu Yi 1,2 5 * Correspondence: 596797782@qq.com 1 School of Energy Safety Engineering, Tianjin Chengjian 6 University, Tianjin 300384, China. 7 Full list of author information is available at the end of the article 8 1. Introduction 27 Shallow geothermal resources in China are abundant but uneven. The relevant 28 documents clearly point out that the development and utilization of shallow geothermal 29 energy should follow the principle of adaption to local conditions (National Development and 30 Reform Commission 2017; China Geological Survey 2018). Ground source heat pump (GSHP) 31 system, as one of the main techniques of shallow geothermal production (Richard 2014; 32 Richard and Marvin 2011), should take full account of local conditions in the application process. 33 Moreover, borehole heat exchangers (BHEs) constitute a core part of the technology and the 34 magnitude of its heat exchange quantity directly affects the efficiency of the overall unit. 35 Therefore, it is particularly important to enhance heat transfer around BHEs. 36 The heat transfer performance of BHEs is studied usually by four methods, i.e., analytical 37 method, numerical method, and in-situ thermal response test (TRT) and laboratory test. In 38 most related fields, one or two of the above methods are adopted commonly to study the effect 39 of natural groundwater seepage on the heat transfer performance of BHEs (A. Angelotti and L. 40 Alberti 2014; Jinzhong 2017; Jung and Joonsang 2013; Huajun and Chengying 2009). 41 Numerical simulations give accurate solutions that are conducive to theoretical analysis 42 but need extensive computational time. Hence, numerous numerical simulation softwares are 43 Page 2 of 22 Page 2 of 22 often applied to the research and design of BHEs, but the correctness and accuracy of these 44 softwares should be verified before (Alessandro and Rajandrea 2014; Yanling and Xiaoli 2017). 45 Angelotti et al. (A. Angelotti and L. Alberti 2014) explore the influence of groundwater 46 seepage velocity on the heat transfer characteristics of rock and soil layer by establishing a 47 single U-pipe heat transfer model through the codes MODFLOW/MT3DMS. Meanwhile, the 48 solution of numerical model is validated through the analytical solution of moving line source 49 model (MLS) on the premise that the U-pipe is simplified into an infinite line source. Hecht et 50 al. (Jozsef and Michael 2013) use FEFLOW, a finite element numerical simulation software, to 51 perform the transient heat transport simulations for the fifteen scenarios of 25 boreholes and 52 study the distribution of hydrodynamic field and temperature field in the homogeneous 53 confined aquifer. Li et al. 1. Introduction 27 (Chaofeng and Peter 2018) analyze the effect of unsaturated soil 54 properties and groundwater flow on the performance of GSHP system by the simulation 55 software COMSOL Multiphysics. The numerical model is validated by combining 56 experimental test results with the analytical model that takes the multiple-layer substrates and 57 groundwater flow into consideration. 58 Analytical models are preferred in most practical applications because of their excellent 59 computational time and flexibility for parameterized design. The method of moving 60 heat-source is applied in most analytical models to solve the problem of heat transfer under 61 the impact of groundwater seepage (Min and Alvin 2015). Sutton et al. (Matthew and Darin 2003) 62 and Diao et al. (Nairen and Qinyun 2004) present an analytical model considering groundwater 63 flow. They both concluded that groundwater flow could change considerably the temperature 64 distribution in the vicinity of the boreholes. Molina-Giraldo et al. (Nelson and Peter 2011) 65 evaluate the influence of thermal dispersion on temperature plumes of geothermal systems by 66 using analytical models. In the above analytical models, the axial effect is not considered and 67 the borehole is considered to be a moving infinite line heat source. Molina-Giraldo et al. 68 (Nelson and Philipp 2011) propose a moving finite line source model (MFLS) which considers 69 the combined effect of groundwater flow and axial effects but does not take the influence of 70 thermal dispersion into account. Groundwater seepage affects heat transfer by involving gross 71 heat convection and thermal dispersion, which is significant for the long-term temperature 72 response of BHEs (Jin and Joachim 2016). Therefore, an optimized analytical model, which 73 considers the thermal dispersion effect in the MFLS model, is proposed in this paper. 74 p p p p p TRTs are applied to some real environments that can truly reflect the heat transfer process 75 of BHEs under the specific regional climatic characteristics and hydrogeological conditions. 76 Most TRTs are performed on a test borehole to estimate the thermo-physical properties and 77 borehole thermal resistance (Huajun and Chengying 2009; A. Michopoulos and T. Zachariadis 2013; 78 Wonjun and Ryozo 2015). 1. Introduction 27 However, solar-assisted GSHP system and cooling 105 tower assisted GSHP system occupy a large land area and are greatly affected by seasonal and 106 environmental factors. 107 Thus, this paper proposes a coupling ground source heat pump (CGSHP) system which 108 sets up pumping and injection wells on both sides of BHEs' well group by basing on GSHP 109 system in Tianjin, China and combining with the local special hydrogeological conditions. 110 According to CGSHP system proposed in this paper, a laboratory experiment system is 111 established and a convection-dispersion analytical model of excess temperature that considers 112 groundwater forced seepage and axial effects and thermal dispersion effects is proposed. The 113 correctness and accuracy of the numerical simulation software FEFLOW7.1 is validated with 114 the laboratory sandbox test and the proposed convection-dispersion analytical model. 115 Meanwhile, the purpose of this paper is to study the enhancement effect of heat transfer of 116 BHEs and reveal the evolution law of the temperature field and hydrodynamic field of the 117 aquifer under different pumping-injection flow rate by the numerical simulation. 118 2. Methods 119 Geological survey 120 China's Bohai Rim region includes the plains of the four provinces of Liaoning, Hebei, 121 Tianjin, Shandong and the surrounding mountainous areas (including the two peninsulas). 122 The Bohai Rim plain is formed in the middle Pleistocene (Q2) and the late Pleistocene (Q3) to 123 the Holocene (Q4) in the Quaternary period. The final formation of the Bohai Rim region is the 124 result of the accumulation of sediment caused by the accumulation of rivers (Rong 2012). 125 having the positive effect that it should have. Therefore, it is necessary to optimize the 94 traditional GSHP system for this special hydrogeological condition. 95 For the long-term application of GSHP system, the cold and heat accumulation around 96 BHEs and the heat transfer quantity of BHEs decay year-by-year, domestic and foreign 97 scholars have proposed some optimized methods and improved measures to regulate heat 98 balance of the soil. Such as, solar assisted GSHP system (Poul and Neven 2019;Giti and Younes 99 2019; H. Biglarian and M. H. Saidi 2019), and cooling tower assisted GSHP system (Zhijian and 100 Yuanwei 2018; Xuemei and Lei 2018). Giti Nouri et al. (Giti and Younes 2019) and Zhijian Liu et 101 al. 1. Introduction 27 From the viewpoint of model validation, the data from TRTs is not 79 suitable because TRTs’ test times are short (usually 48-72 hours) and are susceptible to 80 uncontrollable factors such as weather conditions during testing (Min and Alvin 2015; David and 81 A.C 2018; Ali and Michel 2014). The indoor sandbox experiments enable to change some 82 parameters, which is conducive to improve the speed and test accuracy of the experiment 83 (Guozhu and Yimu 2016; Linlin and Lei 2015) because the experimental requirements have well 84 controllability comparing with the TRT. 85 Most studies (Selcuk and Bertrand 2018; Martin and Jonathan 2018; Junye and Gui 2017; Wenke 86 and Hongxing 2014) point out that the presence of groundwater significantly affects the heat 87 transfer between BHE and its surrounding aquifer. However, most of these study conclusions 88 are obtained under the condition of high natural seepage velocity, which is generally higher 89 than 10-8 m∙s-1. However, many areas such as the Bohai Rim plain have abundant groundwater 90 reserves but the natural seepage velocity there is generally lower than 10-8 m∙s-1. In this case, it 91 is usually considered that groundwater seepage has almost no effect on heat transfer of BHEs. 92 At this time, using the traditional GSHP system would render groundwater idle without 93 Page 3 of 22 Page 3 of 22 having the positive effect that it should have. Therefore, it is necessary to optimize the 94 traditional GSHP system for this special hydrogeological condition. 95 For the long-term application of GSHP system, the cold and heat accumulation around 96 BHEs and the heat transfer quantity of BHEs decay year-by-year, domestic and foreign 97 scholars have proposed some optimized methods and improved measures to regulate heat 98 balance of the soil. Such as, solar assisted GSHP system (Poul and Neven 2019;Giti and Younes 99 2019; H. Biglarian and M. H. Saidi 2019), and cooling tower assisted GSHP system (Zhijian and 100 Yuanwei 2018; Xuemei and Lei 2018). Giti Nouri et al. (Giti and Younes 2019) and Zhijian Liu et 101 al. (Zhijian and Yuanwei 2018) study solar assisted GSHP system and cooling tower assisted 102 GSHP system, respectively. Their research results show that the application of these hybrid 103 systems could be helpful to reach considerable savings of energy through using free resources 104 of stored heat in the ground and sun or air. 1. Introduction 27 (Zhijian and Yuanwei 2018) study solar assisted GSHP system and cooling tower assisted 102 GSHP system, respectively. Their research results show that the application of these hybrid 103 systems could be helpful to reach considerable savings of energy through using free resources 104 of stored heat in the ground and sun or air. However, solar-assisted GSHP system and cooling 105 tower assisted GSHP system occupy a large land area and are greatly affected by seasonal and 106 environmental factors. 107 Thus, this paper proposes a coupling ground source heat pump (CGSHP) system which 108 sets up pumping and injection wells on both sides of BHEs' well group by basing on GSHP 109 system in Tianjin, China and combining with the local special hydrogeological conditions. 110 According to CGSHP system proposed in this paper, a laboratory experiment system is 111 established and a convection-dispersion analytical model of excess temperature that considers 112 groundwater forced seepage and axial effects and thermal dispersion effects is proposed. The 113 correctness and accuracy of the numerical simulation software FEFLOW7.1 is validated with 114 the laboratory sandbox test and the proposed convection-dispersion analytical model. 115 Meanwhile, the purpose of this paper is to study the enhancement effect of heat transfer of 116 BHEs and reveal the evolution law of the temperature field and hydrodynamic field of the 117 aquifer under different pumping-injection flow rate by the numerical simulation. 118 2. Methods 119 China's Bohai Rim region includes the plains of the four provinces of Liaoning, Hebei, 121 Tianjin, Shandong and the surrounding mountainous areas (including the two peninsulas). 122 The Bohai Rim plain is formed in the middle Pleistocene (Q2) and the late Pleistocene (Q3) to 123 the Holocene (Q4) in the Quaternary period. The final formation of the Bohai Rim region is the 124 result of the accumulation of sediment caused by the accumulation of rivers (Rong 2012). 125 Page 4 of 22 126 Figure 1. Depth Isolines Map of Shallow Aquifer Embedment of Plain Country in Tianjin, China 127 Tianjin is located in China's Bohai Rim plain, having abundant underground water and 128 diverse hydrogeological structures. The whole Tianjin plain can be divided into the freshwater 129 area, brackish water distribution underlying shallow freshwater area and brackish water area 130 from north to south (Figure 1). In the structure of aquifer, the sandy layer transforms into 131 medium coarse sand, medium sand, medium fine sand, fine sand and silty sand from north to 132 south. 133 126 Figure 1. Depth Isolines Map of Shallow Aquifer Embedment of Plain Country in Tianjin, China 127 Ti ji i l t d i Chi ' B h i Ri l i h i b d t d d t 128 Figure 1. Depth Isolines Map of Shallow Aquifer Embedment of Plain Country in Tianjin, China Tianjin is located in China's Bohai Rim plain, having abundant underground water and 128 diverse hydrogeological structures. The whole Tianjin plain can be divided into the freshwater 129 area, brackish water distribution underlying shallow freshwater area and brackish water area 130 from north to south (Figure 1). In the structure of aquifer, the sandy layer transforms into 131 medium coarse sand, medium sand, medium fine sand, fine sand and silty sand from north to 132 south. 133 The distribution area of shallow underground brackish water in China's Binhai plain of 134 Tianjin is 6,922 km2, of which the brackish water area with a mineralization content of 2-3 g·L-1 135 and 3-10 g·L-1 are 3,753 km2 and 3169 km2 respectively, accounting for more than 2/3 of the 136 city's total area (Zaiming 2012). Groundwater resources are rich in reserves and convenient for 137 exploitation, but the hydraulic gradient and the natural seepage generally range from 1.3×10-2 138 m∙a-1 to 12×10-1 m∙a-1. Therefore, CGSHP system is suitable in Tianjin plain. 2. Methods 119 139 Underground thermo-physical properties 140 Underground thermo-physical properties 140 Underground thermo-physical properties 140 N 0 1500 1000 500 2000 Meters 0 150 100 50 200Meters Borehole heat exchanger (BHE) Study site School districts N w Ti1 di ψ To1 To2 Ti2 141 Figure 2. Location of study site in China, Tianjin, 2016 and plan view of BHEs 142 The GSHP system that installed in Tianjin Binhai New Area, China, 2016 is taken as the 143 project prototype (Figure 2). The project has a research area of 150×120 m2 and a vertical depth 144 of -83m that it is divided into 5 geotechnical layers. Among whole study area, the fine sand 145 layer has stronger permeability that is regarded as a well-developed confined aquifer, which 146 Borehole heat exchanger (BHE) 141 Figure 2. Location of study site in China, Tianjin, 2016 and plan view of BHEs 142 The GSHP system that installed in Tianjin Binhai New Area, China, 2016 is taken as the 143 project prototype (Figure 2). The project has a research area of 150×120 m2 and a vertical depth 144 of -83m that it is divided into 5 geotechnical layers. Among whole study area, the fine sand 145 layer has stronger permeability that is regarded as a well-developed confined aquifer, which 146 Page 5 of 22 interacts with silty sand layer to form shallow-groundwater accumulation section. The 147 geotechnical distribution and physical parameters are shown in Table 1. 148 Table 1. The physical parameters of the underground rock-soil layers 149 The project, which is mainly responsible for the energy supply of the adjacent school, 150 contains 219 BHEs with a center-to-center spacing of 4-5 m. The vertical depth of the borehole 151 is 83 m and the 2U-DN32-HDPE-BHEs with the length of 80 m is arranged in the well. The 152 expansive soil with sand is selected as the grout materials. Water is selected as the circulating 153 refrigerant in BHEs. The design parameters of BHEs are shown in Table 2. 154 Table 2. 2. Methods 119 Design Parameters of 2U-Type BHE 155 Parameter/Symbol (Unit) Value BHE depth /H (m) 80 borehole diameter /ψ (m) 0.13 outer diameter of branch pipe-in(out)/di(o) (m) 0.032 wall thickness of branch pipe-in(out)/bi(o) (m) 0.0029 adjacent branch pipe distance /w (m) 0.05 thermal conductivity of pipe-in(out) material/λpi(o) (W·m-1·K-1) 0.6 thermal conductivity of refrigerant (water)/ λr (W·m-1·K-1) 0.65 volumetric heat capacity of refrigerant (water) /crρr (J·m-3·K-1) 4.18·106 volumetric heat capacity of grout/ cgρg (J·(m-3·K-1)) 2.19·106 thermal conductivity of grout/ λg (W·m-1·K-1) 1.9 Heat-seepage coupling model 156 soil media Depth of distribution  (m) horizontal permeability kXY (m2) porosity εs volumetric heat capacity of the soil csρs (J·m-3·K-1) thermal conductivity λs (W·m-1·K-1) longitudinal thermal dispersivity αL (m) transverse thermal dispersivity αT (m) Initial temperature of soil layer TS0/℃ clay 0-9 5.0·10-14 0.45 2.7·106 1.1 0.3 0.03 16 silty clay 9-24 5.0·10-14 0.4 3.2·106 1.2 0.3 0.03 fine sand 24-44 1.2·10-12 0.35 1.4·106 0.95 3.0 0.3 silty sand 44-80 8·10-13 0.38 2.4·106 1.6 0.1 0.01 silty clay 80-83 5.0·10-14 0.4 3.2·106 1.2 0.3 0.03 interacts with silty sand layer to form shallow-groundwater accumulation section. The 147 geotechnical distribution and physical parameters are shown in Table 1. 148 Table 1. The physical parameters of the underground rock-soil layers 149 Heat-seepage coupling model 156 Heat-seepage coupling model 156 190 0 f f f 0 x x y ( ) ( , , , ) exp ( , , , ) ( , , , ) 2 2 H H c u x x q T x y z t f x y z t dz f x y z t dz λ λ λ                     191 (6) 192 2 2 2 2 2 2 2 2 2 f f f f f f e e x x x y x e x x x y x e x e e e 2 f f f x x 1 ( , , , ) 4 ( ') ( ') ( ') ( ') ( ') ( ') ( ') ( ') ( ') exp 2 2 ( ') exp f x y z t x x y y z z c u t x x y y z z x x y y z z c u c λ λ λ λ λ λ λ λ λ λ λ λ λ erfc c t λ x x c u λ λ                                                              2 2 2 2 2 f f f x y x e x x x y x e x e e e ( ') ( ') ( ') ( ') ( ') 2 2 e e c u t y y z z x x y y z z c λ λ λ λ λ λ λ λ λ λ λ erfc c t λ                                             （7） 193 194 0 f f f 0 x x y ( ) ( , , , ) exp ( , , , ) ( , , , ) 2 2 H H c u x x q T x y z t f x y z t dz f x y z t dz λ λ λ                     191 192 (6) 2 2 2 2 2 2 2 2 2 f f f f f f e e x x x y x e x x x y x e x e e e 2 f f f x x 4 ( ') ( ') ( ') ( ') ( ') ( ') ( ') ( ') ( ') exp 2 2 ( ') exp x x y y z z c u t x x y y z z x x y y z z c u c λ λ λ λ λ λ λ λ λ λ λ λ λ erfc c t λ x x c u λ λ                                                             2 2 2 2 2 f f f x y x e x x x y x e x e e e ( ') ( ') ( ') ( ') ( ') 2 2 e e c u t y y z z x x y y z z c λ λ λ λ λ λ λ λ λ λ λ erfc c t λ                                             （7） In the analytic model, ρece is the volumetric heat capacity of the porous medium (Eq.8). Heat-seepage coupling model 156 172 In which, the conductive part of thermodispersion tensor Λcond and the dispersive part of 171 thermodispersion tensor Λdisp are determined by equation (4) and (5) respectively. 172 cond s f s s ij [ +(1 ) ]   Λ   (4) fi disp f f T f ij L T f ( ) u u c u u               Λ fj (5) (4) fi disp f f T f ij L T f ( ) u u c u u               Λ fj (5) (5) The problem for determining solution of seepage flow is associated with the problem of 175 heat transfer by the momentum equation (2), so the heat-seepage coupling model is 176 constructed in the aquifer. The discharge (suction) heat process of the borehole can be 177 considered as the source (sink) term in the aquifer thermal migration model. Because the 178 diameter of the vertical borehole is much smaller than its depth, in order to consider 179 effectively the influence of groundwater seepage on the heat transfer of BHE, the heat transfer 180 process of BHE is simplified to the heat transfer process of the moving finite line heat source in 181 the semi-infinite medium. 182 On the basis of considering the thermal dispersion effect and the spatial position of BHE, 183 the moving finite line heat source model (MFLS) (A. Michopoulos; T. Zachariadis 2013) is 184 improved. Under the premise of constant heat flow rate per unit length of BHE, the optimized 185 analytical model (Eq.6-7) is obtained by applying the method of images and the moving source 186 theory. The optimized analytical model takes into account the heat convection, heat 187 conduction and thermal dispersion effects in the process of groundwater seepage to determine 188 the transient temperature in the aquifer caused by heat from the heat exchanger and is coupled 189 with the internal heat transfer process of the borehole. Heat-seepage coupling model 156 It is assumed that the seepage process of aquifer satisfies the following conditions: the 157 physical parameters of aquifer and groundwater do not change with temperature; the seepage 158 direction of groundwater is single and the vertical seepage process is ignored. According to 159 the continuity equation of seepage flow and Darcy law (Yujin and Ryozo 2008), the continuity 160 governing equation (1) and momentum governing equation (2) in the anisotropic and 161 homogeneous aquifers are established: 162 s f s s f [ (1 ) ] h u Q t          (1) f f f k g h u x      (2) s f s s f [ (1 ) ] h u Q t          (1) 63 f f f k g h u x      (2) 64 (1) (2) In order to describe the change of heat transfer characteristics of BHEs in the aquifer 165 under complex space-time conditions accurately on the basis of the convective-conduction 166 equation (Antonio and Michele 2013), the control equation of three-dimensional unsteady 167 convective - thermal dispersion is established by taking the thermal dispersion effect into 168 account. 169 Page 6 of 22 s f f s s s f f f T [ (1 ) ] + [( ) ] T c c c u T T Q t             cond disp Λ + Λ 170 s f f s s s f f f T [ (1 ) ] + [( ) ] T c c c u T T Q t             cond disp Λ + Λ (3) s f f s s s f f f T [ (1 ) ] + [( ) ] T c c c u T T Q t             cond disp Λ + Λ (3) 170 (3) In which, the conductive part of thermodispersion tensor Λcond and the dispersive part of 171 thermodispersion tensor Λdisp are determined by equation (4) and (5) respectively. Heat-seepage coupling model 156 195 When the thermal conductivity in the aquifers is the same in each direction, the thermal 196 conductivity components of the Λcond are determined by equation (9). λx and λy is the effective 197 longitudinal and transverse thermal conductivity coefficient, respectively, which are 198 determined by Eq.10 and Eq.11. r is the distance to the source located on the z-axis at the (x0, 199 y0, z’) coordinates (Eq.12). 200 e e s f f s s s (1 ) c c c       (8) 201 cond cond cond x y z e s f s s = (1 ) λ λ λ λ        (9) 202 x e L f f f u c       (10) 203 (8) (9) 203 (10) Page 7 of 22 Page 7 of 22 y e T f f f u c       (11) 2 2 ' 2 0 0 ( ) ( ) ( ) r x x y y z z       (12) y e T f f f u c       (11) 2 2 ' 2 0 0 ( ) ( ) ( ) r x x y y z z       (12) (11) 204 2 2 ' 2 0 0 ( ) ( ) ( ) r x x y y z z       (12 (12) ( ) When the thermal dispersion effect in the aquifer is ignored (λx=λy=λe), the excess temperature 206 analysis model (Eq.6) and (Eq.7) can be simplified to Eq. 13 and Eq.14. 207 ( ) When the thermal dispersion effect in the aquifer is ignored (λx=λy=λe), the excess temperature 206 analysis model (Eq.6) and (Eq.7) can be simplified to Eq. 13 and Eq.14. 207 ( ) When the thermal dispersion effect in the aquifer is ignored (λx=λy=λe), the excess temperature 206 analysis model (Eq.6) and (Eq.7) can be simplified to Eq. 13 and Eq.14. Heat-seepage coupling model 156 207 0 f f f 0 e e ( ) ( , , , ) exp '( , , , ) ' '( , , , ) ' 2 2 H H c u x x q T x y z t f x y z t dz f x y z t dz λ λ                     (13) 208 e e f f f e e f f f f f f f f f e e e e e e e e '( , , , ) exp exp 2 2 2 2 r c c u t r c c u t c u r c u r f x y z t erfc erfc λ λ c λ t c λ t                                          (14) 209 (13) (14) 3.Experimental System 210 3.Experimental System 210 In order to ensure the reasonable use of the limited laboratory indoor space, the fine sand 211 layer, which was a typical hydrogeological medium in shallow the aquifer, with the area of 212 20×16m2 and the buried depth of 24~44m in Figure 2 were selected as the engineering 213 prototype for designing and building a complete laboratory experimental system. The 214 engineering prototype contained 6 ordinals arranged BHEs with the tube pitch of 4m. 215 The experimental system consists of a sandbox, a heating apparatus, a flow conditioning 217 apparatus and a data-acquisition apparatus (Figure 3). Since the seepage and heat transfer 218 processes in the seepage sandbox and the engineering prototype followed the same form of 219 governing equations (Eq.1-3), the similitude relation ratio of basic design parameters was 220 determined by equation analysis method according to the principle of similarity criteria (Min 221 and Alvin 2015;X. Mao and H. Prommer 2006). 222 Sandbox Data acquisition device Graduated cylinder Constant temperature water tank Piezometer Filter Valve Reservoir Storage tank Circulating water pump Valve Buried pipe Sandbox Data acquisition device Graduated cylinder Constant temperature water tank Piezometer Filter Valve Reservoir Storage tank Circulating water pump Valve Buried pipe 223 Figure 3. Schematic diagram of the experimental system 224 Reservoir Figure 3. Schematic diagram of the experimental system The proportional relationship between hydrogeology and thermophysical parameters of 225 aquifers was 1:1 due to the use of equal volumetric weight filling of raw sand and seepage of 226 raw water. In order to shorten the experimental period, the proportional relationship between 227 the heat intensity of BHEs and the difference of water head was set as 1:1. The geometric size 228 proportional relationship between the actual and experimental was determined as 20:1. The 229 Pr (Eq.15), Fo (Eq.16) and Pe (Eq.17) in the engineering prototype and the sandbox were 230 required to be equal in order to ensure that the experimental system can reproduce effectively 231 the heat-seepage migration process of the aquifer. Thus, the operation time proportional 232 relationship between the actual and the experimental was determined to be 400:1 and the 233 seepage velocity proportional relationship between the actual and the experimental was 234 determined to be 1:20, so as to determine other design parameters, as shown in Table 3. 3.Experimental System 210 235 f e e fm em em m f e fm em c c Pr Pr        236 f e e fm em em m f e fm em c c Pr Pr        （15） 236 Page 8 of 22 e em m m 2 2 e e em em m = = t t Fo Fo c l c l      （16） 237 f f fm fm m m e em m c ul c u l Pe Pe        （17） 238 239 （16）（17） 239 Table 3. Engineering prototype and experimental system design parameters 240 classify volume x∙y∙z (m3) pressure diaphragm thickness 1 (m) confined aquifer (fine sand layer) thickness 2 (m) tube pitch l(m) heat transfer rates per unit borehole depth q (W ∙ m-1) Darcy velocity uf (m ∙ s-1) operatio n time t prototype 20×16×20 4 12 4 50 1.2×10-7 90d test-bed 1.0×0.8×1 0.2 0.6 0.2 50 2.4×10-6 5.4h According to the geometric similarity ratio, the sandbox was set to 1.2×0.8×1.2 m3 while 241 the seepage region was 1×0.8×1 m3 and the confined aquifuge region was 1×0.8×0.2 m3. The 242 liquid supply/discharge region was 0.1 ×0.8 ×1.2 m3, which was symmetrically set at both 243 ends. Then five overflow holes (Φ20 mm) with spacing of 0.2m were drilled in the centerline 244 of plexiglas’s plate on the outsides of the liquid supply/discharge region. During the 245 experiment, rubber plugs of overflow holes at different heights were opened to control the 246 hydraulic difference between the liquid supply/drainage region, so as to change the seepage 247 velocity in the seepage region. Plexiglas’s plate with the equally distributed holes (Φ5 mm) 248 was installed between liquid supply/discharge region and the seepage area to ensure that the 249 seepage solution flows horizontally and evenly in the aquifer. 250 The K-type (±0.1℃) thermocouple treated with waterproof and anti-corrosion package 251 was selected to measure aquifer temperature. Nine K-type thermocouples were embedded 252 0 5m away from the bottom of the sandbox The data acquisition apparatus was used to 253 Table 3. Engineering prototype and experimental system design parameters 240 p g y y q The K-type (±0.1℃) thermocouple treated with waterproof and anti-corrosion package 251 was selected to measure aquifer temperature. 3.Experimental System 210 Nine K-type thermocouples were embedded 252 0.5m away from the bottom of the sandbox. The data acquisition apparatus was used to 253 record temperatures of the aquifer. Then, six PPR pipes with 1.5m-long, which fixed on the 254 bottom plate of the sandbox, were wound evenly with electric heating wire (50W/m) to 255 simulate the BHEs as the heat source device of the sandbox. The plane layout of BHEs and 256 temperature observation points was shown as Figure 4. 257 p p g 200 200 200 200 300 300 300 300 300 1000 800 ~ Borehole heat exchanger(BHE) Groundwater flow 300 1* 2* 4* 5* 6* 100 200 200 1~9 Temperature observation point 100 100 100 100 100 100 3* 9* 7* 8* 258 Figure 4. Layout of BHEs and temperature observation points (unit: mm) 259 / 258 Figure 4. Layout of BHEs and temperature observation points (unit: mm) 259 The p ecisio bath ci culato THD 3015 as selected as the cold/heat so 260 igure 4. Layout of BHEs and temperature observation points (unit: mm) 258 Figure 4. Layout of BHEs and temperature observation points (unit: mm) 259 of BHEs and temperature observation points (unit: mm The precision bath circulator THD-3015 was selected as the cold/heat source equipment 260 in order to ensure that the temperature of the circulating groundwater meets the 261 requirements of the test. Furthermore, the thermal insulation materials with the thickness of 262 0.15 m were attached to the outside of the sandbox device, connecting pipes, storage tank and 263 reservoir. After pasting thermal insulation materials, six K-type thermocouples were fixed 264 respectively on all six sides of the sandbox to measure the heat loss of the experimental 265 system. 266 The precision bath circulator THD-3015 was selected as the cold/heat source equipment 260 in order to ensure that the temperature of the circulating groundwater meets the 261 requirements of the test. Furthermore, the thermal insulation materials with the thickness of 262 0.15 m were attached to the outside of the sandbox device, connecting pipes, storage tank and 263 reservoir. After pasting thermal insulation materials, six K-type thermocouples were fixed 264 respectively on all six sides of the sandbox to measure the heat loss of the experimental 265 system. 3.Experimental System 210 Numerical model 294 Compared with analytical and experimental data 295 According to the engineering prototype, FEFLOW 7.1 is used to establish the geometric 296 model, mesh division (triangular element non-equidistant) and set the parameters, and then 297 perform numerical simulation calculations. Meanwhile, MATLAB 2012 is used to calculate 298 the transient temperature response caused by the running BHE in engineering prototype, 299 according to the unsteady analytical model of the excess temperature in the aquifer (Eq.6-7). 300 Then, the experimental result and the analytical solution of the engineering prototype are 301 compared with the numerical solution of the engineering prototype. 302 In the numerical simulation, the clay layer is defined as a confined aquifuge while the 303 fine sand layer with a thickness of 20m exists as a confined aquifer. The clay layer in the 304 Figure 5. Photograph of the infiltration sandbox After the establishment of the experimental system, the room temperature was first 279 maintained at 18℃ by central air conditioning, and then the 16℃ underground raw water 280 was filled into the sandbox for the exhaust gas process of porous media. When all 281 temperature observation points in the sandbox were maintained at 16±0.1℃, and the water 282 level in the piezometer was stable and no bubbles appeared, the porous medium of the 283 sandbox could be considered as saturated aquifer. 284 Before the experiment, it was proved that Darcy velocity was 2.4±0.02×10-6 (Pe≈2.5) when 285 the hydraulic difference was 0.2m, which satisfied the proportional relationship between 286 actual and experimental seepage velocity 1:20. During the experiment, the 16℃ underground 287 raw water with 0.2 m hydraulic difference was filled continuously into the sandbox to ensure 288 a stable Darcy velocity, simultaneously, BHEs ①~ ④ were opened in the test. The 289 experimental run time was set at 5.4 h and the temperature of each observation point was 290 recorded per 1 min. Through the experiment, it was found that the temperature measurement 291 range of the six thermocouples outside the thermal insulation sandbox was 18±0.5℃, so the 292 heat loss from the sandbox was in negligible level. 293 3.Experimental System 210 266 Page 9 of 22 Experimental scheme 267 The sand sample and the groundwater were collected in the aquifer at the underground 268 depth of 24~44m in the relevant project. The sandbox was filled with equal volumetric weight 269 by layered wet filling method. Each raw sand layer with a thickness of 50mm was filled while 270 the raw underground water was sprayed evenly and the sand layer was compacted to ensure 271 that the porous medium in the sandbox has unit weight of 1.68±0.1 kgL-1, which was similar 272 to that of the underground aquifer. The filling height of raw sand was 1m, and then the upper 273 part of the sandbox was laid with a 0.2m clay-gravel layer as the confined aquifuge to isolate 274 the aquifer from the external environment. The photograph of the sandbox without the 275 thermal insulation material was shown as Figure 5. 276 Filter plate Liquid discharge region Aquifer (raw sand) Confined aquifuge Lens Overflow hole Piezometer Steel frame Liquid supply region 277 Confined aquifuge Liquid supply region Liquid discharge region 277 Figure 5. Photograph of the infiltration sandbox 278 After the establishment of the experimental system, the room temperature was first 279 maintained at 18℃ by central air conditioning, and then the 16℃ underground raw water 280 was filled into the sandbox for the exhaust gas process of porous media. When all 281 temperature observation points in the sandbox were maintained at 16±0.1℃, and the water 282 level in the piezometer was stable and no bubbles appeared, the porous medium of the 283 sandbox could be considered as saturated aquifer. 284 Before the experiment, it was proved that Darcy velocity was 2.4±0.02×10-6 (Pe≈2.5) when 285 the hydraulic difference was 0.2m, which satisfied the proportional relationship between 286 actual and experimental seepage velocity 1:20. During the experiment, the 16℃ underground 287 raw water with 0.2 m hydraulic difference was filled continuously into the sandbox to ensure 288 a stable Darcy velocity, simultaneously, BHEs ①~ ④ were opened in the test. The 289 experimental run time was set at 5.4 h and the temperature of each observation point was 290 recorded per 1 min. Through the experiment, it was found that the temperature measurement 291 range of the six thermocouples outside the thermal insulation sandbox was 18±0.5℃, so the 292 heat loss from the sandbox was in negligible level. 293 4. Compared with analytical and experimental data 295 321 e 4 = T q    (18) 322 x L l  (19) 323 (18) e 4 = T q    (18) x L l  (19) n 2 N(i)/A(i) E(i) i=1( ) RMSE = n θ θ   (20) 323 (19) (20) n n (20) 324 in which θN(i)/A(i) corresponds to the numerical solutions and analytical solutions respectively, 325 and θE(i) corresponds to the experimental result. 326 ( 0) in which θN(i)/A(i) corresponds to the numerical solutions and analytical solutions respectively, 325 and θE(i) corresponds to the experimental result. 326 ( in which θN(i)/A(i) corresponds to the numerical solutions and analytical solutions respectively and θE(i) corresponds to the experimental result. Figure 6. Comparison of multi-tube (Four-tube) analytical solution and numerical solution with 327 experimental result（y=0m,z=0.5m）:(a) temperature response θ over Fo (the observation points 1~6); (b) 328 temperature response θ over dimensionless distance L (Fo=0.232) 329 Th RMSE b t th l ti l l ti d th i t l lt i Fi 6( ) 330 0.00 0.05 0.10 0.15 0.20 0.25 0.0 0.5 1.0 1.5 2.0 2.5 3.0 point 5* point 6* point 1* θ Fo Analytical solution Numerical solution Test result point 2* point 3* point 4* (a) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 point 9* point 8* point 7* point 4* point 6* point 5* point 3* point 2* point 1* θ L Analytical solution Numerical solution Test result (b) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 point 9* point 8* point 7* point 4* point 6* point 5* point 3* point 2* point 1* θ L Analytical solution Numerical solution Test result (b) 0.00 0.05 0.10 0.15 0.20 0.25 0.0 0.5 1.0 1.5 2.0 2.5 3.0 point 5* point 6* point 1* θ Fo Analytical solution Numerical solution Test result point 2* point 3* point 4* (a) (b) (a) Figure 6. Compared with existing studied data 337 Compared with analytical and experimental data 295 According to the engineering prototype, FEFLOW 7.1 is used to establish the geometric 296 model, mesh division (triangular element non-equidistant) and set the parameters, and then 297 perform numerical simulation calculations. Meanwhile, MATLAB 2012 is used to calculate 298 the transient temperature response caused by the running BHE in engineering prototype, 299 according to the unsteady analytical model of the excess temperature in the aquifer (Eq.6-7). 300 Then, the experimental result and the analytical solution of the engineering prototype are 301 compared with the numerical solution of the engineering prototype. 302 In the numerical simulation, the clay layer is defined as a confined aquifuge while the 303 fine sand layer with a thickness of 20m exists as a confined aquifer. The clay layer in the 304 Page 10 of 22 upper part of the study area is defined as the impervious and adiabatic boundary while the 305 external environmental impact is ignored. Moreover, the four flanks of the 20×16×83 m3 306 model are defined as the fixed hydraulic head and constant temperature boundary. Then, the 307 parameters of the six ordinals arranged BHEs in this area are set according to Table 2. In 308 addition, the triangular element non-equidistant mesh generation is adopted in the entire 309 geotechnical layer. The physical model has a total number of nodes of 51740 and a grid 310 number of 93156. The fixed-time step method is used in the solution process. The total 311 number of time steps is 324 when the time-step length is set to 1 min and the maximum 312 iteration is 2500 times per step. 313 For facilitating the comparison, the time t, the excess temperature ΔT and the coordinate 314 displacement x are all in a dimensionless form. Due to the different proportional relationship 315 between the engineering prototype and the experimental system in operation time and 316 geometric size, Fo is taken as dimensionless time (Eq.16), θ is taken as dimensionless excess 317 temperature (Eq.18) and L is taken as dimensionless coordinate displacement (Eq.19). 318 Meanwhile, the root mean square error (RMSE) of dimensionless excess temperature θ (Eq.20) 319 is selected as the similarity index between experimental results, analytical solutions and 320 numerical solutions. Compared with analytical and experimental data 295 Comparison between the numerical solutions and the Hecht's solution in: θ and relative error for Fo Fo  θ Fit standard mean deviation θ relative error (%) 0.05153 0.17937 0.248 4.9% 0.07729 0.37967 0.2846 -6.7% 0.10306 0.59666 0.62288 1.9% 0.12882 0.81075 0.7164 -6.7% 0.15459 1.01956 1.165 10.3% 0.18035 1.21858 1.1348 -5.9% 0.20612 1.40478 1.5009 6.8% 0.23188 1.59073 1.48792 -7.3% 0.00 0.05 0.10 0.15 0.20 0.25 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Fo Numerical solution in this paper Hecht's solution  Figure 7. Comparison of multi-tube (Four-tube) numerical solution with Hecht's solution (Jozsef and Michael 2013): dimensionless mean temperature ( θ =4πλeΔT/q) change over Fo Table 4. Comparison between the numerical solutions and the Hecht's solution in: θ and relative error for Fo Fo  θ Fit standard mean deviation θ relative error (%) 0.05153 0.17937 0.248 4.9% 0.07729 0.37967 0.2846 -6.7% 0.10306 0.59666 0.62288 1.9% 0.12882 0.81075 0.7164 -6.7% 0.15459 1.01956 1.165 10.3% 0.18035 1.21858 1.1348 -5.9% 0.20612 1.40478 1.5009 6.8% 0.23188 1.59073 1.48792 -7.3% Table 4. Comparison between the numerical solutions and the Hecht's solution in: θ and relative error for Fo 0.00 0.05 0.10 0.15 0.20 0.25 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Fo Numerical solution in this paper Hecht's solution  Figure 7. Comparison of multi-tube (Four-tube) numerical solution with Hecht's solution (Jozsef and Michael 2013): dimensionless mean temperature ( θ =4πλeΔT/q) change over Fo After comparing Pe, it is found that the “scenario 12” in Hecht's research (Jozsef and 343 Michael 2013) are close to the working conditions in this paper (Figure 7). The results show 344 that the maximum error is 10.3% and the other errors are less than 10%. Therefore, using 345 FEFLOW7.1 to simulate the heat transfer process of the BHEs under forced seepage is 346 effective and correct. 347 Compared with analytical and experimental data 295 Comparison of multi-tube (Four-tube) analytical solution and numerical solution with 27 experimental result（y=0m,z=0.5m）:(a) temperature response θ over Fo (the observation points 1~6); (b) 28 temperature response θ over dimensionless distance L (Fo=0.232) 29 The RMSE between the analytical solutions and the experimental results in Figure 6(a) 330 does not exceed 5% while the RMSE between the numerical solution and the experimental 331 result does not exceed 1%. Besides, the RMSE between the analytical solution and the 332 experimental result is 3.8% and the RMSE of the numerical solutions and experimental results 333 is 0.5% in Figure 6(b). The results show that the analytical solution and experimental results 334 are consistent with the numerical solution, so FEFLOE 7.1 can simulate effectively and 335 accurately the heat transfer process of BHEs in the aquifer. 336 The RMSE between the analytical solutions and the experimental results in Figure 6(a) 330 does not exceed 5% while the RMSE between the numerical solution and the experimental 331 result does not exceed 1%. Besides, the RMSE between the analytical solution and the 332 experimental result is 3.8% and the RMSE of the numerical solutions and experimental results 333 is 0.5% in Figure 6(b). The results show that the analytical solution and experimental results 334 are consistent with the numerical solution, so FEFLOE 7.1 can simulate effectively and 335 accurately the heat transfer process of BHEs in the aquifer. 336 337 Page 11 of 22 The numerical solutions regarding the mean temperature change of the aquifer are then 338 compared to available data derived from (Jozsef and Michael 2013). Since the studies in 339 literature refer to different ground properties and groundwater velocities, as well as to 340 different dimensions, the comparison is performed by calculating for each condition in the 341 papers the Pe, Fo, θ. 342 0.00 0.05 0.10 0.15 0.20 0.25 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Fo Numerical solution in this paper Hecht's solution  Figure 7. Comparison of multi-tube (Four-tube) numerical solution with Hecht's solution (Jozsef and Michael 2013): dimensionless mean temperature ( θ =4πλeΔT/q) change over Fo Table 4. 5. Results and Discussion 348 The triangular element non-equidistant 370 mesh division is adopted for each rock-soil layer. Local refinement of the mesh is set in the 371 location of BHEs and the pumping-injection wells. 372 The number of total nodes and grid in the physical model is 248,752 and 413,295. The 373 fixed-time step method is used in the solution process. The time step, the total time step and 374 the maximum iteration are set to 1d, 3650 and 2500(times per step) respectively. Based on the 375 established model, the simulation calculations are carried out for eight kinds of operation 376 modes respectively (Table 5). 377 Table 5. Operation scenarios of the coupling pumping-injection wells and BHEs 378 Scenario Total flow rate of refrigerant per borehole (m3∙d-1) Pumping quantity of single well (m3∙d-1) Rated power of the submersible pump kW Recharge quantity of single well (m3∙d-1) Rated power of the pressure pump kW 1 30 0 0 0 0 2 30 100 1.5 40 0.75 3 30 200 2.2 80 1.1 4 30 300 4 120 2.2 5 30 400 5.5 160 2.2 6 30 500 5.5 200 3 7 30 600 7.5 240 3 8 30 700 7.5 280 3 To divide one operation cycle (1 year) into four stages that are followed by summer 379 cooling stage (4 months), autumn intermittent stage 1 (2 months), winter heating stage (4 380 months) and spring intermittent stage 2 (2 months). The system runs five operation cycles 381 and BHEs operate continuously for 10 hours per day in both the cooling and heating stage. 382 The inlet water temperature of BHEs during cooling/heating stage is constant at 31℃ /6℃ is 383 respectively. 384 Analysis and discussion 385 Because of the complex and diverse layout of the on-site well group and a large number 355 of boreholes, the BHEs (Figure 2) are generalized into five groups of BHEs in Figure 8. For 356 each group, there are seven boreholes arranged according to a staggered layout while the 357 space between each borehole is 4~5 m. To ensure the groundwater synchronous recharge, two 358 pumping wells and five injection wells are set up. Moreover, the design parameters of BHE 359 are shown in Table 2. The seven working wells are all incomplete diving wells with a depth of 360 60 m and a diameter of 0.4 m. 5. Results and Discussion 348 This section explores the influence of the pumping-injection flow rate of CGSHP system 349 compared with the established traditional GSHP system (Figure 2) on enhancing the heat 350 transfer effect. 351 Generalized model and Operation scheme 352 e e a i e o e a Ope a io e e staggered type C D A B 15 45 30 10 30 40 2* A ~ E Borehole heat exchangers Injection well Pumping well 30 30 E 35 40 40 15 45 15 45 1~3 Temperature observation point 5 3* 5 1* b c d e f g a Groundwater flow 35 353 Figure 8. Geometric arrangement of BHEs and pumping - injection wells (unit: m) 354 Figure 8. Geometric arrangement of BHEs and pumping - injection wells (unit: m) 354 Page 12 of 22 Page 12 of 22 Page 12 of 22 Because of the complex and diverse layout of the on-site well group and a large number 355 of boreholes, the BHEs (Figure 2) are generalized into five groups of BHEs in Figure 8. For 356 each group, there are seven boreholes arranged according to a staggered layout while the 357 space between each borehole is 4~5 m. To ensure the groundwater synchronous recharge, two 358 pumping wells and five injection wells are set up. Moreover, the design parameters of BHE 359 are shown in Table 2. The seven working wells are all incomplete diving wells with a depth of 360 60 m and a diameter of 0.4 m. Meanwhile, the filter is placed at a depth of 40~50 m. 361 On the grounds of the layout of the well group, the horizontal calculation area is 150×120 362 m2 and the vertical calculation range is 0~-83 m. From the top to the bottom, the rock-soil 363 layer with a thickness of 83 m is separated into five types of horizontal fault. The spatial 364 distribution and physical properties of that layer are shown in Table 1. 365 In the numerical calculation, the silty clay layer in the upper and lower part of the study 366 area is defined as the impervious and adiabatic boundary as the effects of atmospheric 367 rainfall and evaporation are ignored. The four flanks of the physical model are defined as the 368 fixed hydraulic head and constant temperature boundary. In addition, the pumping-injection 369 wells are defined as the constant flow boundary. 5. Results and Discussion 348 Meanwhile, the filter is placed at a depth of 40~50 m. 361 On the grounds of the layout of the well group, the horizontal calculation area is 150×120 362 m2 and the vertical calculation range is 0~-83 m. From the top to the bottom, the rock-soil 363 layer with a thickness of 83 m is separated into five types of horizontal fault. The spatial 364 distribution and physical properties of that layer are shown in Table 1. 365 In the numerical calculation, the silty clay layer in the upper and lower part of the study 366 area is defined as the impervious and adiabatic boundary as the effects of atmospheric 367 rainfall and evaporation are ignored. The four flanks of the physical model are defined as the 368 fixed hydraulic head and constant temperature boundary. In addition, the pumping-injection 369 wells are defined as the constant flow boundary. The triangular element non-equidistant 370 mesh division is adopted for each rock-soil layer. Local refinement of the mesh is set in the 371 location of BHEs and the pumping-injection wells. 372 The number of total nodes and grid in the physical model is 248,752 and 413,295. The 373 fixed-time step method is used in the solution process. The time step, the total time step and 374 the maximum iteration are set to 1d, 3650 and 2500(times per step) respectively. Based on the 375 established model, the simulation calculations are carried out for eight kinds of operation 376 modes respectively (Table 5). 377 p y Table 5. Operation scenarios of the coupling pumping-injection wells and BHEs 378 Scenario Total flow rate of refrigerant per borehole (m3∙d-1) Pumping quantity of single well (m3∙d-1) Rated power of the submersible pump kW Recharge quantity of single well (m3∙d-1) Rated power of the pressure pump kW 1 30 0 0 0 0 2 30 100 1.5 40 0.75 3 30 200 2.2 80 1.1 4 30 300 4 120 2.2 5 30 400 5.5 160 2.2 6 30 500 5.5 200 3 7 30 600 7.5 240 3 8 30 700 7.5 280 3 Table 5. Operation scenarios of the coupling pumping-injection wells and BHEs To divide one operation cycle (1 year) into four stages that are followed by summer 379 cooling stage (4 months), autumn intermittent stage 1 (2 months), winter heating stage (4 380 months) and spring intermittent stage 2 (2 months). 5. Results and Discussion 348 The system runs five operation cycles 381 and BHEs operate continuously for 10 hours per day in both the cooling and heating stage. 382 The inlet water temperature of BHEs during cooling/heating stage is constant at 31℃ /6℃ is 383 respectively. 384 To divide one operation cycle (1 year) into four stages that are followed by summer 379 cooling stage (4 months), autumn intermittent stage 1 (2 months), winter heating stage (4 380 months) and spring intermittent stage 2 (2 months). The system runs five operation cycles 381 and BHEs operate continuously for 10 hours per day in both the cooling and heating stage. 382 The inlet water temperature of BHEs during cooling/heating stage is constant at 31℃ /6℃ is 383 respectively. 384 Page 13 of 22 Page 13 of 22 The Darcy velocity and the hydrodynamic distribution have a significant difference in 386 the same aquifer since the total pumping-injection flow rate (∑G) is different (Figure 9). Darcy 387 velocity, which increases approximately 10 time, increases from 2.4 ~ 3.2×10-7 m∙s-1 to 388 2.0~3.0×10-6 m∙s-1 When the total pumping-injection flow rate increases from 200 m3∙d-1to 1200 389 m3∙d-1. 390 y / m 0 10 20 30 40 50 60 70 80 90 100110120130140150 0 10 20 30 40 50 60 70 80 90 100 110 120 Injection 1 Injection 2 Injection 3 Injection 4 Injection 5 Pumping 1 Pumping 2 x / m (a) x / m y / m 0 10 20 30 40 50 60 70 80 90 100110120130140150 0 10 20 30 40 50 60 70 80 90 100 110 120 2.2E-007 4.0E-007 4.0E-007 Injection 1 Injection 2 Injection 3 Injection 4 Injection 5 Pumping 1 Pumping 2 (b) Figure 9. 5. Results and Discussion 348 Spatial hydrodynamic distribution in the fine sand layer on the 120d under the different total 391 pumping-injection flow rate ∑G: (a) ∑G=200 m3∙d-1; (b) ∑G=600 m3∙d-1; (c) ∑G=1200 m3∙d-1 392 y / m 0 10 20 30 40 50 60 70 80 90 100110120130140150 0 10 20 30 40 50 60 70 80 90 100 110 120 Injection 1 Injection 2 Injection 3 Injection 4 Injection 5 Pumping 1 Pumping 2 x / m (a) x / m y / m 0 10 20 30 40 50 60 70 80 90 100110120130140150 0 10 20 30 40 50 60 70 80 90 100 110 120 2.2E-007 4.0E-007 4.0E-007 Injection 1 Injection 2 Injection 3 Injection 4 Injection 5 Pumping 1 Pumping 2 (b) y / m 0 10 20 30 40 50 60 70 80 90 100110120130140150 0 10 20 30 40 50 60 70 80 90 100 110 120 Injection 1 Injection 2 Injection 3 Injection 4 Injection 5 Pumping 1 Pumping 2 x / m (c) (b) (a) (a) y / m 0 10 20 30 40 50 60 70 80 90 100110120130140150 0 10 20 30 40 50 60 70 80 90 100 110 120 Injection 1 Injection 2 Injection 3 Injection 4 Injection 5 Pumping 1 Pumping 2 x / m (c) (c) Figure 9. Spatial hydrodynamic distribution in the fine sand layer on the 120d under the different total 391 pumping-injection flow rate ∑G: (a) ∑G=200 m3∙d-1; (b) ∑G=600 m3∙d-1; (c) ∑G=1200 m3∙d-1 392 pumping-injection flow rate ∑G: (a) ∑G=200 m3∙d-1; (b) ∑G=600 m3∙d-1; (c) ∑G=1200 m3∙d-1 pumping-injection flow rate ∑G: (a) ∑G=200 m3∙d-1; (b) ∑G=600 m3∙d-1; (c) ∑G=1200 m3∙d-1 392 Due to the difference of pumping-injection flow rate, profile temperature fields have a 393 significant difference in the same aquifer (Figure 10). Taking BHEs' well group E as the 394 research object, the injection well and the pumping well with a spacing of 120 m are defined 395 as the upstream and downstream boundaries, respectively. 5. Results and Discussion 348 405 By the end of the cooling stage (120d), the thermal radius of BHEs in both upstream and 406 downstream areas is 11 m. 407 The range of thermal diffusion in the downstream area of BHEs significantly expands 408 with the pumping-injection flow rate increases from 200 m3∙d-1to 1200 m3∙d-1. At the 120 d, the 409 thermal radius along the direction of pumping reaches 19 m, 35 m and 49 m, so the thermal 410 radius of 1200 m3∙d-1 is 2.6 times of that of 200 m3∙d-1. With the increase of the flow rate, the 411 migration speed of the temperature fronts accelerates and the thermal radius enlarges 412 continuously, moreover, the thermal radius in the upstream zone is smaller than 11m. 413 Figure 10. Profile temperature field in the fine sand aquifer under the different total pumping-injection 397 flow rate: (a) the end of the cooling stage (120 d); (b) the end of the heating stage (300 d) 398 Figure 10. Profile temperature field in the fine sand aquifer under the different total pumping-injection 397 flow rate: (a) the end of the cooling stage (120 d); (b) the end of the heating stage (300 d) 398 To describe accurately the evolution process of the aquifer's temperature field under 399 different operation modes, the calculation area with a temperature change of ±0.5 ℃ is 400 defined as the thermal diffusion range of the BHEs. Besides, the heat-influencing radius is 401 defined as the coordinate distance between E (g) and the farthest acting position. 402 When the pumping-injection flow rate is 0 m3∙d-1, there is only the heat conduction 403 between BHEs and the aquifer as well as between the aqueous medium units. The heat 404 transfer process is slow and the heat influence range is diffused symmetrically around BHEs. 405 By the end of the cooling stage (120d), the thermal radius of BHEs in both upstream and 406 downstream areas is 11 m. 407 The range of thermal diffusion in the downstream area of BHEs significantly expands 408 with the pumping-injection flow rate increases from 200 m3∙d-1to 1200 m3∙d-1. At the 120 d, the 409 thermal radius along the direction of pumping reaches 19 m, 35 m and 49 m, so the thermal 410 radius of 1200 m3∙d-1 is 2.6 times of that of 200 m3∙d-1. 5. Results and Discussion 348 With the increase of the flow rate, the 411 migration speed of the temperature fronts accelerates and the thermal radius enlarges 412 continuously, moreover, the thermal radius in the upstream zone is smaller than 11m. 413 Figure 11. Temperature response dynamic curve in the fine sand aquifer at the first cooling stage: (a) the 414 upstream area of BHEs (the observation point 1); (b) the inside area of BHEs (the observation point 2); 415 (c) the downstream area of BHEs (the observation point 3) 416 According to the theory of heat and mass transfer, it is precisely because of the increase 417 of Darcy velocity that the convective heat transfer intensity and thermomechanical dispersion 418 effect are improved correspondingly, thereby expanding the range of thermal diffusion in the 419 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.90 0.95 1.00 1.05 1.10 1.15 1.20 observation point 1 T disp / T cond Fo Pe=0.97 Pe=2.93 Pe=4.88 Pe=9.74 (a) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.8 1.0 1.2 1.4 1.6 1.8 2.0 observation point 2* T disp / T cond Fo Pe=0.97 Pe=2.93 Pe=4.88 Pe=9.74 (b) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.8 1.0 1.2 1.4 1.6 1.8 2.0 observation point 3* T disp / T cond Fo Pe=0.97 Pe=2.93 Pe=4.88 Pe=9.74 (c) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.90 0.95 1.00 1.05 1.10 1.15 1.20 observation point 1* T disp / T cond Fo Pe=0.97 Pe=2.93 Pe=4.88 Pe=9.74 (a) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.8 1.0 1.2 1.4 1.6 1.8 2.0 observation point 2* T disp / T cond Fo Pe=0.97 Pe=2.93 Pe=4.88 Pe=9.74 (b) (b) (a) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.8 1.0 1.2 1.4 1.6 1.8 2.0 observation point 3* T disp / T cond Fo Pe=0.97 Pe=2.93 Pe=4.88 Pe=9.74 (c) (c) Figure 11. 5. Results and Discussion 348 396 x / m T / pumping well injection well BHE E(b) BHE E(g) BHE E(e) 0 10 20 30 40 50 60 70 80 90 100110120 14 15 17 19 21 23 25 27 28 29 G (m3 ·d-1 ) 200 0 600 1200 (a) x / m T / pumping well injection well BHE E(b) BHE E(g) BHE E(e) 0 10 20 30 40 50 60 70 80 90 100110120 7 8 10 12 14 15 16 18 19 20 21 22 G (m3 ·d-1 ) 200 0 600 1200 (b) x / m T / pumping well injection well BHE E(b) BHE E(g) BHE E(e) 0 10 20 30 40 50 60 70 80 90 100110120 7 8 10 12 14 15 16 18 19 20 21 22 G (m3 ·d-1 ) 200 0 600 1200 (b) x / m T / pumping well injection well BHE E(b) BHE E(g) BHE E(e) 0 10 20 30 40 50 60 70 80 90 100110120 14 15 17 19 21 23 25 27 28 29 G (m3 ·d-1 ) 200 0 600 1200 (a) x / m T / pumping well injection well BHE E(b) BHE E(g) BHE E(e) 0 10 20 30 40 50 60 70 80 90 100110120 14 15 17 19 21 23 25 27 28 29 G (m3 ·d-1 ) 200 0 600 1200 (a) x / m 60 70 x / m 60 70 (a) (b) Page 14 of 22 Figure 10. Profile temperature field in the fine sand aquifer under the different total pumping-injection 397 flow rate: (a) the end of the cooling stage (120 d); (b) the end of the heating stage (300 d) 398 To describe accurately the evolution process of the aquifer's temperature field under 399 different operation modes, the calculation area with a temperature change of ±0.5 ℃ is 400 defined as the thermal diffusion range of the BHEs. Besides, the heat-influencing radius is 401 defined as the coordinate distance between E (g) and the farthest acting position. 402 When the pumping-injection flow rate is 0 m3∙d-1, there is only the heat conduction 403 between BHEs and the aquifer as well as between the aqueous medium units. The heat 404 transfer process is slow and the heat influence range is diffused symmetrically around BHEs. 5. Results and Discussion 348 Temperature response dynamic curve in the fine sand aquifer at the first cooling stage: (a) the 414 upstream area of BHEs (the observation point 1); (b) the inside area of BHEs (the observation point 2); 415 (c) the downstream area of BHEs (the observation point 3*) 416 According to the theory of heat and mass transfer, it is precisely because of the increase 417 of Darcy velocity that the convective heat transfer intensity and thermomechanical dispersion 418 effect are improved correspondingly, thereby expanding the range of thermal diffusion in the 419 According to the theory of heat and mass transfer, it is precisely because of the increase 417 of Darcy velocity that the convective heat transfer intensity and thermomechanical dispersion 418 effect are improved correspondingly, thereby expanding the range of thermal diffusion in the 419 Page 15 of 22 Page 15 of 22 Page 15 of 22 downstream region and alleviating the thermal accumulation phenomenon of BHE. So, in 420 order to obtain the difference between the temperature response ∆Tdisp with forced 421 groundwater seepage and the temperature response ∆Tcond without groundwater seepage at 422 different temperature observation points, the dynamic variation of ∆Tdisp/∆Tcond with time is 423 calculated (Figure 11). 424 As shown in Figure 11 (a), the mutative extent of temperature decreases gradually with the 425 running time. When the running time exceeds 50% of the whole period (Fo≥0. 2), ∆Tdisp/∆Tcond＜1 . As 426 shown in Figure 11 (b), ∆Tdisp/∆Tcond tends to be stable gradually with time. At the end of the cooling 427 stage(120d), ∆Tdisp/∆Tcond＞1. The change of Darcy velocity has no obvious influence on the change 428 rate and amplitude of excess temperature because the thermal convection and thermal dispersion 429 enhances thermal interference between BHEs. As shown in Figure 11 (c), with the passage of time, the 430 excess temperature tends to be stable and the curve of ∆Tdisp/∆Tcond tends to be smooth. The mutative 431 degree of excess temperature enhances with the increase of Darcy velocity but the difference between 432 ∆Tcond and ∆Tdisp tends to decrease when Darcy velocity increases to a certain extent. 433 Figure 12. 5. Results and Discussion 348 Dynamic changes of the average outlet water temperature (TO) and the average heat transfer 435 efficiency (E=Q/Q’=\|Ti-To\|/\|Ti-Ts0\|) of BHEs in the fifth operation cycle: (a) the cooling stage; (b) 436 the heating stage 437 1460 1480 1500 1520 1540 1560 1580 24 25 26 27 28 29 30 31 32 To/℃ operation time/d To: ∑G= 0 m3d-1 ∑G= 200 m3d-1 ∑G= 400 m3d-1 ∑G= 600 m3d-1 ∑G=1200 m3d-1 E : ∑G= 0 m3d-1 ∑G= 200 m3d-1 ∑G= 400 m3d-1 ∑G= 600 m3d-1 ∑G=1200 m3d-1 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 E (a) 1640 1660 1680 1700 1720 1740 1760 6 7 8 9 10 11 To/℃ operation time/d E : ∑G= 0 m3d-1 ∑G= 200 m3d-1 ∑G= 400 m3d-1 ∑G= 600 m3d-1 ∑G=1200 m3d-1 To: ∑G= 0 m3d-1 ∑G= 200 m3d-1 ∑G= 400 m3d-1 ∑G= 600 m3d-1 ∑G=1200 m3d-1 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 E (b) 1640 1660 1680 1700 1720 1740 1760 6 7 8 9 10 11 To/℃ operation time/d E : ∑G= 0 m3d-1 ∑G= 200 m3d-1 ∑G= 400 m3d-1 ∑G= 600 m3d-1 ∑G=1200 m3d-1 To: ∑G= 0 m3d-1 ∑G= 200 m3d-1 ∑G= 400 m3d-1 ∑G= 600 m3d-1 ∑G=1200 m3d-1 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 E (b) 1460 1480 1500 1520 1540 1560 1580 24 25 26 27 28 29 30 31 32 To/℃ operation time/d To: ∑G= 0 m3d-1 ∑G= 200 m3d-1 ∑G= 400 m3d-1 ∑G= 600 m3d-1 ∑G=1200 m3d-1 E : ∑G= 0 m3d-1 ∑G= 200 m3d-1 ∑G= 400 m3d-1 ∑G= 600 m3d-1 ∑G=1200 m3d-1 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 E (a) (b) (a) Figure 12. Dynamic changes of the average outlet water temperature (TO) and the average heat transfer 435 efficiency (E=Q/Q’=\|Ti-To\|/\|Ti-Ts0\|) of BHEs in the fifth operation cycle: (a) the cooling stage; (b) 436 the heating stage 437 At the early stage of various operation modes, due to the large temperature difference in 438 heat transfer between the boreholes and the rock-soil layer, the temperature difference 439 between the inlet with outlet water of BHEs is higher and the corresponding energy efficiency 440 coefficient also rise, (Figure 12). The heat exchange rate reduces because of the decreasing 441 temperature difference between the boreholes and the surrounding medium. As a result, the 442 temperature difference of the inlet and outlet water of BHEs decreases rapidly. 5. Results and Discussion 348 At the end of 443 the fifth operation cycle, the average heat transfer efficiency of the five types of operation 444 modes in the cooling stage is 7.1%, 8.5%, 11.1%, 13.4% and 18.6% while the average heat 445 transfer efficiency in the heating stage is 10.6%, 11.9%, 12.8%, 14.6% and 18.1% respectively. 446 447 Page 16 of 22 Figure 13. Cumulative distributions of the temperature difference of the inlet and outlet water of BHEs: 448 (a) the cooling stage; (b) the heating stage 449 Th l i di ib i b d h l i i d d ib 0 0 10 20 30 40 50 60 70 80 90 100 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 ∑G= 0 m3d-1 ∑G= 200 m3d-1 ∑G= 400 m3d-1 ∑G= 600 m3d-1 ∑G=1200 m3d-1 cumulate time/ % ｜△T ｜/℃ (a) 0 10 20 30 40 50 60 70 80 90 100 1.5 2.0 2.5 3.0 3.5 4.0 ｜△T ｜/℃ cumulate time/ % ∑G= 0 m3d-1 ∑G= 200 m3d-1 ∑G= 400 m3d-1 ∑G= 600 m3d-1 ∑G=1200 m3d-1 (b) 0 10 20 30 40 50 60 70 80 90 100 1.5 2.0 2.5 3.0 3.5 4.0 ｜△T ｜/℃ cumulate time/ % ∑G= 0 m3d-1 ∑G= 200 m3d-1 ∑G= 400 m3d-1 ∑G= 600 m3d-1 ∑G=1200 m3d-1 (b) 0 10 20 30 40 50 60 70 80 90 100 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 ∑G= 0 m3d-1 ∑G= 200 m3d-1 ∑G= 400 m3d-1 ∑G= 600 m3d-1 ∑G=1200 m3d-1 cumulate time/ % ｜△T ｜/℃ (a) (b) (a) (b) Figure 13. Cumulative distributions of the temperature difference of the inlet and outlet water of BHEs: 448 (a) the cooling stage; (b) the heating stage 449 Figure 13. Cumulative distributions of the temperature difference of the inlet and outlet water of BHEs: 448 (a) the cooling stage; (b) the heating stage 449 The cumulative distribution curve can be used as another evaluation index to describe 450 the duration of a certain heat exchange efficiency of BHEs (Figure 13). The absolute values of 451 the inlet and outlet temperature difference of BHEs is arranged in descending order, and the 452 cumulative average temperature difference distribution in the cooling stages and heating 453 stages over the whole simulation stages (5 years) is calculated. 5. Results and Discussion 348 When the cumulate time 454 exceeds 20% of the total operation stage, the temperature difference tends to be moderate, 455 when the cumulate time reaches 50% and the temperature difference reaches a steady state. 456 p y The inlet and outlet temperature difference of BHEs corresponding to the median time is 457 used as another evaluation index to compare the heat transfer performance of BHEs under 458 different modes. In cooling stage, the median of temperature difference is 2.34℃ when the 459 pumping-injection flow rate is 0 m3∙d-1. With the pumping-injection flow rate rises from 200 460 m3∙d-1 to 1200 m3∙d-1, the growth rate of the median temperature difference varies from 11.5% 461 to 73.9%. In heating stage, the median temperature difference is 1.76℃ when BHEs run 462 individually.With the pumping-injection flow rate increases from 200 m3∙d-1 to 1200 m3∙d-1, the 463 growth rate of the median temperature difference increases from 10.2% to 34.1%. As the 464 pumping-injection flow rate, Darcy velocity and the heat exchange intensity of BHEs all 465 increase, thereby the decline rate of the temperature difference slows down while the time 466 required reaching a steady state increase. 467 The heat transfer rates per unit borehole depth of BHEs (q ) rise gradually when 468 pumping and injection flow rate increases (Figure 14). Taking the fifth year as an example, 469 theq in cooling and heating stages with the increase of the pumping-injection flow rate from 470 0 m3∙d-1 to 1200 m3∙d-1 varies from 30.3 W∙m-1 and 25.4 W∙m-1 to 82.2 W∙m-1 and 39.6 W∙m-1 471 respectively. 472 Page 17 of 22 Figure 14. Average heat transfer rates per unit borehole depth of BHEs (q=(ρrcrMΔT)/H) in the fine sand layer over the whole operation period under different pumping-injection flow rate ∑G: (a) ∑G=0 m3∙d-1; (b) ∑G=200 m3∙d-1; (c) ∑G=400 m3∙d-1; (d) ∑G=600 m3∙d-1; (e) ∑G=800 m3∙d-1; (f) ∑G=1200 m3∙d-1 Thermal accumulation becomes serious with the continuous operation of the system, then the average heat transfer efficiency gets lower on the condition that the pumping and injection well ceases to work or the pumping-injection flow rate come to be smaller (200 m3∙d-1). As a result, cooling and heating quantity is declining every year. 5. Results and Discussion 348 Average heat transfer rates per unit borehole depth of BHEs (q=(ρrcrMΔT)/H) in the fine 473 sand layer over the whole operation period under different pumping-injection flow rate ∑G: (a) ∑G=0 474 m3∙d-1; (b) ∑G=200 m3∙d-1; (c) ∑G=400 m3∙d-1; (d) ∑G=600 m3∙d-1; (e) ∑G=800 m3∙d-1; (f) ∑G=1200 m3∙d-1 475 Thermal accumulation becomes serious with the continuous operation of the system, 476 then the average heat transfer efficiency gets lower on the condition that the pumping and 477 injection well ceases to work or the pumping-injection flow rate come to be smaller (200 478 m3∙d-1). As a result, cooling and heating quantity is declining every year. However, the 479 rock-soil layer is not a single cold or heat source while an energy storage body has certain 480 thermal storage and rejection. Therefore, thermal accumulation in the wells group’s area 481 enhances the heat storage capacity of the rock-soil layer across the season in the summer. 482 During the heating stage, the temperature of the rock-soil layer is higher than the initial stage. 483 igure 14. Average heat transfer rates per unit borehole depth of BHEs (q=(ρrcrMΔT)/H) in the fine and layer over the whole operation period under different pumping-injection flow rate ∑G: (a) ∑G=0 m3∙d-1; (b) ∑G=200 m3∙d-1; (c) ∑G=400 m3∙d-1; (d) ∑G=600 m3∙d-1; (e) ∑G=800 m3∙d-1; (f) ∑G=1200 m3∙d-1 Figure 14. Average heat transfer rates per unit borehole depth of BHEs (q=(ρrcrMΔT)/H) in the fine 473 sand layer over the whole operation period under different pumping-injection flow rate ∑G: (a) ∑G=0 474 m3∙d-1; (b) ∑G=200 m3∙d-1; (c) ∑G=400 m3∙d-1; (d) ∑G=600 m3∙d-1; (e) ∑G=800 m3∙d-1; (f) ∑G=1200 m3∙d-1 475 Thermal accumulation becomes serious with the continuous operation of the system, 476 th th h t t f ffi i t l th diti th t th i d 477 Thermal accumulation becomes serious with the continuous operation of the system, 476 then the average heat transfer efficiency gets lower on the condition that the pumping and 477 injection well ceases to work or the pumping-injection flow rate come to be smaller (200 478 m3∙d-1). As a result, cooling and heating quantity is declining every year. However, the 479 rock-soil layer is not a single cold or heat source while an energy storage body has certain 480 thermal storage and rejection. 5. Results and Discussion 348 However, the rock-soil layer is not a single cold or heat source while an energy storage body has certain thermal storage and rejection Therefore thermal accumulation in the wells group’s area 1st cooling season 1st heating season 2nd cooling season 2nd heating season 3rd cooling season 3rd heating season 4th cooling season 4th heating season 5th cooling season 5th heating season 16 18 20 22 24 26 28 ∑G= 0 m3d-1 q /W·m-1 operation period (a) 1st cooling season 1st heating season 2nd cooling season 2nd heating season 3rd cooling season 3rd heating season 4th cooling season 4th heating season 5th cooling season 5th heating season 20 22 24 26 28 30 32 34 36 ∑G=200 m3d-1 q /W·m-1 operation period (b) 1st cooling season 1st heating season 2nd cooling season 2nd heating season 3rd cooling season 3rd heating season 4th cooling season 4th heating season 5th cooling season 5th heating season 20 25 30 35 40 45 50 55 ∑G=400 m3d-1 q /W·m-1 operation period (c) 1st cooling season 1st heating season 2nd cooling season 2nd heating season 3rd cooling season 3rd heating season 4th cooling season 4th heating season 5th cooling season 5th heating season 25 30 35 40 45 50 55 60 65 70 ∑G=600 m3d-1 operation period q /W·m-1 (d) 1st cooling season 1st heating season 2nd cooling season 2nd heating season 3rd cooling season 3rd heating season 4th cooling season 4th heating season 5th cooling season 5th heating season 25 30 35 40 45 50 55 60 65 70 75 80 85 ∑G=800 m3d-1 operation period q /W·m-1 (e) 1st cooling season 1st heating season 2nd cooling season 2nd heating season 3rd cooling season 3rd heating season 4th cooling season 4th heating season 5th cooling season 5th heating season 30 35 40 45 50 55 60 65 70 75 80 85 90 95 ∑G=1200 m3d-1 q /W·m-1 operation period (f) 1st cooling season 1st heating season 2nd cooling season 2nd heating season 3rd cooling season 3rd heating season 4th cooling season 4th heating season 5th cooling season 5th heating season 16 18 20 22 24 26 28 ∑G= 0 m3d-1 q /W·m-1 operation period (a) 1st cooling season 1st heating season 2nd cooling season 2nd heating season 3rd cooling season 3rd heating season 4th cooling season 4th heating season 5th cooling season 5th heating season 20 22 24 26 28 30 32 34 36 ∑G=200 m3d-1 q /W·m-1 operation period (b) operation period (b) 1st cooling season 1st heating season 2nd cooling season 2nd heating season 3rd cooling season 3rd heating season 4th cooling season 4th heating season 5th cooling season 5th heating season 25 30 35 40 45 50 55 60 65 70 ∑G=600 m3d-1 operation period q /W·m-1 (d) (a) (b) 1st cooling season 1st heating season 2nd cooling season 2nd heating season 3rd cooling season 3rd heating season 4th cooling season 4th heating season 5th cooling season 5th heating season 20 25 30 35 40 45 50 55 ∑G=400 m3d-1 q /W·m-1 operation period (c) (c) 1st cooling season 1st heating season 2nd cooling season 2nd heating season 3rd cooling season 3rd heating season 4th cooling season 4th heating season 5th cooling season 5th heating season 25 30 35 40 45 50 55 60 65 70 75 80 85 ∑G=800 m3d-1 operation period q /W·m-1 (e) (d) (c) 1st cooling season 1st heating season 2nd cooling season 2nd heating season 3rd cooling season 3rd heating season 4th cooling season 4th heating season 5th cooling season 5th heating season 30 35 40 45 50 55 60 65 70 75 80 85 90 95 ∑G=1200 m3d-1 q /W·m-1 operation period (f) 4 5 operation period operation period (f) (e) Figure 14. 5. Results and Discussion 348 Therefore, thermal accumulation in the wells group’s area 481 enhances the heat storage capacity of the rock-soil layer across the season in the summer. 482 During the heating stage, the temperature of the rock-soil layer is higher than the initial stage. 483 Page 18 of 22 Page 18 of 22 Page 18 of 22 The temperature difference between the soil and the circulating solution increases so that the 484 heat exchange quantity of the BHEs is improved at this stage. 485 0 2 4 6 8 10 12 0 10 20 30 40 50 60 70 80 90 100 110 Energy consumption of pump / kWh·a-1 Pe 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 △q/ W·m-1 Cooling period (R2=0.87) △q =0.93+17.17exp[-0.5(Pe-3.61)/1.87)2]; Heating period (R2=0.85) △q =0.31+4.1exp[-0.5(Pe-3.74)/2.2)2]; Pump's energy consumption 486 Figure 15. The total energy consumption of pumps and the increment of heat transfer rate per unit 487 d h f BHE (Δ   ) h P 488 0 2 4 6 8 10 12 0 10 20 30 40 50 60 70 80 90 100 110 Energy consumption of pump / kWh·a-1 Pe 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 △q/ W·m-1 Cooling period (R2=0.87) △q =0.93+17.17exp[-0.5(Pe-3.61)/1.87)2]; Heating period (R2=0.85) △q =0.31+4.1exp[-0.5(Pe-3.74)/2.2)2]; Pump's energy consumption 486 Figure 15. The total energy consumption of pumps and the increment of heat transfer rate per unit 487 depth of BHEs (Δq= qt+1-qt ) change over Pe 488 Figure 15. The total energy consumption of pumps and the increment of heat transfer rate per unit 487 depth of BHEs (Δq= qt+1-qt ) change over Pe 488 Under the cooling and heating stages, the relation between Δq（known as the increment 489 of average heat transfer rate per unit depth of BHEs）and Pe is determined by fitting which 490 shows that Δq is distributed as a Gaussian function with the uptrend of Pe (Figure 15). 491 Although the effect of convective heat transfer and thermal dispersion between BHEs and the 492 rock-soil layer can be enhanced effectively by strengthening the velocity of groundwater 493 seepage, the average heat transfer rates per unit borehole depth of BHEs q does not raise 494 linearly with the increase of the pumping-injection flow rate. 5. Results and Discussion 348 495 The research shows that Darcy velocity is only 0.6×10-6~1.4×10-6 m∙s-1 when the pumping 496 and injection flow rate is 400~600m3∙d-1, but the Δq respectively reaches 12.8~17.9W∙m-1 and 497 3.6~4.2 W∙m-1 during the cooling stage and heating stage which are located on both sides of 498 the extremum point of the distribution curve. As the designed Pumping-injection flow rate 499 further increases, not only the Δq decreases gradually, but also the energy consumption of 500 pumping and injection pumps increase that leads to the increase of operation cost of the 501 system. Furthermore, the change of aquifer spatial structure will be irreversible if the forced 502 seepage velocity is too high. Therefore, in order to obtain the best heat transfer enhancement 503 effect, system environment and economic benefits, the pumping-injection flow rate when 504 Darcy velocity reaches 0.6×10-6~1.4×10-6 m∙s-1 is taken as the best reference range for CGSHP 505 system, so the best flow rate range of pumping-injection wells is 400~600 m3∙d-1 in this paper. 506 6. Conclusion 507 560 Page 19 of 22 Page 19 of 22 sandbox experiment and analytical model are used to verify the numerical simulation of 516 FEFLOW7.1. The results show that the numerical simulation software FEFLOW7.1 can 517 simulate effectively and correctly the evolution process of the aquifer temperature field 518 during the taking heat of BHEs. 519 sandbox experiment and analytical model are used to verify the numerical simulation of 516 FEFLOW7.1. The results show that the numerical simulation software FEFLOW7.1 can 517 simulate effectively and correctly the evolution process of the aquifer temperature field 518 during the taking heat of BHEs. 519 (b) When the pumping-injection flow rate from 200 m3∙d-1 to 1200 m3∙d-1, Darcy velocity 520 in the fine sand layer increases approximately 10 times and the effect of convective heat 521 transfer and thermal dispersion is enhanced. In the downstream region of BHEs, the thermal 522 action radius is 2.6 times of the original radius. Hence, the cold and heat accumulation of 523 CGSHP system can be alleviated effectively in the aquifer. 524 (b) When the pumping-injection flow rate from 200 m3∙d-1 to 1200 m3∙d-1, Darcy velocity 520 in the fine sand layer increases approximately 10 times and the effect of convective heat 521 transfer and thermal dispersion is enhanced. In the downstream region of BHEs, the thermal 522 action radius is 2.6 times of the original radius. Hence, the cold and heat accumulation of 523 CGSHP system can be alleviated effectively in the aquifer. 524 (c) For long-term running BHEs, the cumulative distribution curve of the inlet and outlet 525 water average temperature difference is introduced to describe the duration of a certain heat 526 exchange efficiency of BHEs. The average temperature difference corresponding to the 527 median time is used as a new parameter for evaluating the heat transfer performance of BHEs. 528 When the flow rate of pumping-injection wells increases, the average temperature difference 529 and the average energy efficiency coefficient of BHEs as well as the heat exchange quantity of 530 BHEs are improved substantially. 531 (d) In cooling and heating stages, the relation curve between the Δq and Pe has a 532 Gaussian function distribution. Therefore, the heat exchange quantity of BHEs cannot be 533 increased continuously by increasing the pumping-injection flow rate infinitely. 6. Conclusion 507 528 When the flow rate of pumping-injection wells increases, the average temperature difference 529 and the average energy efficiency coefficient of BHEs as well as the heat exchange quantity of 530 BHEs are improved substantially. 531 (d) In cooling and heating stages, the relation curve between the Δq and Pe has a 532 Gaussian function distribution. Therefore, the heat exchange quantity of BHEs cannot be 533 increased continuously by increasing the pumping-injection flow rate infinitely. The 534 pumping-injection flow rate that can make Darcy velocity reaches 0.6×10-6~1.4×10-6 m∙s-1 is the 535 best reference range of CGSHP system，so the best pumping-injection flow rate is 400~600 536 m3∙d-1 in this paper. 537 (e) CGSHP system is proposed for the areas where the shallow-groundwater reserves 538 are abundant but the seepage velocity is weak, such as the Bohai Plain in Tianjin, China. The 539 results show that the system can control the seepage velocity of groundwater by changing the 540 pumping-injection flow rate to enhance heat transfer, so CGSHP system has good 541 controllability and predictability. In addition, CGSHP system should also be applicable to 542 other countries with the same hydrogeological conditions as the region in this paper. The 543 system has certain universality. 544 7. Declarations 545 Author contributions: JM and QJ performed conceptualization; JM and QJ performed data curation; JM 546 and QJ performed formal analysis; JM and QJ provided methodology; QZ provided resources; YX and 547 YW provided software simulation; FY validated data; QJ writed the original draft; JM and QJ improved 548 and revised the manuscript. All authors read and approved the final manuscript. 549 Funding: This research was funded by National Natural Science Foundation of China of Funder, grant 550 number 41402228; This research was funded by Enterprise science and technology commissioner project 551 of Tianjin of Funder, grant number 19JCTPJC48100. 552 Availability of data and materials: The datasets generated and analyzed during the current study are 553 available from the corresponding author on reason-able request. 554 Acknowledgments: Thanks to the Tianjin Institute of Geotechnical Investigation Surveying for the 555 hydrogeological parameters and relevant data provided. 556 Conflicts of Interest: The authors declare that they have no competing interests. 557 Author details: 1 School of Energy Safety Engineering, Tianjin Chengjian University, Tianjin 300384, 558 China. 2 Research Center for Efficient Utilization Technology of Geothermal Energy, Tianjin Chengjian 559 University, Tianjin 300384, China. 6. Conclusion 507 In this paper, CGSHP system is proposed as the same as the effect of the flow rate of 508 pumping and injection wells on heat transfer characteristic of BHEs is studied for this system. 509 The main conclusions of this study can be summarized as follows: 510 (a) For CGSHP system, an indoor sandbox is built by equation analysis method 511 according to the principle of similarity criteria. At the same time, the equal volumetric weight 512 by layered wet filling method is adopted to ensure that the porous medium in the sandbox is 513 similar to that of the underground aquifer. So, the sandbox experiment could effectively 514 reproduce the process of forced seepage and convection heat transfer in the aquifer. The 515 (a) For CGSHP system, an indoor sandbox is built by equation analysis method 511 according to the principle of similarity criteria. At the same time, the equal volumetric weight 512 by layered wet filling method is adopted to ensure that the porous medium in the sandbox is 513 similar to that of the underground aquifer. So, the sandbox experiment could effectively 514 reproduce the process of forced seepage and convection heat transfer in the aquifer. The 515 Page 19 of 22 sandbox experiment and analytical model are used to verify the numerical simulation of 516 FEFLOW7.1. The results show that the numerical simulation software FEFLOW7.1 can 517 simulate effectively and correctly the evolution process of the aquifer temperature field 518 during the taking heat of BHEs. 519 (b) When the pumping-injection flow rate from 200 m3∙d-1 to 1200 m3∙d-1, Darcy velocity 520 in the fine sand layer increases approximately 10 times and the effect of convective heat 521 transfer and thermal dispersion is enhanced. In the downstream region of BHEs, the thermal 522 action radius is 2.6 times of the original radius. Hence, the cold and heat accumulation of 523 CGSHP system can be alleviated effectively in the aquifer. 524 (c) For long-term running BHEs, the cumulative distribution curve of the inlet and outlet 525 water average temperature difference is introduced to describe the duration of a certain heat 526 exchange efficiency of BHEs. The average temperature difference corresponding to the 527 median time is used as a new parameter for evaluating the heat transfer performance of BHEs. References 564 1. National Development and Reform Commission. The 13th Five-Year Plan for the Exploitation and 565 Utilization of Geothermal Energy. N.D.R.C. Publishing: Beijing, China, 2017,Available online: 566 http://www.ndrc.gov.cn/zcfb/zcfbtz/201702/t20170204_837203.html.( in Chinese) 567 2. China Geological Survey. China Geothermal Energy Development Report. Beijing, China 568 Petrochemical Press: Beijing, China, 2018; pp.5-10. (in Chinese) 569 3. Richard A.B. Transient Heat Transfer in a U-tube Borehole Heat Exchanger. Applied Therma Engineering. 2014, 62, 256-266. 4. Richard A.B., Marvin D.S. Reference Data Sets for Vertical Borehole Ground Heat Exchange Models and Thermal Response Test Analysis. Geothermics. 2011, 40, 79-85. 5. A. Angelotti, L. Alberti. Energy Performance and Thermal Impact of a Borehole Heat Exchanger in 574 a sandy aquifer: Influence of the groundwater velocity. Energy Conversion and Management. 2014, 575 77, 700-708. 576 6. Jinzhong H. An Improved Analytical Model for Vertical Borehole Ground Heat Exchanger with Multiple-Layer Substrates and Groundwater Flow. Applied Energy. 2017, 202, 537-549. 7. Jung C.C., Joonsang P. Numerical Evaluation of the Effects of Groundwater Flow on Borehole Hea Exchanger Arrays. Renewable Energy. 2013, 52, 230-240. 8. Huajun W., Chengying Q. Thermal Performance of Borehole Heat Exchanger under Groundwater 581 Flow: A Case Study from Baoding. Energy and Buildings. 2009, 41, 1368-1373. 582 9. Alessandro C., Rajandrea S. Efficiency of Closed Loop Geothermal Heat Pumps: A Sensitivity 583 Analysis. Renewable Energy. 2014, 62, 737-746. 584 10. Yanling G., Xiaoli Z. 3D Dynamic Numerical Programming and Calculation of Vertical Buried Tube 585 Heat Exchanger Performance of Ground-Source Heat Pumps under Coupled Heat Transfer Inside 586 and Outside of Tube. Energy and Buildings. 2017, 139, 186-196. 587 11. Jozsef H.M., Michael d.P. Optimization of Energy Extraction for Vertical Closed-Loop Geothermal 588 Systems Considering Groundwater Flow. Energy Conversion and Management. 2013, 66, 1–10. 589 12. Chaofeng L., Peter J.C. Numerical Simulation of Ground Source Heat Pump Systems Considering 590 Unsaturated Soil Properties and Groundwater Flow Applied. Thermal Engineering. 2018, 139, 591 307-316. 592 13. Min L., Alvin C.K. Lai. Review of Analytical Models for Heat Transfer by Vertical Ground Hea Exchangers (GHEs): A Perspective of Time and Space Scales. Applied Energy. 2015, 151, 178–191. 14. Matthew G.S., Darin W.N. A Ground Resistance for Vertical Bore Heat Exchangers with Groundwater Flow. Energy Resources Technology-Transactions of the ASME. 2003, 125, 183-189. 15. Nairen D., Qinyun L. Heat Transfer in Ground Heat Exchangers with Groundwater Advection. 597 Thermal Sciences. 2004, 43, 1203–1211. 598 16. Nelson M.G., Peter B. 6. Conclusion 507 A Moving Finite Line Source Model to Simulate Borehole Heat Exchangers 601 with Groundwater Advection. Thermal Sciences. 2011, 50, 2506-2513. 602 18. Jin L., Joachim R. Review of Ground Investigations for Ground Source Heat Pump (GSHP) Systems. 603 Energy and Buildings. 2016, 117, 160-175. 604 19. A. Michopoulos, T. Zachariadis. Operation Characteristics and Experience of a Ground Source Heat 605 Pump System with a Vertical Ground Heat Exchanger. Energy. 2013, 51, 349-357. 606 20. Wonjun C., Ryozo O. Interpretation of Disturbed Data in Thermal Response Tests Using the Infinite 607 Line Source Model and Numerical Parameter estimation method. Applied Energy. 2015, 148, 608 476–488. 609 21 D id C S A C El Th Ob d Eff f Ch i G d Fl B h l H 610 Received: date Accepted: date 561 Published: date 562 563 References 564 1. National Development and Reform Commission. The 13th Five-Year Plan for the Exploitation and 565 Utilization of Geothermal Energy. N.D.R.C. Publishing: Beijing, China, 2017,Available online: 566 http://www.ndrc.gov.cn/zcfb/zcfbtz/201702/t20170204_837203.html.( in Chinese) 567 2. China Geological Survey. China Geothermal Energy Development Report. Beijing, China 568 Petrochemical Press: Beijing, China, 2018; pp.5-10. (in Chinese) 569 3. Richard A.B. Transient Heat Transfer in a U-tube Borehole Heat Exchanger. Applied Thermal 570 Engineering. 2014, 62, 256-266. 571 4. Richard A.B., Marvin D.S. Reference Data Sets for Vertical Borehole Ground Heat Exchanger 572 Models and Thermal Response Test Analysis. Geothermics. 2011, 40, 79-85. 573 5. A. Angelotti, L. Alberti. Energy Performance and Thermal Impact of a Borehole Heat Exchanger in 574 a sandy aquifer: Influence of the groundwater velocity. Energy Conversion and Management. 2014, 575 77, 700-708. 576 6. Jinzhong H. An Improved Analytical Model for Vertical Borehole Ground Heat Exchanger with 577 Multiple-Layer Substrates and Groundwater Flow. Applied Energy. 2017, 202, 537-549. 578 7. Jung C.C., Joonsang P. Numerical Evaluation of the Effects of Groundwater Flow on Borehole Heat 579 Exchanger Arrays. Renewable Energy. 2013, 52, 230-240. 580 8. Huajun W., Chengying Q. Thermal Performance of Borehole Heat Exchanger under Groundwater 581 Flow: A Case Study from Baoding. Energy and Buildings. 2009, 41, 1368-1373. 582 9. Alessandro C., Rajandrea S. Efficiency of Closed Loop Geothermal Heat Pumps: A Sensitivity 583 Analysis. Renewable Energy. 2014, 62, 737-746. 584 10. Yanling G., Xiaoli Z. 6. Conclusion 507 3D Dynamic Numerical Programming and Calculation of Vertical Buried Tube 585 Heat Exchanger Performance of Ground-Source Heat Pumps under Coupled Heat Transfer Inside 586 and Outside of Tube. Energy and Buildings. 2017, 139, 186-196. 587 11. Jozsef H.M., Michael d.P. Optimization of Energy Extraction for Vertical Closed-Loop Geothermal 588 Systems Considering Groundwater Flow. Energy Conversion and Management. 2013, 66, 1–10. 589 12. Chaofeng L., Peter J.C. Numerical Simulation of Ground Source Heat Pump Systems Considering 590 Unsaturated Soil Properties and Groundwater Flow Applied. Thermal Engineering. 2018, 139, 591 307-316. 592 13. Min L., Alvin C.K. Lai. Review of Analytical Models for Heat Transfer by Vertical Ground Heat 593 Exchangers (GHEs): A Perspective of Time and Space Scales. Applied Energy. 2015, 151, 178–191. 594 14. Matthew G.S., Darin W.N. A Ground Resistance for Vertical Bore Heat Exchangers with 595 Groundwater Flow. Energy Resources Technology-Transactions of the ASME. 2003, 125, 183-189. 596 15. Nairen D., Qinyun L. Heat Transfer in Ground Heat Exchangers with Groundwater Advection. 597 Thermal Sciences. 2004, 43, 1203–1211. 598 16. Nelson M.G., Peter B. Evaluating the Influence of Thermal Dispersion on Temperature Plumes from 599 Geothermal Systems Using Analytical Solutions. Thermal Sciences. 2011, 50, 1223-1231. 600 17. Nelson M.G., Philipp B. A Moving Finite Line Source Model to Simulate Borehole Heat Exchangers 601 with Groundwater Advection. Thermal Sciences. 2011, 50, 2506-2513. 602 18. Jin L., Joachim R. Review of Ground Investigations for Ground Source Heat Pump (GSHP) Systems. 603 Energy and Buildings. 2016, 117, 160-175. 604 19. A. Michopoulos, T. Zachariadis. Operation Characteristics and Experience of a Ground Source Heat 605 Pump System with a Vertical Ground Heat Exchanger. Energy. 2013, 51, 349-357. 606 20 Wonjun C Ryozo O Interpretation of Disturbed Data in Thermal Response Tests Using the Infinite 607 6. Conclusion 507 The 534 pumping-injection flow rate that can make Darcy velocity reaches 0.6×10-6~1.4×10-6 m∙s-1 is the 535 best reference range of CGSHP system，so the best pumping-injection flow rate is 400~600 536 m3∙d-1 in this paper. 537 (e) CGSHP system is proposed for the areas where the shallow-groundwater reserves 538 are abundant but the seepage velocity is weak, such as the Bohai Plain in Tianjin, China. The 539 results show that the system can control the seepage velocity of groundwater by changing the 540 pumping-injection flow rate to enhance heat transfer, so CGSHP system has good 541 controllability and predictability. In addition, CGSHP system should also be applicable to 542 other countries with the same hydrogeological conditions as the region in this paper. The 543 system has certain universality. 544 Author contributions: JM and QJ performed conceptualization; JM and QJ performed data curation; JM 546 and QJ performed formal analysis; JM and QJ provided methodology; QZ provided resources; YX and 547 YW provided software simulation; FY validated data; QJ writed the original draft; JM and QJ improved 548 and revised the manuscript. All authors read and approved the final manuscript. 549 Funding: This research was funded by National Natural Science Foundation of China of Funder, grant 550 number 41402228; This research was funded by Enterprise science and technology commissioner project 551 of Tianjin of Funder, grant number 19JCTPJC48100. 552 Availability of data and materials: The datasets generated and analyzed during the current study are 53 available from the corresponding author on reason-able request. 54 Availability of data and materials: The datasets generated and analyzed during the current study are vailable from the corresponding author on reason-able request. Acknowledgments: Thanks to the Tianjin Institute of Geotechnical Investigation Surveying for the 555 hydrogeological parameters and relevant data provided. 556 Conflicts of Interest: The authors declare that they have no competing interests. 557 Author details: 1 School of Energy Safety Engineering, Tianjin Chengjian University, Tianjin 300384, 558 China. 2 Research Center for Efficient Utilization Technology of Geothermal Energy, Tianjin Chengjian 559 University, Tianjin 300384, China. 560 Page 20 of 22 Received: date Accepted: date 561 Published: date 562 563 References 564 1. National Development and Reform Commission. The 13th Five-Year Plan for the Exploitation and 565 Utilization of Geothermal Energy. N.D.R.C. Publishing: Beijing, China, 2017,Available online: 566 http://www.ndrc.gov.cn/zcfb/zcfbtz/201702/t20170204_837203.html.( in Chinese) 567 2. China Geological Survey. China Geothermal Energy Development Report. 6. Conclusion 507 Beijing, China 568 Petrochemical Press: Beijing, China, 2018; pp.5-10. (in Chinese) 569 3. Richard A.B. Transient Heat Transfer in a U-tube Borehole Heat Exchanger. Applied Thermal 570 Engineering. 2014, 62, 256-266. 571 4. Richard A.B., Marvin D.S. Reference Data Sets for Vertical Borehole Ground Heat Exchanger 572 Models and Thermal Response Test Analysis. Geothermics. 2011, 40, 79-85. 573 5. A. Angelotti, L. Alberti. Energy Performance and Thermal Impact of a Borehole Heat Exchanger in 574 a sandy aquifer: Influence of the groundwater velocity. Energy Conversion and Management. 2014, 575 77, 700-708. 576 6. Jinzhong H. An Improved Analytical Model for Vertical Borehole Ground Heat Exchanger with 577 Multiple-Layer Substrates and Groundwater Flow. Applied Energy. 2017, 202, 537-549. 578 7. Jung C.C., Joonsang P. Numerical Evaluation of the Effects of Groundwater Flow on Borehole Heat 579 Exchanger Arrays. Renewable Energy. 2013, 52, 230-240. 580 8. Huajun W., Chengying Q. Thermal Performance of Borehole Heat Exchanger under Groundwater 581 Flow: A Case Study from Baoding. Energy and Buildings. 2009, 41, 1368-1373. 582 9. Alessandro C., Rajandrea S. Efficiency of Closed Loop Geothermal Heat Pumps: A Sensitivity 583 Analysis. Renewable Energy. 2014, 62, 737-746. 584 10. Yanling G., Xiaoli Z. 3D Dynamic Numerical Programming and Calculation of Vertical Buried Tube 585 Heat Exchanger Performance of Ground-Source Heat Pumps under Coupled Heat Transfer Inside 586 and Outside of Tube. Energy and Buildings. 2017, 139, 186-196. 587 11. Jozsef H.M., Michael d.P. Optimization of Energy Extraction for Vertical Closed-Loop Geothermal 588 Systems Considering Groundwater Flow. Energy Conversion and Management. 2013, 66, 1–10. 589 12. Chaofeng L., Peter J.C. Numerical Simulation of Ground Source Heat Pump Systems Considering 590 Unsaturated Soil Properties and Groundwater Flow Applied. Thermal Engineering. 2018, 139, 591 307-316. 592 13. Min L., Alvin C.K. Lai. Review of Analytical Models for Heat Transfer by Vertical Ground Heat 593 Exchangers (GHEs): A Perspective of Time and Space Scales. Applied Energy. 2015, 151, 178–191. 594 14. Matthew G.S., Darin W.N. A Ground Resistance for Vertical Bore Heat Exchangers with 595 Groundwater Flow. Energy Resources Technology-Transactions of the ASME. 2003, 125, 183-189. 596 15. Nairen D., Qinyun L. Heat Transfer in Ground Heat Exchangers with Groundwater Advection. 597 Thermal Sciences. 2004, 43, 1203–1211. 598 16. Nelson M.G., Peter B. Evaluating the Influence of Thermal Dispersion on Temperature Plumes from 599 Geothermal Systems Using Analytical Solutions. Thermal Sciences. 2011, 50, 1223-1231. 600 17. Nelson M.G., Philipp B. References 564 Investigations on the in 648 ground-source heat pump operation. Applied En 649 38. X. Mao, H. Prommer. Three-dimensional Mode 650 Variable Density Groundwater flow. Environmen 651 652 Nomenclature r distance to the source /sink(m) t time(s) l tube pitch of each BHE(m) q heat transfer rates per unit borehole depth of the BHEs (W∙m-1) α H borehole depth(m) T temperature (K) k permeability(m2) Sub g gravitational acceleration x Qρ flow intensity of source (sink) term of flow field (m3∙ m-3·s-1) QT heat intensity of heat source (sink) (W∙m-3) x, y, z Cartesian coordinates 23. Guozhu Z., Yimu G. Experimental Study on the Thermal Performance of Tunnel Lining GHE unde Groundwater Flow. Applied Thermal Engineering. 2016, 106, 784-795. 23. Guozhu Z., Yimu G. Experim 614 Groundwater Flow. Applied T 615 24. Linlin Z., Lei Z. Analyses on 616 in Soils with Groundwater Ad 617 25. Selcuk E., Bertrand F. Mult 618 Groundwater Flow. Geotherm 619 26. Martin S., Jonathan D. Influen 620 Heat Pump Systems. Geother 621 27. Junye H., Gui L. The Hot S 622 with/without Intensification 623 Buildings. 2017, 150, 558-566. 624 28. Wenke Z., Hongxing Y. The 625 Ground Heat Exchangers in 626 115-128. 627 29. Poul A.Ø., Neven D. Sustain 628 Energy. 2019, 146, 2430-2437. 629 30. Giti N., Younes N. Designin 630 System to Supply Heating, Co 631 31. H. Biglarian., M. H. 632 a Solar-assisted Ground Sourc 633 Environmental Science and T 634 32. Zhijian L., Yuanwei L. Perfo 635 System Assisted with Cooling 636 Conversion and Management 637 33. Xuemei G., Lei X. Investigatio 638 Assisted Ground Source Heat 639 34. Rong M. Dealing with the spa 640 study of Luancheng County 641 Geological Sciences. Beijing, C 642 35. Zaiming Z. Spatial Variability 643 Bohai Sea. Doctoral Dissertati 644 Chinese) 645 36. Yujin N., Ryozo O. Develop 646 ground-source heat pump sys 647 37. Antonio C., Michele D.C. Inv 648 ground-source heat pump op 649 38. X. Mao, H. Prommer. Three 650 Variable Density Groundwate 651 652 Nomenclature r distance to the source /sink(m) t time(s) l tube pitch of each BHE(m) q heat transfer rates per unit boreho the BHEs (W∙m-1) H borehole depth(m) T temperature (K) k permeability(m2) g gravitational acceleration Qρ flow intensity of source (sink) term field (m3∙ m-3·s-1) QT heat intensity of heat source (sink) (W x, y, z Cartesian coordinates 24. Linlin Z., Lei Z. References 564 Evaluating the Influence of Thermal Dispersion on Temperature Plumes from 599 Geothermal Systems Using Analytical Solutions. Thermal Sciences. 2011, 50, 1223-1231. 600 17. Nelson M.G., Philipp B. A Moving Finite Line Source Model to Simulate Borehole Heat Exchanger with Groundwater Advection. Thermal Sciences. 2011, 50, 2506-2513. 18. Jin L., Joachim R. Review of Ground Investigations for Ground Source Heat Pump (GSHP) Systems. 603 Energy and Buildings. 2016, 117, 160-175. 604 19. A. Michopoulos, T. Zachariadis. Operation Characteristics and Experience of a Ground Source Heat 605 Pump System with a Vertical Ground Heat Exchanger. Energy. 2013, 51, 349-357. 606 20. Wonjun C., Ryozo O. Interpretation of Disturbed Data in Thermal Response Tests Using the Infinite 607 Line Source Model and Numerical Parameter estimation method. Applied Energy. 2015, 148, 608 476–488. 609 21. David C.S., A.C Elmore. The Observed Effects of Changes in Groundwater Flow on a Borehole Heat 610 Exchanger of a Large Scale Ground Coupled Heat pump system. Geothermics. 2018, 74, 240-246. 611 22. Ali S.S., Michel B. A Small-scale Experimental Apparatus to Study Heat Transfer in the Vicinity of 612 Geothermal Boreholes. HVAC&R Research. 2014, 20, 819-827. 613 Page 21 of 22 23. Guozhu Z., Yimu G. Experimental Study on the Thermal Performance of Tunnel Lining GHE under 614 Groundwater Flow. Applied Thermal Engineering. 2016, 106, 784-795. 615 24. Linlin Z., Lei Z. Analyses on Soil Temperature Response to Intermittent Heat Rejection from BHEs 616 in Soils with Groundwater Advection. Energy and Buildings. 2015, 107, 355–365. 617 25. Selcuk E., Bertrand F. Multilayer Analytical Model for Vertical Ground Heat Exchanger with 618 Groundwater Flow. Geothermics. 2018, 71, 294-305. 619 26. Martin S., Jonathan D. Influence of Groundwater Flow on Cost Minimization of Ground Coupled 620 Heat Pump Systems. Geothermics. 2018, 73, 100-110. 621 27. Junye H., Gui L. The Hot Stack Performance of the Shallow Geothermal Heat Pump System 622 with/without Intensification State of Groundwater Seepage in Nanjing (China). Energy and 623 Buildings. 2017, 150, 558-566. 624 28. Wenke Z., Hongxing Y. The Research on Ring-coil Heat Transfer Models of Pile Foundation 625 Ground Heat Exchangers in the Case of Groundwater Seepage. Energy and Buildings. 2014, 71, 626 115-128. 627 29. Poul A.Ø., Neven D. Sustainable Development Using Renewable Energy Technology. Renewable 628 Energy. 2019, 146, 2430-2437. 629 30. Giti N., Younes N. 623 References 564 Analyses on Soil Temperature Response to Intermittent Heat Rejection from BHE in Soils with Groundwater Advection. Energy and Buildings. 2015, 107, 355–365. 25. Selcuk E., Bertrand F. Multilayer Analytical Model for Vertical Ground Heat Exchanger with Groundwater Flow. Geothermics. 2018, 71, 294-305. 26. Martin S., Jonathan D. Influence of Groundwater Flow on Cost Minimization of Ground Coupled Heat Pump Systems. Geothermics. 2018, 73, 100-110. 27. Junye H., Gui L. The Hot Stack Performance of the Shallow Geothermal Heat Pump System with/without Intensification State of Groundwater Seepage in Nanjing (China). Energy and Buildings. 2017, 150, 558-566. 28. Wenke Z., Hongxing Y. The Research on Ring-coil Heat Transfer Models of Pile Foundation Ground Heat Exchangers in the Case of Groundwater Seepage. Energy and Buildings. 2014, 71, 115-128. 29. Poul A.Ø., Neven D. Sustainable Development Using Renewable Energy Technology. Renewable Energy. 2019, 146, 2430-2437. 30. Giti N., Younes N. Designing and Optimization of Solar Assisted Ground Source Heat Pump System to Supply Heating, Cooling and Hot Water Demands. Geothermics. 2019, 82, 212–231. 31. H. Biglarian., M. H. Saidi. Economic and Environmental Assessment of a Solar-assisted Ground Source Heat Pump System in a Heating-dominated Climate. Environmental Science and Technology. 2019, 16, 3091-3098. 32. Zhijian L., Yuanwei L. Performance and Feasibility Study of Hybrid Ground Source Heat Pump System Assisted with Cooling Tower for one Office Building Based on one Shanghai Case. Energy Conversion and Management. 2018, 173, 28-37. 33. Xuemei G., Lei X. Investigation on the Optimal Cooling Tower Input Capacity of a Cooling Tower Assisted Ground Source Heat Pump System. Energy and Buildings. 2018, 174, 239-253. 34. Rong M. Dealing with the spatial synthetic heterogeneity of aquifers in the north china plain: A case 640 study of Luancheng County in Hebei Province. Doctoral Dissertation, Chinese Academy of 641 Geological Sciences. Beijing, China, 2012. (in Chinese) 642 35. Zaiming Z. Spatial Variability and its Effect Mechanism of Soil Salinity in the Low Plain Around the 643 Bohai Sea. Doctoral Dissertation, Chinese Academy of Geological Sciences. Beijing, China, 2012. (in 644 Chinese) 645 36. Yujin N., Ryozo O. Development of a numerical model to predict heat exchange rates for a ground-source heat pump system. Energy and Buildings. 2008.40:2133–2140. 36. Yujin N., Ryozo O. Development of a numerical model to predict heat exchange rates for a 646 ground-source heat pump system. Energy and Buildings. 2008.40:2133–2140. 647 g p p y gy g 37. References 564 Designing and Optimization of Solar Assisted Ground Source Heat Pump 630 System to Supply Heating, Cooling and Hot Water Demands. Geothermics. 2019, 82, 212–231. 631 31. H. Biglarian., M. H. Saidi. Economic and Environmental Assessment of 632 a Solar-assisted Ground Source Heat Pump System in a Heating-dominated Climate. 633 Environmental Science and Technology. 2019, 16, 3091-3098. 634 32. Zhijian L., Yuanwei L. Performance and Feasibility Study of Hybrid Ground Source Heat Pump 635 System Assisted with Cooling Tower for one Office Building Based on one Shanghai Case. Energy 636 Conversion and Management. 2018, 173, 28-37. 637 33. Xuemei G., Lei X. Investigation on the Optimal Cooling Tower Input Capacity of a Cooling Tower 638 Assisted Ground Source Heat Pump System. Energy and Buildings. 2018, 174, 239-253. 639 34. Rong M. Dealing with the spatial synthetic heterogeneity of aquifers in the north china plain: A case 640 study of Luancheng County in Hebei Province. Doctoral Dissertation, Chinese Academy of 641 Geological Sciences. Beijing, China, 2012. (in Chinese) 642 35. Zaiming Z. Spatial Variability and its Effect Mechanism of Soil Salinity in the Low Plain Around the 643 Bohai Sea. Doctoral Dissertation, Chinese Academy of Geological Sciences. Beijing, China, 2012. (in 644 Chinese) 645 36. Yujin N., Ryozo O. Development of a numerical model to predict heat exchange rates for a 646 ground-source heat pump system. Energy and Buildings. 2008.40:2133–2140. 647 37. Antonio C., Michele D.C. Investigations on the influence of aquifers on the ground temperature in 648 ground-source heat pump operation. Applied Energy. 2013.107: 35. 649 38. X. Mao, H. Prommer. Three-dimensional Model for Multi-component Reactive Transport with 650 Variable Density Groundwater flow. Environmental Modelling & Software. 2006, 21, 615-628. References 564 651 652 Nomenclature Λ tensor of thermal hydrodynamic dispersion(W∙m-1·K r distance to the source /sink(m) δij Kronecker tensor (i=j, δij =1; i≠j ,δij =0;) t time(s)  compressibility coefficient l tube pitch of each BHE(m) λ thermal conductivity (W∙m-1∙K-1) q heat transfer rates per unit borehole depth of the BHEs (W∙m-1) αL, αT longitudinal and transverse thermal dispersivit respectively, of fluid (m) H borehole depth(m) ρc volumetric heat capacity (J·m-3·K-1) T temperature (K) θ dimensionless temperature k permeability(m2) Subscripts g gravitational acceleration x, y longitudinal and transverse direction, respectiv Qρ flow intensity of source (sink) term of flow field (m3∙ m-3·s-1) r refrigerant fluid QT heat intensity of heat source (sink) (W∙m-3) s soil x, y, z Cartesian coordinates f fluid 23. Guozhu Z., Yimu G. Experimental Study on the T 614 Groundwater Flow. Applied Thermal Engineerin 615 24. Linlin Z., Lei Z. Analyses on Soil Temperature Re 616 in Soils with Groundwater Advection. Energy an 617 25. Selcuk E., Bertrand F. Multilayer Analytical M 618 Groundwater Flow. Geothermics. 2018, 71, 294-30 619 26. Martin S., Jonathan D. Influence of Groundwater 620 Heat Pump Systems. Geothermics. 2018, 73, 100-1 621 27. Junye H., Gui L. The Hot Stack Performance 622 with/without Intensification State of Groundw 623 Buildings. 2017, 150, 558-566. 624 28. Wenke Z., Hongxing Y. The Research on Ring 625 Ground Heat Exchangers in the Case of Groun 626 115-128. 627 29. Poul A.Ø., Neven D. Sustainable Development U 628 Energy. 2019, 146, 2430-2437. 629 30. Giti N., Younes N. Designing and Optimizatio 630 System to Supply Heating, Cooling and Hot Wate 631 31. H. Biglarian., M. H. Saidi. Econom 632 a Solar-assisted Ground Source Heat Pump Syste 633 Environmental Science and Technology. 2019, 16, 634 32. Zhijian L., Yuanwei L. Performance and Feasibi 635 System Assisted with Cooling Tower for one Off 636 Conversion and Management. 2018, 173, 28-37. 637 33. Xuemei G., Lei X. Investigation on the Optimal C 638 Assisted Ground Source Heat Pump System. Ene 639 34. Rong M. Dealing with the spatial synthetic hetero 640 study of Luancheng County in Hebei Provin 641 Geological Sciences. Beijing, China, 2012. (in Chin 642 35. Zaiming Z. Spatial Variability and its Effect Mech 643 Bohai Sea. Doctoral Dissertation, Chinese Academ 644 Chinese) 645 36. Yujin N., Ryozo O. Development of a numeri 646 ground-source heat pump system. Energy and Bu 647 37. Antonio C., Michele D.C. 621 624 References 564 Antonio C., Michele D.C. Investigations on the influence of aquifers on the ground temperature in 648 ground-source heat pump operation. Applied Energy. 2013.107: 35. 649 g p p y gy g 37. Antonio C., Michele D.C. Investigations on the influence of aquifers on the ground temperature in 648 ground-source heat pump operation. Applied Energy. 2013.107: 35. 649 g p p p pp gy 38. X. Mao, H. Prommer. Three-dimensional Model for Multi-component Reactive Transport with 650 Variable Density Groundwater flow. Environmental Modelling & Software. 2006, 21, 615-628. 651 Nomenclature Λ tensor of thermal hydrodynamic dispersion(W∙m-1·K-1) r distance to the source /sink(m) δij Kronecker tensor (i=j, δij =1; i≠j ,δij =0;) t time(s)  compressibility coefficient l tube pitch of each BHE(m) λ thermal conductivity (W∙m-1∙K-1) q heat transfer rates per unit borehole depth of the BHEs (W∙m-1) αL, αT longitudinal and transverse thermal dispersivity, respectively, of fluid (m) H borehole depth(m) ρc volumetric heat capacity (J·m-3·K-1) T temperature (K) θ dimensionless temperature k permeability(m2) Subscripts g gravitational acceleration x, y longitudinal and transverse direction, respectively Qρ flow intensity of source (sink) term of flow field (m3∙ m-3·s-1) r refrigerant fluid QT heat intensity of heat source (sink) (W∙m-3) s soil x, y, z Cartesian coordinates f fluid Page 22 of 22 Page 22 of 22 u absolute Darcy velocity(m∙s-1) s0 initial state of rock soil layer L dimensionless distance m experimental system model E the average heat transfer efficiency coefficient i, o pipe-in or internal, pipe-out or outer, respectively Q actual average heat exchange quantity (J) Superscripts Q’ theoretical average heat exchange quantity (J) cond conduction M circulating flow flux of refrigerant (m3∙s-1) disp dispersion Greek symbols  dynamic viscosity (Pa·s) ε porosity 653 654 © 2020 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). u absolute Darcy velocity(m∙s-1) s0 initial state of rock soil layer L dimensionless distance m experimental system model E the average heat transfer efficiency coefficient i, o pipe-in or internal, pipe-out or outer, respectively Q actual average heat exchange quantity (J) Superscripts Q’ theoretical average heat exchange quantity (J) cond conduction M circulating flow flux of refrigerant (m3∙s-1) disp dispersion Greek symbols  dynamic viscosity (Pa·s) ε porosity 653 654 © 2020 by the authors. References 564 Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2020 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). 654
https://openalex.org/W2328767963	https://sciforum.net/paper/download/2818/manuscript	English	null	Multitude and Internet: Cooperation and Domination	null	2,015	cc-by	1,232	Universidad Pedagógica Nacional (Adress: Calle 79 No. 106-33, Bogotá, Colombia) E-Mails: rruedaortiz@yahoo.com Tel.: 0057 1 2275389; Fax: 0057 12275389 Tel.: 0057 1 2275389; Fax: 0057 12275389 Accepted: Key Words: Multitude, Internet, qualitative research, co-operation, domination, digital technologies ccepted: Key Words: Multitude, Internet, qualitative research, co-operation, domination, digital p Key Words: Multitude, Internet, qualitative research, co-operation, domination, digital technologies Multitude and Internet: cooperation and domination Rueda, Rocío Methods The methodology used was of a qualitative, ethnographic nature (participant observations, in-depth interviews, life stories). Firstly, a review was made of collectives who are connected to the Internet in Colombia, of which six were selected: one in the Valle del Cauca, in Santander de Quilichao: El tejido de Comunicaciones of the NASA-ACIN indigenous community; one in Medellín: Corporación Vamos Mujer, and four in Bogotá: Niuton, Mefisto, La Cápsula and Chicas Linux. These collectives were chosen for their social, political and cultural critiques of the established and dominant culture. Monitoring was then made for a period of one year of the different actions realized by these collectives in the cities where they are located, as well as on the Internet through their respective webpages. Introduction This presentation is the product of reflections arising from qualitative research realized with five countercultural groups in Colombia that intensively use information and communication technologies. The findings are analysed in relationship with new ways of sharing and disseminating knowledge and new forms of social and political organization. These sociotechnical practices are considered as potential forms of resistance against dominant and homogenous political and cultural models. They also show alternative forms of community and the creation of knowledge. Nevertheless they are a novelty that is observed in relation to the ambiguous power and inequality that exist on different levels within these collectives (Rueda, 2012). For this reason, the concept of multitude as introduced by Spinoza and elaborated by Negri and Hardt (2000) and Lazzarato (2006), can be useful in understanding that while this socius is an unstable and volatile social energy, it constitutes a collective voice that resists the cultural and political order with surprising political potential. The multitude articulates affects and experiences that are the basis for political action. It is something located in between, it is multiple and at the same time conforms a singular body made up of diverse interests, experiences, feelings and relations, without a homogenous unity. Relationality and cooperation establish what is common, which in turn faces the political challenge of difference. But “modern society is characterized by antagonism of co-operation and competition. …Characteristics of late-modern society such as the colonization of the life-world and the whole society by economic logic are again reproduced in cyberspace” (Fuchs & Zimmerman, 2009:119-125). 2 This multitude is then also unpredictable and unstable and we believe it faces the challenge of critically confronting the inequalities and exercises of power that exist within it. In fact, this new condition of the subjective experience – individual and collective – requires us to be alert to certain social and technological determinisms that inflate the reach of both the technology as well as the movements and collectives, and suggest that, for example, connectivity immediately means collectiveness and democratisation. We cannot underestimate the complexity of this socius, which is always faced by an array of possibilities ranging from social cooperation and creativity, to new forms of domination and the capturing of desire in the service of capital. Results and Discussion. These cases of new forms of sociality, of multitude, that we found in the five countercultural groups are far from conforming a coherent whole, but they can be – and are – functional bodies of knowledge, valuable for inventing for us better and more just political forms of the everyday. These invite us to unlearn social, political, cultural forms that in the past were sole, colonial, homogenising. At the same time, however, they remind us that, despite libertarian, rebellious and non-conformist outlooks regarding the establishment, these collectives contain within them questions related to relationships of power, of gender, of race, of social class and profession that appear in an ambiguous way, that become invisible and seem to coexist in a not always harmonious way in this social context, as is the case with markedly masculine and competitive practices in certain collectives of free software, or the hierarchical relations in countercultural collectives of women who oppose authoritarian political models. In effect, we see a complex condition, of hybridity, of non- contemporary contemporaneity of cultural times. Of the ways in which dimensions of subjectivity that were excluded from modern thought (such as affectivity, desire) are rescued while simultaneously exclusions of gender, race, social class and region are maintained intact, as is notorious, we might say, in those collectives that are principally urban and middle class. Similarly, technologies are not sufficient or determinant per se regarding social or cultural agencements, because collective dimensions of use are what constitute settings of communication, spaces for the dissemination of the sensitive and places where diagrams of social creativity and desire are described. For this reason, we examinated the power possessed by the collectives we have selected, 3 because we believe that friendship as politics, peer learning, sharing, donating, expression and the free circulation of commons, places us before new forms of understanding the formation of subjectivity and social practices that is still not easy to define. It is in these practices that technologies and their settings for participation and collaboration – such as the social network or Web 2.0 – are of interest for their political potency. Conclusions While discursive categories exist that give an identity (women, young people, indigenous people) to the five counterculture collectives we’ve considered in this study, internal differences also exist within these collectives, different subject positions and partial forms of articulation to the collectives and to the social practices of resistance to power, in which individual aspirations and dreams are also in tension. We need to find metaphors to make evident the complexity of this sociotechnical vitality and the coexistence of traditional and novel forms of social and political organisation, as well as new and traditional cultural forms and practices. Another challenge we face is to try and better understand how that tactical combination is produced between connectivity and the conformation of common(ity), network, (multitude) and how the forms of control and diverse dynamics of power (co)exist even in alternative forms of networks and multitudes. © 2015 by the authors; licensee MDPI and ISIS. This abstract is distributed under the terms and conditions of the Creative Commons Attribution license. References and Notes Fuchs, C.; Zimmerman, R. Practical civil virtues in cyberspace. Towards the utopian identitiy of civitas and Multitudo. Schaker Verlagt, München; 2009. Fuchs, C.; Zimmerman, R. Practical civil virtues in cyberspace. Towards the utopian identitiy of civitas and Multitudo. Schaker Verlagt, München; 2009. Lazzarato, M. Políticas del Acontecimiento. 1ª. ed.; Tinta Limón, Buenos Aires; 2006. Lazzarato, M. Políticas del Acontecimiento. 1ª. ed.; Tinta Limón, Buenos Aires; 2006 Negri, T.; Hardt, M. Imperio. Harvard University Press, Cambridge; 2000. ; Hardt, M. Imperio. Harvard University Press, Cambridge; 2000. Rueda, R. “Sociedades de la información y el conocimiento: tecnicidad, pharmakon e invención social”. Nómadas, 2012, 36, 43-55. Rueda, R. “Sociedades de la información y el conocimiento: tecnicidad, pharmakon e invención social”. Nómadas, 2012, 36, 43-55. © 2015 by the authors; licensee MDPI and ISIS. This abstract is distributed under the terms and conditions of the Creative Commons Attribution license.
https://openalex.org/W2335927372	https://revistas.ufg.br/VISUAL/article/download/17914/10692	Spanish; Castilian	null	Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos	Visualidades	2,012	cc-by	9,956	This paper focuses on the analysis of the educational method known as “Visual Thinking Strategies” which, after being implemented and evaluated in the United States and countries of East Europe, it is being adop- ted by many art institutions in Spain and South Ameri- ca. The article goes over the history of this educational method and explores from a critical point of view its advantages and limitations. Furthermore, it critically questions its current implementation and analyzes its theorethical roots, in order to invite the readers to re- flect on this educational methodology. This paper focuses on the analysis of the educational method known as “Visual Thinking Strategies” which, after being implemented and evaluated in the United States and countries of East Europe, it is being adop- ted by many art institutions in Spain and South Ameri- ca. The article goes over the history of this educational method and explores from a critical point of view its advantages and limitations. Furthermore, it critically questions its current implementation and analyzes its theorethical roots, in order to invite the readers to re- flect on this educational methodology. This paper focuses on the analysis of the educational method known as “Visual Thinking Strategies” which, after being implemented and evaluated in the United States and countries of East Europe, it is being adop- ted by many art institutions in Spain and South Ameri- ca. The article goes over the history of this educational method and explores from a critical point of view its advantages and limitations. Furthermore, it critically questions its current implementation and analyzes its theorethical roots, in order to invite the readers to re- flect on this educational methodology. a b s t ra c t Keywords: museum art education, constructivism, educational methodology. Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos Eneritz LÓPEZ Magali KIVATINETZ Eneritz LÓPEZ Magali KIVATINETZ Este artigo constitui uma análise do método educati- vo conhecido como “Estratégias de Pensamento Visu- al” que, após haver sido implementado e avaliado em Estados Unidos e países de Europa do Este, está sendo adotado em numerosos centros de arte de Espanha e Sudamérica. O texto apresenta a história deste método educativo analisando críticamente suas vantagens e limi- tações. Além disso, problematiza sua implementação e revisa suas raízes teóricas, com o objetivo de convidar os leitores a refletirem sobre esta metodologia educativa. r e s u m o r e s u m o Palavras-chave: museu, ensino de Arte, constructivist- mo, metodologia educativa. VISUALIDADES. RE VISTA DO PROGR AMA DE MESTR ADO EM CULTUR A VISUAL - FAV I UFG Eneritz López, Magali Kivatinetz Introducción En el contexto en el que vivimos, caracterizado por la inme- diatez de las comunicaciones, la complejidad y la incertidum- bre, los museos necesitan estar alerta ante la tentación de ac- tuar como instituciones estáticas y cerradas. Ahora al espacio museístico se le pide ser un campo para la problematización y el cuestionamiento, la ruptura y el cambio (ROBERTS, 1997). En la actualidad los museos necesitan implicarse con la socie- dad y por ello, han de enfocarse hacia diferentes públicos y trabajar de manera que todos los visitantes puedan encontrar en ellos no sólo un lugar para el disfrute estético, sino también un hueco donde aprender sobre sí mismos, sobre los demás y donde se les autorice a generar e intercambiar visiones críticas y contrahegemónicas (PADRÓ, 2005). Los nuevos planteamien- tos para la educación artística en museos que se están intro- duciendo en los últimos años se deben en parte a un crecien- te interés por encontrar alternativas a la educación tradicional –basada en la simple transmisión de información–, y por ir más allá de la idea de educación como comunicación tan vinculada a la noción de museo instructor. En este contexto de tránsito y replanteamiento, aparecen métodos como el Visual Thinking Strategies (V.T.S.) que, debido a que su principal propuesta educativa es el diálogo, se presenta para los museos como una buena alternativa a los programas educativos tradicionales. En este artículo exploraremos específicamente el caso de este método educativo, también conocido en su origen como Visual Thinking Currículum (V.T.C.), que ha ido mutando sus acepciones para adaptarse a progresivos cuestionamientos y replanteamientos 1. Este método se considera actualmente inno- vador en España y países de latinoamérica, aunque tiene larga tradición de implementación y evaluación en Estados Unidos y países del este de Europa. Uno de los atractivos principales que tiene V.T.S. para los museos es que solventa, en cierta medida, su preocupación por la educación. Esto se debe a que se presenta como una propuesta dinámica y como una “filosofía” educativa lista para ser implementada en la institución, ya que viene acompañada Eneritz López, Magali Kivatinetz 82 Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos –no sólo del prestigio de la institución en la que inicialmente se originó (el Museum of Modern Art de Nueva York)–, sino también de unas instrucciones que, de seguirlas con fidelidad, garantizan su eficacia. Por ello, V.T.S. Introducción encaja muy bien con las expectativas del museo, a pesar de no ser más que el empa- quetamiento de unas ideas teóricas que hoy se encuentran en revisión, junto con las indicaciones pertinentes para llevarlo a la práctica en las salas del museo. En este sentido, no debemos pasar por alto la disociación general de tradición teórica sobre educación museística en Latinoamérica, aspecto que permite que los museos se vean sacudidos y cegados por este tipo de ideas que provienen del extranjero, especialmente de Estados Unidos. Estas ideas –basadas en investigaciones desde otros contextos y vinculadas con valores culturales e históricos espe- cíficos– son muchas veces adoptadas sin ser cuestionadas y sin ser adaptadas a cada contexto y necesidades. 2 Este texto está inspirado precisamente en la observación de cómo ha sido implementado V.T.S. en España y en el diá- logo con educadores de museos que lo utilizan en su práctica diaria. Además, lo que nos mueve a escribir este artículo es el hecho de haber percibido que se está realizando en distintos países una intensa propaganda sobre este método. Esta pu- blicidad se ha visto acompañada de un creciente interés por adoptarlo en numerosos museos (explícitamente o dándole otro nombre) sin reflexionar sobre cómo podría integrarse en el entorno, y sin tener en cuenta que la educación museística no consiste en la adopción de modelos como si de aplicación de fórmulas se tratara. Para contribuir a la necesaria proble- matización de las propuestas educativas para los museos, a lo largo de este artículo analizaremos desde varios ángulos este método educativo. Partiendo de que los museos no son espacios neutrales de representación objetiva de hechos indiscutibles, ya que los va- lores culturales, sociales y políticos siempre están implicados en las exposiciones (MACDONALD, 1998), apostamos por una educación museística basada en la pregunta, la indagación y preocupada por difundir una nueva noción de museo como zona de controversia y contestación (PADRÓ, 2005). En esta 83 VISUALIDADES. RE VISTA DO PROGR AMA DE MESTR ADO EM CULTUR A VISUAL - FAV I UFG línea, el planteamiento crítico desde el que abordamos V.T.S. nos conduce a desvelar sus bases y fundamentaciones, que consideramos alejadas de la educación transmisora en su for- ma pero no en el fondo. Introducción Con ello, queremos introducir una perspectiva alternativa a la que ofrece como predominante este método, para invitar tanto a responsables de departamentos de educación, como a educadores de sala, maestros y profesores a reflexionar sobre esta propuesta. Buceando en los orígenes del método Para contextualizar la corriente en la que se enmarca V.T.S. y ver sus contribuciones en la educación artística en los años 80, haremos un breve recorrido por su historia. En un principio, este método fue propuesto por el MoMA de Nueva York, gra- cias a las ideas de la psicóloga cognitiva Abigail Housen –de- dicada a estudiar en desarrollo del pensamiento estético– y al entonces responsable de educación de este museo, Philip Yena- wine. En 1988 se asociaron firmando un contrato dirigido a la creación de programas para estudiantes y profesores visitantes del MoMA. A partir de 1991 comenzaron a ensayar maneras de organizar sus hallazgos en base a un método secuencial que posibilitara a los profesores llevar a cabo discusiones sobre obras de arte con sus estudiantes 3. En este momento se unieron con el Bard College, el Institute of Contemporary Art en Bos- ton, y el Boston Museum of Fine Arts para producir el primer boceto de lo que sería después el “Visual Thinking Strategies”. Mientras tanto, Housen dejó su puesto en el Mass College of Art y Yenawine cesó de su cargo en el MoMA. Posteriormente, en 1995, formaron una consultora educativa llamada Develo- pment Through Art, Inc. (D’Art) para continuar su trabajo de diseminación del programa que habían ensayado en el MoMA. Paralelamente constituyen la “Visual Understanding in Educa- tion” (V.U.E.), una organización sin fines de lucro, cuya finali- dad es extender la investigación en educación basada en el de- sarrollo desde un sentido cognitivo y evolucionista. Desde 1994 V.T.S. se empieza a implementar en escuelas de Europa del Este y Rusia y, a partir de 1998, inician la publicación de libros de es- Eneritz López, Magali Kivatinetz 84 Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos trategias educativas que ponen en práctica en diversos centros de Estados Unidos (VUE, 2001a). Después de años de estudio, Housen demostró que la prác- tica de V.T.S. produce un crecimiento en el pensamiento pura- mente estético y contemplativo, así como la mejora de otras habilidades cognitivas en un periodo corto de tiempo. Concre- tamente, esto se logra observando, especulando y razonando en base a evidencias. Buceando en los orígenes del método Esta concepción parte de que el apren- dizaje secuencial puede ser fácil, rápido y llevar a resultados espectaculares, sirviendo de base a una noción de educación artística que pretende un resultado inmediato y unívoco, más que una aproximación dialógica, reflexiva y divergente. En resumen, a pesar de que las originarias ideas de Hou- sen hayan estado siempre presentes, este método, como ya hemos mencionado, ha pasado por diversas metamorfosis, adoptando en cada una de ellas un nombre distinto: V.T.C., V.T.S., Pensamiento Visual, entre otras denominaciones. Esto nos hace ver que a pesar de que se cambie el formato de pre- sentación, las pautas del método están presentes en todas la reinterpretaciones. 4 Poniendo el zoom en el contexto El “Visual Thinking Curriculum”, tal y como fue concebido y desarrollado por Housen y Yenawine, se podría asociar con el constructivismo piagetiano, ya que es un método que no presta atención a los contenidos (lo que se aprende), sino so- bre todo a los procesos del funcionamiento cognitivo (COLL y GÓMEZ, 1995). En este sentido, es importante recordar que las aportaciones de Jean Piaget fueron fundamentales para la elaboración de una concepción constructivista en el ámbito educativo, ya que contestó al empirismo y al asociacionismo –teorías imperantes en la primera mitad del siglo XX– con el constructivismo, argumentando que el sujeto es activo e inter- preta la información del entorno 5. Esto significa que el cono- cimiento nuevo se genera a partir del previo porque construir conocimiento incluye un proceso de acomodación de lo nuevo a lo existente, que puede terminar o no por asimilarse. “Una de El “Visual Thinking Curriculum”, tal y como fue concebido y desarrollado por Housen y Yenawine, se podría asociar con el constructivismo piagetiano, ya que es un método que no presta atención a los contenidos (lo que se aprende), sino so- bre todo a los procesos del funcionamiento cognitivo (COLL y GÓMEZ, 1995). En este sentido, es importante recordar que las aportaciones de Jean Piaget fueron fundamentales para la elaboración de una concepción constructivista en el ámbito educativo, ya que contestó al empirismo y al asociacionismo –teorías imperantes en la primera mitad del siglo XX– con el constructivismo, argumentando que el sujeto es activo e inter- preta la información del entorno 5. Esto significa que el cono- cimiento nuevo se genera a partir del previo porque construir conocimiento incluye un proceso de acomodación de lo nuevo a lo existente, que puede terminar o no por asimilarse. “Una de 85 VISUALIDADES. RE VISTA DO PROGR AMA DE MESTR ADO EM CULTUR A VISUAL - FAV I UFG las características de esta perspectiva cognitivista (…) es que la progresión de la inteligencia se vincula al concepto de estadio o etapa”; en otras palabras, para Piaget, la inteligencia atravie- sa fases cualitativamente distintas 6. Sin embargo, “en los años setenta esta forma de concebir el desarrollo intelectual entra en crisis y la caracterización de los estadios pierde su carácter estable, progresivo e intelectual” (AGIRRE, 2000, p. 55). Poniendo el zoom en el contexto En suma, según Piaget y sus seguidores, el proceso de cons- trucción del conocimiento es sobre todo de orden interno e indi- vidual. Entonces, a la hora de acercarnos a métodos educativos elaborados desde esta postura teórica, como V.T.S., podemos encontrarnos con varios inconvenientes: en primer lugar, la en- señanza que se plantea prioriza que los educandos construyan estructuras de pensamiento que, supuestamente, les permiti- rán comprender todo tipo de contenidos. En segundo lugar, el conocimiento se construye a través de un proceso de descu- brimiento autónomo, derivado de la relación de cada sujeto epistemológico con el entorno inmediato, sin prestar atención a la interacción social. Así, se da a entender que los individu- os generan conocimientos desde sí mismos, olvidándose de los factores externos que influyen esta construcción. De este modo, uno de los principales problemas que pre- senta V.T.S. es que no considera el componente sociocultural y contextual de la construcción del conocimiento, obviando tam- bién que éste es mediado por otros factores, como las rela- ciones de poder constituidas históricamente. No debemos olvidar que los hechos, los conocimientos, los saberes nunca son neutrales ni objetivos, sino que están producidos e inclui- dos en contextos de relaciones sociales (ROGERS, 2005, citado en HERNÁNDEZ, 2006). Por ello, una crítica fundamental a este método es que pasa por alto que cualquier conocimiento está inevitablemente ligado a contextos y a las circunstancias biográficas de cada uno, ejes del constructivismo crítico 7. Esto quiere decir que se ignora que nuestros conocimientos se pro- ducen en interrelación con los ambientes en los que se usan, y que los aspectos cognitivos, emocionales y sociales forman parte de cómo somos producidos como sujetos. En relación a esta idea, ya desde principios del siglo XX, la voz de Lev S. Eneritz López, Magali Kivatinetz 86 Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos Vygotsky puso de relieve que “las posibilidades de aprendizaje guardan estrecha relación con el nivel de desarrollo alcanzado y subrayan, en consecuencia, el decisivo papel que el entorno tiene en este proceso de construcción del conocimiento” (AGIR- RE, 2000, p. 67). Por ello, una de sus contribuciones esenciales –que métodos como V.T.S. Poniendo el zoom en el contexto no tienen en cuenta– es la de con- cebir al sujeto como un ser eminentemente social y al conoci- miento mismo como un producto social. Por otra parte, V.T.S. da más importancia al hecho de es- tablecer un diálogo que a los propios contenidos del mismo. El énfasis del método reside en la forma de llegar al mensaje preestablecido –vinculado a la meta de progresar de un estadio a otro–, y no de prestar atención al aprendizaje que los partici- pantes puedan obtener. Es decir, no se repara en los contenidos (alternativos, paralelos, divergentes) sino en la forma en la que son transmitidos aquellos que se adaptan a la concepción del educador que sigue el V.T.S. Habrá momentos en que [el profesor] piense que un comentario está equivocado, pero los conceptos de correcto/incorrecto aquí no son válidos. Lo que importa es que los alumnos piensen. Es mejor [no] corregir ni apostillar (VUE, 2001c). (...) aceptar cada comentario de un modo imparcial. Recuerda que este proceso no está dirigido a conseguir respuestas correctas sino a establecer unas pautas útiles para pensar (VUE, 2001d). (...) aceptar cada comentario de un modo imparcial. Recuerda que este proceso no está dirigido a conseguir respuestas correctas sino a establecer unas pautas útiles para pensar (VUE, 2001d). A pesar de que siempre se establece un diálogo entre el educador y su grupo de visitantes, este diálogo utiliza normal- mente la “vía socrática”, que consiste en poner a los partici- pantes en una situación argumentativa no para aprender cómo pensar por ellos mismos, sino para llegar al lugar en el que el educador estaba inicialmente, es decir, para que consigan aprender lo que el educador tenía previsto desde el principio. Por ello, podemos decir que se trata de un proceso de en- señanza tradicional que concibe que los estudiantes o visi- tantes aprenderán pensando o actuando del modo en el que el educador les guía. Esto no permite a los participantes abrir posibilidades de divergencia ni hacer conexiones alternativas, 87 VISUALIDADES. RE VISTA DO PROGR AMA DE MESTR ADO EM CULTUR A VISUAL - FAV I UFG convirtiendo el proceso de razonamiento en un simulacro de lo que el pensamiento independiente y personal podría llegar a ser (HERNÁNDEZ, 2006). De este modo, la diferencia esencial entre un método de estas características y la educación museís- tica transmisora es su forma pero no el fondo, ya que se sigue partiendo de unos contenidos fijos que los participantes deben aprender. Eneritz López, Magali Kivatinetz Releyendo la receta de v.t.s. Retomando los orígenes de este método, vemos que el proyecto inicial llamado “Visual Thinking Curriculum” es fruto de una revisión constructivista de la perspectiva del arte para la comprensión (art for understanding) impulsado por el grupo Zero de Harvard. Partiendo de una misma base teórica y de problemáticas parecidas vinculadas a la apreciación estética, y casi en paralelo con Housen, Michael Parsons desarrolla, a partir de las ideas de Kohlberg, una metodología similar a la de su colega aunque poniendo el acento en el juicio estético del arte, en vez de en las formas de comprensión. De cualquier modo, la propuesta de Parsons tiene mucho en común con la teoría del desarrollo cognitivo de Piaget, aunque sostiene que aprender arte es sólo una cuestión estética, estableciendo que su significado es distinto del correspondiente a las ciencias o a la moral (PARSONS, 2002). Un problema que tiene este modelo de interpretación, es que utiliza un marco de referencia estética de base formalista (HERNÁNDEZ, 2000); es decir, que únicamente tiene en cuenta el análisis formal de la obra de arte, sin atender a la incidencia del contexto cultural en los significados estéticos y en los proce- sos psicológicos. Además, la noción de “estadio de desarrollo” de Piaget difiere de la de Parsons en que la entiende en eta- pas evolutivo/cognitivas y ve los estadios como agrupaciones de ideas y no como logros de las personas, evitando además cualquier identificación de los estadios con el desarrollo crono- lógico (MORALES, 2001). De este modo, Parsons establece cinco estadios del desar- rollo del juicio estético que se suceden secuencialmente, de for- Eneritz López, Magali Kivatinetz 88 Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos ma que cada estadio implica una elaboración más compleja que el anterior en los aspectos que considera que están presentes en la respuesta estética (PARSONS, 2002). “La estructuración de estos estadios refleja una evolución que va desde la depen- dencia en relación con lo que se aprecia en cada obra, hasta la autonomía interpretativa con respecto a ella” (HERNÁNDEZ, 2000, p. 121). Releyendo la receta de v.t.s. Se parte de una visión de progresión donde se considera que el final es la autonomía en vez de pensar que en las artes la ambigüedad, las múltiples versiones e interpretacio- nes no sólo dependen de la autonomía a la que el espectador llegue, sino que hay que tener en cuenta las cuestiones que la misma obra pueda generar en el observador. Autores como Hargreaves (1991) argumentan que la te- oría de Parsons tiene puntos flojos tales como que se limita al arte visual; que todas las obras que se utilizan en su expe- rimento son propias de las “bellas artes”; y que su método de entrevistas semiestructuradas ofrece desventajas de rigor experimental. A pesar de ello, el estudio de Parsons tiene el mérito de haber acercado el tema del conocimiento estético y artístico a las investigaciones que desde la psicología del desarrollo de tendencia cognitiva comienzan a realizarse en Estados Unidos en los años 70. “Además, ha permitido elabo- raciones posteriores que han introducido nuevos criterios de ordenación de las apreciaciones sobre las obras de arte. Una de estas revisiones ha sido el estudio dirigido por Housen et al.” (HERNÁNDEZ, 2000, p. 122). En este caso, los estadios hacen referencia al “tipo” de construcción de sentido, o de dotación de significado de cada sujeto, en un recorrido que va desde la narración, basada en preferencias individuales, hasta la actividad reconstructiva. 8 En síntesis, la propuesta “Visual Thinking Curriculum” per- mitió “ordenar las apreciaciones de los individuos en torno a las obras de arte, ejemplo de cómo la psicología del desarrollo de orientación cognitiva –a la que se adscribe Housen–, ha abor- dado su relación con el conocimiento artístico y estético” (HER- NÁNDEZ, 2000, p. 123). Este método educativo está vinculado teóricamente a la noción de desarrollo estético progresivo y se enmarca dentro de la psicología evolutiva, manteniendo 89 VISUALIDADES. RE VISTA DO PROGR AMA DE MESTR ADO EM CULTUR A VISUAL - FAV I UFG una concepción del aprendizaje como un camino de progreso, haciendo pervivir así una noción moderna del conocimiento. Siguiendo esta tendencia, se cree que las “Estrategias de Pensamiento Visual” incrementan en gran medida la capacidad de los estudiantes de apreciar el arte, extendiéndose a sus habi- lidades para hacer arte, respondiendo así al principal interés de los profesores en las escuelas 9. De este modo V.T.S. Eneritz López, Magali Kivatinetz Releyendo la receta de v.t.s. ayuda a los especialistas en la tarea de desarrollar la alfabetización visual entre los estudiantes y llegar así a los estándares de la educaci- ón artística. Este método educativo constructivista partía de la idea de usar el arte para enseñar a pensar, a desarrollar habi- lidades comunicativas y para alfabetizar visualmente a niños y jóvenes. Esta noción de “alfabetización visual” está relacionada con la idea del “arte como lenguaje”, concepción que lleva a pensar que una imagen se puede leer como si de un texto se tratase. 10 En este sentido, al V.T.S. le corresponde enseñar a leer la obra de arte. Si no aprendemos a leer, si no damos este primer paso, que es fundamental, nunca podremos desarrollar nuestras capaci- dades como espectador y como interesados en arte (YENAWINE, 2001). Sin embargo, consideramos que aunque una imagen con- tenga códigos, no es un código en sí misma; por lo tanto, al no ser solamente un sistema de signos, no se puede articular como un lenguaje y, consecuentemente, no se puede leer de la misma forma que un texto. Por otro lado, según los defensores de V.T.S., el crecimiento y desarrollo mental es estimulado de tres modos: mirando dete- nidamente las obras de arte incrementando progresivamente su complejidad, respondiendo a preguntas basadas en la percep- ción visual y el desarrollo, y participando en discusiones grupa- les cuidadosamente coordinadas por educadores. Así, durante cada sesión, utilizando las “Estrategias de Pensamiento Visual” se pretende que cada estudiante pueda señalar lo que ve en la obra de arte que está observando y expresar su opinión so- bre ella, argumentando sus interpretaciones. De este modo, los participantes son conscientes de que sus ideas se escuchan y se valoran, comprobando que cada comentario contribuye al pro- Eneritz López, Magali Kivatinetz 90 Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos ceso del grupo en su búsqueda de los diversos significados de la obra de arte (VUE, 2001c). Superando una educación transmisora Una vez presentado el método y problematizados sus funda- mentos, destacaremos a continuación sus ventajas, y más ade- lante, plantearemos algunos de sus inconvenientes. La adopci- ón de V.T.S. por parte de numerosos museos se puede explicar por sus marcados beneficios y avances respecto a la educación transmisora imperante, ya que no toma como punto de partida la idea de un educador transmisor que comunica el discurso del museo al público, sino que propone al educador como un mediador, un facilitador que, a través del diálogo, estimula en los participantes la capacidad de observación y de reflexión, no- ción vinculada a la nueva museología. Según datos una evalua- ción de un programa que utiliza esta metodología, su punto de partida es “ser un instrumento para facilitar procesos de apren- dizaje a distintos niveles y una herramienta para el desarrollo de las habilidades cognitivas de los alumnos” 11. Sumado a esta finalidad, la propuesta (...) facilita la mejora de capacidades deductivas, especulativas y argumentativas de los alumnos. Favorece la generación de inter- pretaciones basadas en las imágenes de menor a mayor comple- jidad. Permite la mejora de la expresión oral. Fomenta una nueva relación profesor-alumno, reafirmando el desarrollo personal del alumno ya que, al redefinir sus roles, se resituan en un plano de igualdad. Su objetivo principal es completar los objetivos del currí- culo y potenciar el aprendizaje activo frente a la recepción pa- siva tradicional. Para su correcto funcionamiento se requiere que el profesor haga bien su papel de mediador y que tenga buena relación con los alumnos, también es necesario que haya una ac- titud receptiva por parte de los participantes y que las normas del método se apliquen correctamente (POU, 2002). Como hemos mencionado anteriormente, los educadores son considerados los facilitadores del proceso, nunca la fuen- te principal de información u opinión. Por ello su tarea consiste en posibilitar que los estudiantes debatan distintas alternativas y dejan que el proceso conducido con las “Estrategias de Pen- 91 91 VISUALIDADES. RE VISTA DO PROGR AMA DE MESTR ADO EM CULTUR A VISUAL - FAV I UFG samiento Visual” fortalezca su habilidad de examinar, articular y reflexionar. El profesor desempeña el papel de moderador, sin aportar informa- ción u opinión algunas [sic]. El profesor permite que los alumnos discutan las diversas posibilidades y que el proceso de percepción mismo incremente su capacidad de analizar, relacionar, escuchar y reflexionar. 92 Eneritz López, Magali Kivatinetz Superando una educación transmisora La participación de los alumnos por turno fomenta la curiosidad y la búsqueda de información (VUE, 2001c). De esta forma, el profesor o el educador del museo se con- vierte en el protagonista del hecho educativo. Si el profesor sabe las normas del método, es decir, sabe estructurar el diá- logo, parafrasear a los alumnos para mejorar su expresión oral y conducir debates de interés común, ya están conseguidos los objetivos propuestos. El currículo se aplicará de modo plenamente satisfactorio si el pro- fesor sigue una serie de pautas básicas, lógicas y comprobadas científicamente, aunque al principio le parezcan algo restrictivas. Estas sencillas reglas abren todo un mundo de posibilidades (VUE, 2001c). Sin embargo, queremos destacar que, a pesar de mostrar a los educadores como líderes del proceso de aprendizaje, su formación se relega a un segundo plano, ya que es entendida como meramente práctica 12. Esto significa, en palabras de Pa- dró, que la profesión del educador en los museos es compren- dida según una concepción tradicional y artesana. Con ello nos referimos a que muchos departamentos de educación se consi- deran meramente prácticos, sin atender a una formación muse- ológica y/o educativa específica. En esta tendencia se considera que la educación es más una vocación que una profesión y por tanto, el educador es visto como un didacta (PADRÓ, 2005). A pesar de esto, es destacable que durante la práctica de V.T.S. se permita que los visitantes observen detenidamente las obras, se facilite la discusión y se estimule el pensamiento críti- co y creativo. El profesor considera las aportaciones de todos los alumnos para que se sientan respaldados e indica al grupo el valor de las ideas de cada persona. (…) Como parte del proceso de adquirir un pen- 92 Eneritz López, Magali Kivatinetz 92 Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos samiento crítico, se necesita subrayar la importancia de considerar todas las posibilidades. Hay que mantenerse abierto mientras los alumnos aportan ideas y considerar todos los comentarios igual- mente válidos (VUE, 2001c). Sin embargo, la noción de pensamiento crítico a la que se refieren no incluye una reflexión por parte de los estudiantes o visitantes del proceso seguido, y tampoco se les ofrece el contexto social, cultural, religioso ni político de las obras. Superando una educación transmisora Por ello, desde nuestro punto de vista el tipo de pensamiento que se estimula, se trataría más de un pensamiento tolerante que de una postura crítica. Esto es debido a que fomenta que todos se expresen y valoren por igual las opiniones de los demás par- ticipantes, pero no se anima a que se pongan en tela de juicio las diferentes opiniones, incluyendo tanto las propias como las de compañeros, educadores o expertos que hablan sobre las obras. Por estas razones, consideramos que V.T.S. puede ser ade- cuado como un método para utilizarse en un primer contacto con el arte, ya que sin duda, permite que todos los visitantes hablen, rompiendo con algunos mitos que encierra la idea tra- dicional de museo de centrarse en la contemplación de la obra en la búsqueda de una experiencia estética. Sin embargo, tras haber comentado formalmente la obra y haber satisfecho las necesidades de todos los participantes de hablar, consideramos que se deberían tratar temas candentes, polémicos y críticos. Pero, contrario a esta postura, Yenawine aboga por que no se indague en temas no convencionales a la hora de establecer un diálogo frente a las imágenes, perdiéndose la oportunidad de abordar temas de actualidad. Existen temas que, en general, deberán evitarse pues, a pesar de ser importantes o se presenten satisfactoriamente a un debate bajo ciertas circunstancias, las situaciones que presentan conlle- van demasiadas variables para asegurar una experiencia positiva. Las imágenes que ilustran escenas de violencia, posturas políticas específicas, imaginería religiosa determinada, desnudez, sexuali- dad y sensualidad manifiestas, así como temas grotescos o maca- bros pueden perjudicar a los observadores inexpertos si los valo- res expresados en ellas entran en conflicto con los suyos propios (YENAWINE, 2001). 93 VISUALIDADES. RE VISTA DO PROGR AMA DE MESTR ADO EM CULTUR A VISUAL - FAV I UFG De esta manera, al evitar cuestiones tales como la raza, el género o la clase social se pone en evidencia que V.T.S. no con- sidera cómo las condiciones socioeconómicas afectan las vidas y expectativas de las personas. V.T.S. al descubierto: un repaso a sus limitaciones Teniendo en cuenta todo lo anteriormente citado, quere- mos evidenciar nuestra preocupación de que actualmente en España y otros países se trabaje la educación en los museos desde una visión esencialista principalmente. Nos pregun- tamos porqué se implementan métodos que –aunque camu- flados tras un aparente diálogo abierto– son unidireccionales. Esto se materializa, por ejemplo, en el punto de partida tradi- cional que utiliza V.T.S., ya que la pregunta con la que se abre el diálogo es siempre: “¿qué ves?”, cuestión que no favorece un pensamiento relacional ni crítico. Esta pregunta hace supo- ner que en la obra hay un contenido que los visitantes deben encontrar, una respuesta correcta que han de identificar. Ade- más, se concibe que el visitante llega al museo sin ningún co- nocimiento previo y se parte de que aprender significa asimilar información y hechos que son independientes del sujeto. Esta perspectiva tiende hacia una posición conductista, concluyen- do que el aprendizaje consiste en la suma de varias asocia- ciones simples y que todo lo que se conoce se ha adquirido a través de la experiencia (HEIN, 1998). Por otro lado, V.T.S. tampoco explora formas de pensa- miento que no estén sujetas a la noción moderna de raciona- lidad. Estas alternativas de pensamiento serían las que prestan atención a la intuición y al pensamiento no lineal y multilógico, donde el pensar es biográfico, contextual y no es sólo una actividad mental (HERNÁNDEZ, 2006). Asimismo, es importante destacar que, a pesar de que se utilice una metodología basada en la realización de múltiples preguntas, V.T.S. parte de que hay un conocimiento clave que se tiene que aprender. Por ello, a pesar de que se promueve la discusión en el grupo, siempre se incita a llegar a un consenso y a dejar claro cuál es el men- saje predeterminado que se debe asimilar. Eneritz López, Magali Kivatinetz 94 Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos V.T.S. es un proceso de descubrimiento. El debate se inicia me- diante unas preguntas concretas, formuladas para provocar di- versas respuestas con sentido, en función de lo que se ve en las imágenes. (…) Así los alumnos conocen la ambigüedad del arte y sus diversos y ocultos posibles significados. (…) La pregunta reco- mendada (y sus variaciones) les obliga a indagar en busca de un significado. V.T.S. al descubierto: un repaso a sus limitaciones (…) Con el tiempo, la interacción del grupo pondrá en su lugar a la ‘verdad’ (VUE, 2001c). Además, a menudo, este mensaje tiene más que ver con las visiones que los museos fomentan del artista y con las expec- tativas que crean en los visitantes, que con las posibilidades de pensar en otras versiones que estos pueden aportar. La mayor parte de sus interpretaciones coincidirán con el pro- pósito del artista si se buscan las razones indagando en la ima- gen. (...) El hecho de que las imágenes sean accesibles ofrece a los observadores la oportunidad de descubrir por sí mismos los significados pretendidos por el artista. (...) Cuanto más abierta sea la interpretación de una obra, más acertadas podrán ser las opiniones intuitivas del observador, siempre y cuando se enmar- quen dentro del propósito del artista o de la cultura en cuestión (VUE, 2001c). Entonces, V.T.S. hace pensar que hay una verdad única en la obra que debe ser descifrada y, por tanto, no permite nin- gún descentramiento ni problematización, ni mucho menos establece conexión alguna con el sujeto y su mundo (HERNÁN- DEZ, 2006). Debido al tipo de preguntas que se realizan duran- te las sesiones con V.T.S., la importancia se da exclusivamente a la obra con la que se está trabajando y, consecuentemente, al artista que la produjo. Los participantes –sus expectativas, sus circunstancias– quedan excluidos a la hora de llevar a cabo la interpretación de la obra, ya que se entiende que esta inter- pretación es única y se encuentra en la propia obra y nunca en los espectadores. Como afirma el teórico cultural Stuart Hall (1997), debemos enfatizar que no existe una simple o correcta respuesta a preguntas como ‘¿qué quiere decir esta imagen?’, ya que los significados cambian en el tiempo y no hay leyes que garanticen el ‘significado verdadero’ de las cosas; por ello, no se debería establecer un debate entre quien tiene razón y 95 VISUALIDADES. RE VISTA DO PROGR AMA DE MESTR ADO EM CULTUR A VISUAL - FAV I UFG quien está equivocado, sino entre significados e interpretacio- nes igualmente plausibles, aunque en ocasiones compitan y se contesten. Eneritz López, Magali Kivatinetz V.T.S. al descubierto: un repaso a sus limitaciones También es cierto que no sólo la pregunta inicial “¿qué ves?”, sino también el resto de las cuestiones que facilitan el diálogo, priorizan el sentido de la vista, dejando de lado la posibilidad de experimentar o conocer el arte desde los otros sentidos. Por ello, los programas educativos basados en las “Estrategias de Pensa- miento Visual”, marginan a las personas que aprenden de forma no visual o verbal, ya que no suelen estar acompañados por una actividad que requiera la utilización de sentidos y habilidades dis- tintas 13. En este sentido sería un programa “monoalfabetizador” y se distancia de las propuestas multialfabetizadoras actualmen- te vigentes (LANKSHEAR y KNOBEL, 2003). Otra de las ideas fundamentales de V.T.S. es que el edu- cando aprende mientras se expresa oralmente aunque todo ha sido previamente diseñado y decidido por los adultos 14. Se da por sentado que los participantes aprenden desarrollando sus capacidades de pensamiento y razonamiento mediante pro- gramas bien estructurados (diseñados por expertos) de habili- dades para aprender (HERNÁNDEZ, 2006). Si nuestro deseo es que los participantes establezcan una relación sólida con el arte, deberíamos pensar en el tipo de arte más ade- cuado para iniciarlos en la materia. Si pretendemos, además, que aprendan a ‘leerlo’ por si mismos –que se conviertan en obser- vadores autosuficientes– deberíamos pensar la mejor manera de estimular esta habilidad. (...) La contemplación del arte se enseña mejor alentando a los alumnos, ayudándoles a mirar con deteni- miento, a pensar sobre lo que ven, y a articular sus reacciones (YENAWINE, 2001). También consideramos cuestionable de este método educa- tivo su peculiar manera de seleccionar las obras de arte “ade- cuadas” para los observadores. Existe una firme resistencia a incluir en las sesiones de V.T.S. la cultura audiovisual, obviando de este modo manifestaciones artísticas distintas a la pintura o escultura, –tales como videos, performances, instalaciones, pu- blicidad, entre otros–. Al centrar las sesiones en observar obras artísticas realizadas con procesos tradicionales –es decir, pintu- Eneritz López, Magali Kivatinetz 96 Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos ra, escultura, dibujo y grabado–, se parte de una noción de arte elitista, ya que se potencia exclusivamente un acercamiento al arte que se considera culto, elevado y legitimado. VISUALIDADES. RE VISTA DO PROGR AMA DE MESTR ADO EM CULTUR A VISUAL - FAV I UFG VISUALIDADES. RE VISTA DO PROGR AMA DE MESTR ADO EM CULTUR A VISUAL - FAV I UFG Sin embargo, partiendo de que no hay una verdad única en la obra, sino que el espectador la interpreta según su baga- je y circunstancias, una obra nunca podrá ser malinterpretada. Finalmente, también se hace hincapié en el tipo de obras que se deben evitar, como las que requieran una información es- pecializada o aquellas cuyo significado esté determinado por un artista o cultura concretos (dentro de esta categoría se in- cluyen temas históricos, religiosos, mitológicos, y especialmen- te étnicos). Tampoco son adecuadas las escenas de violencia, política, religión, desnudez, sensualidad o sexualidad, los temas grotescos o macabros y las naturalezas muertas (porque pre- sentan una serie de cualidades que, al igual que la pintura abs- tracta, no invitan a investigar) (YENAWINE, 2001). Siguiendo estas pautas de selección, siempre se elegirán imágenes cuyo análisis difícilmente trascenderá los aspectos estéticos y forma- les. En este sentido, notamos que no permiten el tratamiento de ningún tema controvertido ni conectado con la realidad so- ciocultural que los participantes puedan estar viviendo. Desde nuestro punto de vista, para conocer las obras de arte y anali- zarlas en contexto, tendríamos que aprender sobre sus sistemas de producción, mecenazgo, canales de distribución, ideas ex- presadas por artistas, coleccionistas y críticos, y todos aquellos elementos que crean el sistema del arte (EFLAND, FREEDMAN y STUHR, 2003). Eneritz López, Magali Kivatinetz V.T.S. al descubierto: un repaso a sus limitaciones El proceso de selección de obras con el fin de iniciar a los obser- vadores principiantes a las maravillas del arte es una tarea que debe hacerse a conciencia. (…) La pintura resulta muy útil, pues ha existido siempre en todas partes y a menudo ha sido conside- rada la más alta expresión artística de una cultura. (…) Las ilustra- ciones, la mayor parte del fotoperiodismo, los dibujos animados y los anuncios rara vez suponen una elección útil porque permiten pocas interpretaciones. Los temas con una gran carga, las técni- cas experimentales, y los estilos provocadores pueden desviar a los observadores de este objetivo [animar a contemplar, a pensar y a desarrollar unas interpretaciones bien infundadas] (YENAWI- NE, 2001). Así, para elegir las imágenes adecuadas para que los “princi- piantes, mediante la práctica, elaboren su propio entendimien- to del arte incrementando su potencial como observadores”, se recomienda a los educadores seguir unas rigurosas pautas ideadas por Philip Yenawine (2001). Según estas normas, las imágenes seleccionadas tienen que ser reconocibles, reales y deben siempre contar una historia. En este sentido, se sugie- re que sean paisajes, retratos o escenas “de género” (juegos, familia, actividades laborales) y cuya lectura sea fácil de desen- trañar para que cautiven a todo tipo de público. Respecto a las técnicas, las que más se recomiendan son la pintura, la escul- tura figurativa, las reproducciones de dibujos y estampas, y la fotografía. Así, se excluye no sólo toda la pintura abstracta sino también casi todas las manifestaciones artísticas posteriores a las primeras vanguardias, ya que a partir de este momento his- tórico la pintura rompe con la imitación a la realidad. Por otro lado, se considera importante que se conozca la intención del artista o la cultura que ha elaborado la pieza para no caer en malas interpretaciones. Si desconocemos el propósito del artista o la cultura en la que se enmarca, este tipo de arte nos resultará ajeno, y nuestra com- prensión del mismo será limitada, susceptible de ser malinterpre- tada (YENAWINE, 2001). 97 Reflexiones inacabadas Partiendo de que la situación actual de la educación en los museos es de tránsito, y muchos de ellos se encuentran en un proceso de cambio y replanteamiento de su ideología educati- va, es lógico que traten de buscar orientación y referencias en tendencias de otros países. Sin embargo, hemos observado que más que ideas o referentes, lo que se prefiere es la adopción de un determinado método educativo, con criterios teóricos bien definidos y pautas para su implementación claras; en fin, un método que pueda utilizarse con facilidad y aprenderse rápida- mente. Sin embargo, no se considera que cualquier metodolo- gía educativa es creada en un momento y lugar determinados y, 98 Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos consecuentemente, permanece siempre ligada a circunstancias socioculturales concretas. Por ello, a la hora de implementar V.T.S. en cualquier institución no se debería olvidar su contextu- alización y adaptación a los intereses locales. En contraste con esta idea, Yenawine (2001) afirma que no hace falta ninguna adaptación de este método previa a su implementación en cual- quier contexto. – Ha sido necesario algún proceso de adaptación del programa VTS para poder ser aplicado a un medio como éste, tan distinto de Estados Unidos? – De acuerdo con nuestra experiencia, no. No ha habido nece- sidad de introducir nuevas variables a la hora de aplicar en este contexto nuestro proceso de facilitar la discusión que es la clave del método de enseñanza del VTS (GONZÁLEZ, 2006). Creemos que para justificar la adopción de V.T.S. en los museos, se valora que a partir de él pueden desarrollarse pro- gramas educativos atractivos, que posibiliten la llegada de más público a la institución, cometido normalmente asumido por los departamentos de educación. Por estos motivos, se accede a la implementación de este método sin cuestionar sus contenidos y sin reflexionar sobre el tipo de visitante que se está creando. A este respecto, Yenawine comenta en una reciente entrevista: Los educadores de museos de todos los países en los que hemos trabajado están interesados en el VTS por los mismos motivos: El VTS dinamiza y da vida a los programas pedagógicos de los museos. Reflexiones inacabadas El trabajo de investigación que hay detrás del VTS – que corrobora el crecimiento tanto en términos de observación como de habilidades interpretativas por parte de los distintos públicos – ha producido un considerable eco entre todos los profesionales de museos interesados en provocar un impacto sobre los visitantes (GONZÁLEZ, 2006). Sin embargo, en la actualidad, la educación museística ya no se entiende sólo como un proceso lineal en el que el museo le enseña un contenido al visitante, sino que trata más bien de cómo los visitantes usan el museo de maneras significativas para ellos (ROBERTS, 1997); por eso, la esencia de la educación 99 VISUALIDADES. RE VISTA DO PROGR AMA DE MESTR ADO EM CULTUR A VISUAL - FAV I UFG 100 Eneritz López, Magali Kivatinetz Gracias a la Dra. Carla Padró, al Dr. Fernando Hernández y a todas las personas que nos ayudaron a construir, deconstruir y reconstruir este texto. hoy en día es crear conocimiento y posibilitar la construcción de significados. hoy en día es crear conocimiento y posibilitar la construcción de significados. La educación en el museo es concebida como un conjunto de pro- cesos, educación como forma de vida, como algo deseable para asimilar los acontecimientos diarios y como actitud positiva frente al mundo. Además los museos son las únicas instituciones de la sociedad que pueden satisfacer las necesidades de aprender de todo tipo de personas. Son también forma de ocio pero siempre en relación con aprendizaje. Los visitantes esperan cada vez más que una visita a un museo tenga una importancia personal inme- diata, interactiva y que dé lugar a la adquisición clara e identifica- da de conocimientos. La educación en el museo se entiende cada vez más como la fuerza que da forma y que se encuentra detrás de la política y de los objetivos generales del museo (HOOPER- GREENHILL, 1998). De este modo, la educación en el museo no es concebida ya sólo ocio o diversión, sino que es entendida como un acto de reflexión y de creación de nuevas narrativas igualmente válidas y significativas que la propuesta por la propia institución. Estas narrativas alternativas parten de los propios visitantes que, mo- tivados por el educador, construyen sus propias historias tenien- do en cuenta su conocimiento previo, sus experiencias y sus condiciones sociales y biográficas (ROBERTS, 1997). Debemos tener en cuenta, por tanto, que un visitante en un museo per- cibe de manera inteligente, y que cada uno tiene sus experien- cias, expectativas, opiniones, suposiciones. De este modo, se puede decir que el proceso de percepción no es algo ligado ex- clusivamente a los sentidos, sino que implica un proceso activo por parte del sujeto que lo puede conectar con su “sentido de ser”. Siempre percibimos desde lo que sabemos; no extraemos del medio toda la información, sino aquellos indicios coheren- tes con nuestras hipótesis, con lo construido a partir de expe- riencias previas y de acuerdo a un proyecto o interés cognitivo (DÍAZ y UNZU, 2003). Consecuentemente, “el museo tiene que proporcionar al visitante herramientas para interactuar conside- rando que cada uno posee sus propios conocimientos, valores y sentimientos intransferibles” (PADRÒ, 1995, p. 19). Notas 1- A pesar de que en su origen este método se dio a conocer como V.T.C. (Visual Thinking Curriculum), en la actualidad se difunde con las siglas V.T.S. (Visual Thinking Strategies) y, por ello, a lo largo del artículo nos referiremos al método de este modo. 2- Según Carla Padró, “La mayoría de departamentos educativos [de museos] sin tener muy claras sus teorías implícitas fruto de un trabajo que, durante mucho tiempo, ha sido considerado de traducción del discurso oficial o, en los últimos años, de reproducción del currículum escolar” en Padró, C. 2005: Museos y educación artística: redes de paso, encrucijadas difusas, zonas de viraje (Texto sin publicar). 3- Paralelamente a este estudio, Michael Parsons en la Universidad de Ohio, siguiendo también el modelo de desarrollo propuesto por Piaget, llegaba a las mismas conclusio- nes: para comprender la realidad del museo se necesitaba una formación en educación artística que tuviera en cuenta los estadios del desarrollo del juicio estético. 4- Véanse los programas “Mira!” del Caixafòrum de Barcelona (España) o “D.A.P., Didác- tica del Arte y del Patrimonio” -que usa las estrategias de Pensamiento Visual-, comercia- lizado por la Fundación Arte Viva en España, Argentina, Brasil y otros países. 5- El constructivismo sostiene que el conocimiento es resultado de un proceso dinámico e interactivo durante el cual la información es interpretada y reinterpretada por la mente, que va construyendo progresivamente modelos explicativos cada vez más complejos y potentes. Esto significa que conocemos la realidad a través de los modelos que construi- mos para explicarla, y que estos modelos siempre son susceptibles de ser mejorados o cambiados (Coll y Gómez, 1995). 6- Según Piaget, “los niños atraviesan unas etapas o estadios cualitativamente distintos que denomina: sensoriomotriz, preoperacional, operacional concreto y operacional for- mal” (Agirre, 2000: 54). 7- Para profundizar en el constructivismo crítico, ver: Hernández, F. (2000) Educación y cultura visual. Barcelona: Octaedro, pgs. 108-109 8- Para profundizar en los estadios de Parsons y en los de Housen, consultar: Hernández, F. 2000: La investigación sobre la comprensión: la interpretación como clave de la educa- ción escolar. En: Educación y Cultura Visual (págs. 105-132) Barcelona, Octaedro. 9- “Estrategias de Pensamiento Visual” es la traducción literal al castellano de “Visual Thinking Strategies” o V.T.S. Utilizamos el término V.T.S. cuando nos referimos al método en general, y “Estrategias de Pensamiento Visual” al hablar de las metodologías que se utilizan. VISUALIDADES. RE VISTA DO PROGR AMA DE MESTR ADO EM CULTUR A VISUAL - FAV I UFG VISUALIDADES. RE VISTA DO PROGR AMA DE MESTR ADO EM CULTUR A VISUAL - FAV I UFG hoy en día es crear conocimiento y posibilitar la construcción de significados. Por todo ello, no es aceptable la concepción de aprendizaje conductista fomentada desde V.T.S., que considera que la mente del visi- 100 Eneritz López, Magali Kivatinetz 100 Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos tante es una “tabula rasa” y que aprender significa simplemen- te sumar asociaciones simples (HEIN, 1998). Por el contrario, consideramos que la educación museística debe construirse de un modo indagador y crítico; desde esta posición, métodos educativos como V.T.S. deberían ser refor- mulados, ya que en vez de promover la reflexión y el aprendi- zaje significativo en el visitante, siguen reproduciendo formas tradicionales de educación. Para romper con ello, entre otros factores, sería necesario repensar la formación y función de los educadores que llevan a cabo el proceso educativo. En este sen- tido, es importante promover en ellos la indagación sobre su trabajo y estimular su capacidad crítica a través de una formaci- ón continua. Éste sería el primer paso para que se convirtieran en profesionales reflexivos concebidos, no como consumi- dores de los conocimientos producidos por otros, sino como creadores de conocimiento en relación con la enseñanza y el aprendizaje (SCHÖN, 1998). Desde nuestra postura como investigadoras en el campo de la educación en los museos, consideramos necesario promover estrategias de resistencia y acción que permitan reposicionar las formas hegemónicas de educación y posibilitar espacios para el cuestionamiento. Nuestro objetivo es fomentar que se lleven a cabo prácticas educativas en los museos que inciten a los visitantes a interrogarse, y desde las que se asuma que el conocimiento que se construya ayudará a las personas a ser mejores y a vivir más plenamente más allá del mundo académi- co (HOOKS, 1994). 101 Notas 9- “Estrategias de Pensamiento Visual” es la traducción literal al castellano de “Visual Thinking Strategies” o V.T.S. Utilizamos el término V.T.S. cuando nos referimos al método en general, y “Estrategias de Pensamiento Visual” al hablar de las metodologías que se utilizan. 10- Esta idea de que es posible leer una imagen del mismo modo que un texto es actu- almente de uso corriente para los defensores de V.T.S. Por ejemplo, en la página web del Museo Picasso de Málaga (España) podemos leer: “Primavera 2006.Curso para pro- fesores de Primaria basado en las Estrategias de Pensamiento Visual (V.T.S.): Se plantea trabajar con los alumnos para aprender a mirar (al igual que aprendemos a leer) como parte de un proceso de desarrollo del conocimiento basado en el hecho mismo de mirar y tratar de comunicarlo por medio de la palabra y dentro de una discusión abierta del grupo.” 11- La evaluación citada se refiere a: Pou, C. 2002: El programa educativo “Mira!” del Laboratori de les Arts: un instrumento para la escuela primaria. (Texto sin publicar. Inves- tigación financiada por la Fundació “la Caixa”) 12- “El educador de V.T.S. hará mejor su trabajo cuantas más veces lo haga. Es un mé- todo que se va aprendiendo con la práctica” (Linda Duke, durante su conferencia en las XIII Jornadas de los D.E.A.C., Murcia, octubre de 2005) 13- En 1983 Howard Gardner publicó su libro “Frames of Mind: The theory of multiple 102 Eneritz López, Magali Kivatinetz Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos intelligences”, en el que plantea una visión pluralista de la inteligencia, reconociendo en ella muchas facetas diferentes, entendiéndose así que cada persona posee distintos potenciales cognitivos. De esto se deriva que no todas las personas aprenden del mismo modo ya que todos desarrollamos nuestras inteligencias de formas dispares. Esta idea debería tenerse en cuenta a la hora de plantear cualquier actividad educativa. 14- Análogamente al método educativo conocido como “learning by doing”, podríamos considerar al V.T.S. como un método que propone el “learning by telling”, ya que consi- dera que el niño aprende cuando se expresa y, por lo tanto, fomenta exclusivamente su expresión oral. Referências Bibliográficas AGIRRE, I. Teorías y prácticas en educación artística. Ideas para una revisi- ón pragmatista de la experiencia estética. Pamplona: Universidad Pública de Navarra, 2000. BELTRÁN, C. L. La educación artística en centros de arte contemporáneo: una alternativa para la construcción de aprendizajes. En: Huerta, R. y De La Calle, R. (eds.) La mirada inquieta. Educación artística y museo. Valen- cia: PUV, 2005, p. 169-182. COLL, C. y Gómez-Granell, C. De qué hablamos cuando hablamos de cons- tructivismo. Cuadernos de Pedagogía (221), 8-10, 1995. DÍAZ, I. y Unzu, A. La mirada que construye. Competencias y extravíos. En: Lorente, J.P. y ALMAZÁN, D. Museología crítica y arte contemporá- neo. Zaragoza: Universidad de Zaragoza, 2003, p. 185-201. DUNCAN, C. Civilizing rituals. London, Routledge, 1995. EFLAND, A., FREEDMAN, K. y STUHR, P. [1996] La educación en el arte posmoderno. Barcelona: Paidós, 2003. GARDNER, H. Frames of Mind: the theory of multiple intelligences. New York: Basic Books, 1983. GOODSON, I. [1997] El cambio en el currículum. Barcelona: Octaedro, 2000. GONZÁLEZ, F. Estrategias de Pensamiento Visual. Contemporánea. Revista grancanaria de cultura (1), 2006. HALL, S. (Ed.) Representation: cultural representations ans signifying prac- tices. Milton Kynes: Open University, 1997. HARGREAVES, D.J. Infancia y educación artística. Madrid: Morata, 1991. 103 VISUALIDADES. RE VISTA DO PROGR AMA DE MESTR ADO EM CULTUR A VISUAL - FAV I UFG HEIN, G.E. Learning in the museum. New York, Routledge, 1998 HEIN, G.E. Learning in the museum. New York, Routledge, 1998 HERNÁNDEZ, F. Educación y cultura visual. Barcelona, Octaedro, 2000. 2006. HERNÁNDEZ, F. Educación y cultura visual. Barcelona, Octaedro, 2000. HOOKS, B. Teaching to transgress: education as the practice of freedom. New York: Routledge, 1994. HOOPER-GREENHILL, E. [1994] Los museos y sus visitantes. Gijón, TREA, 1998. LANKSHEAR, L. & KNOBEL, M. New Literacies. Changing knowledge and classroom learning. Buckingham: Open University Press, 2003. MACDONALD, S. (Ed.) The politics of display: museums, science, culture. London, Routledge, 1998. MACHÍN, M.F. Museo Picasso de Antibes y Artium de Vitoria-Gasteiz. Dos museos, dos proyectos educativos. En: CALAF, R. y FONTAL, O. Comuni- cación educativa del patrimonio: referentes, modelos y ejemplos. Gijón: Trea, 2004, p. 179-199. MORALES, J.J. La evaluación en el área de educación visual y plástica en la E.S.O. (tesis doctoral, Universitat Autónoma de Barcelona), 2001. PADRÓ, C.¿Lecciones o preguntas? La relación entre el visitante y el pro- ceso expositivo en la museología estadounidense contemporánea. Revista de museología (6), 19-22, 1995. PADRÓ, C. Educación artística en museos y centros de arte. En HUERTA, R. y DE LA CALLE, R. (eds.) La mirada inquieta. Educación artística y museos. Valencia: PUV, 2005, p. 137-152. PARSONS, M. [1987] Cómo entendemos el arte. Una experiencia cogniti- vo-evolutiva de la experiencia estética. Barcelona: Paidós, 2002. PASTOR, I. Pedagogía museística. Nuevas perspectivas y tendencias actu- ales. Barcelona: Ariel Patrimonio, 2004. POU, C. El programa educativo `Mira!´ del Laboratori de les Arts: un ins- trumento para la escuela primaria, 2002 (Texto sin publicar, investigación financiada por la Fundació “la Caixa”). ROBERTS, L. From knowledge to narrative. Educators and the changing museum. Washington y Londres, Smithsonian Institution Press, 1997. SCHÖN, D. El profesional reflexivo. Cómo piensan los profesionales cuan- do actúan. Barcelona, Paidós, 1998. VUE. Background and History, VUE, 2001a. En: www.vue.org (última con- sulta: abril 2006) VUE. Understanding the Basics, VUE, 2001b. En: www.vue.org (última consulta: abril 2006) VUE. HEIN, G.E. Learning in the museum. New York, Routledge, 1998 Comprendiendo los conceptos básicos de VTS, 2001c (Texto divulga- 104 Eneritz López, Magali Kivatinetz Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos Replanteando las estrategias de pensamiento visual: un método controvertido para la educación en museos do y utilizado por VUE en el curso “VTS Estrategias de Pensamiento Visual” en el Centro Atlántico de Arte Moderno de Las Palmas de Gran Canaria, 15-18 de Noviembre de 2005). VUE. Un vistazo al VTS, 2001d. (Texto divulgado y utilizado por VUE en el curso “VTS Estrategias de Pensamiento Visual” en el Centro Atlántico de Arte Moderno de Las Palmas de Gran Canaria, 15-18 de Noviembre de 2005). VYGOTSKY, L. Mind in society: the development of higher psychological processes. Cambridge: Harvard University Press, 1978. VYGOTSKY, L. Mind in society: the development of higher psychological processes. Cambridge: Harvard University Press, 1978. YENAWINE, P. Iniciación al arte: ideas para la selección de imágenes, 2001 (Texto divulgado y utilizado por VUE en el curso “VTS Estrategias de Pen- samiento Visual” en el Centro Atlántico de Arte Moderno de Las Palmas de Gran Canaria, 15-18 de Noviembre de 2005). YENAWINE, P. Iniciación al arte: ideas para la selección de imágenes, 2001 (Texto divulgado y utilizado por VUE en el curso “VTS Estrategias de Pen- samiento Visual” en el Centro Atlántico de Arte Moderno de Las Palmas de Gran Canaria, 15-18 de Noviembre de 2005). Páginas web Artium, Centro-Museo Vasco de Arte Contemporáneo de Vitoria: <http://www.artium.org> Artium, Centro-Museo Vasco de Arte Contemporáneo de Vitoria: <http://www.artium.org> Caixafòrum de Barcelona: <http://www.fundacio.lacaixa.es/caixaforum> Centro Atlántico de Arte Moderno de Las Palmas de Gran Canaria: <http://www.caam.net> Museum of Modern Art de Nueva York: <http://www.moma.org> Museo Picasso de Málaga: <http://www.museopicassomalaga.org> Visual Understanding in Education: <http://www.vue.org> http://www.artium.org Caixafòrum de Barcelona: <http://www.fundacio.lacaixa.es/caixaforum> Centro Atlántico de Arte Moderno de Las Palmas de Gran Canaria: <http://www.caam.net> Museum of Modern Art de Nueva York: <http://www.moma.org> Museo Picasso de Málaga: <http://www.museopicassomalaga.org> Visual Understanding in Education: <http://www.vue.org> Caixafòrum de Barcelona: <http://www.fundacio.lacaixa.es/caixaforum> Centro Atlántico de Arte Moderno de Las Palmas de Gran Canaria: <http://www.caam.net> Museum of Modern Art de Nueva York: <http://www.moma.org> Museo Picasso de Málaga: <http://www.museopicassomalaga.org> Visual Understanding in Education: <http://www.vue.org> ENERITZ LÓPEZ Investigadora en formación, Universidad de Barcelona. Financiación: Go- bierno Vasco. Email: eneritzlop@hotmail.com MAGALI KIVATINETZ Becaria de investigación, Universidad de Barcelona. Coordinadora de moti- vART serveis educatius. Email: maga@motivart.com 105
https://openalex.org/W2180881133	https://europepmc.org/articles/pmc4616229?pdf=render	English	null	Optically secured information retrieval using two authenticated phase-only masks	Scientific reports	2,015	cc-by	6,291	Optically secured information retrieval using two authenticated phase-only masks received: 16 June 2015 accepted: 28 September 2015 Published: 23 October 2015 received: 16 June 2015 accepted: 28 September 2015 Published: 23 October 2015 Xiaogang Wang1, Wen Chen2,3, Shengtao Mei3 & Xudong Chen3 We propose an algorithm for jointly designing two phase-only masks (POMs) that allow for the encryption and noise-free retrieval of triple images. The images required for optical retrieval are first stored in quick-response (QR) codes for noise-free retrieval and flexible readout. Two sparse POMs are respectively calculated from two different images used as references for authentication based on modified Gerchberg-Saxton algorithm (GSA) and pixel extraction, and are then used as support constraints in a modified double-phase retrieval algorithm (MPRA), together with the above- mentioned QR codes. No visible information about the target images or the reference images can be obtained from each of these authenticated POMs. This approach allows users to authenticate the two POMs used for image reconstruction without visual observation of the reference images. It also allows user to friendly access and readout with mobile devices. When designing diffractive optical elements that allow for the encryption of data for security appli- cations, phase retrieval algorithms (PRAs) such as the Gerchberg-Saxton algorithm (GSA)1, Fienup method2 and their derivative3 can be used. In 1996, Johnson and Brasher encrypted a biometric image in two phase-only masks (POMs) that together reconstruct an image although neither diffractive element by itself gives any hints as to what is in the image4. Under the framework of linear double-random-phase encoding (DRPE) scheme5, many modified algorithms in Fourier domain6,7, Fresnel domain8–14 and gyra- tor domain15 have been proposed to generate POMs for data retrieval. Recently, we have also proposed several methods to produce two POMs for single-image retrieval using iterative nonlinear DRPE16,17. Degrees of freedom to manipulate the physical parameters of optical waves can be used in those algo- rithms where an image can be encoded into two or more POMs4–17. Optical information hiding with POMs has now been one of the most popular application areas for PRAs18–20. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Optically secured information retrieval using two authenticated phase-only masks However, the identifying and the capacity of those computer-generated POMs and the deteriorated original inputs reconstructed optically are still the major concerns in regards to the PRA-based data security protocols.f p y j g y p In this paper, a method for jointly designing two diffractive optical elements having quasi-random phases that allow for the authentication of the elements themselves, the encryption and noise-free retrieval of triple images is proposed based on modified PRAs. No visible information can be obtained from each of the POMs. The target images are inserted into QR codes via hyperlinks that allow for flexi- ble readout with mobile devices. This approach allows users to authenticate the two POMs without visual observation of those images used as references for authentication. The target images can be revealed without visible loss of information due to the property of high damage tolerance capability of QR codes. Scientific Reports \| 5:15668 \| DOI: 10.1038/srep15668 Results O i l i Optical image reconstruction scheme. The proposed reconstruction procedure of target images is demonstrates in Fig. 1. When the authenticated POM P1 is illuminated with incident plane wave and then modified by another authenticated POM P2, the approximates of the input QR codes ( ) ′, = , , q i 1 2 3 i can be detected in three different object planes. Thus, we have 1School of Sciences, Zhejiang A&F University, Linan 311300, China. 2Department of Electronic and Information Engineering, The Hong Kong Polytechnic University, Hong Kong China. 3Department of Electrical and Computer Engineering, National University of Singapore, Singapore 117583, Singapore. Correspondence and requests for materials should be addressed to X.W. (email: wxg1201@163.com) Scientific Reports \| 5:15668 \| DOI: 10.1038/srep15668 1 www.nature.com/scientificreports/ Figure 1. Optical image reconstruction procedure with two authenticated POMs. Figure 1. Optical image reconstruction procedure with two authenticated POMs. ′( , ) = ( , ) ×  ( , ) , ( ) λ λ + + , , { } q x y P x y P x y FrT FrT 1 i i i z z 1 1 2 1 1 1 0 0 i 0 ( ) 1 where the operator \| \| denotes a modulus operation and FrT[•] represents Fresnel transform17. Image hiding algorithm with sparsity constrains. The procedure of designing two POMs P1 and P2 that together reconstruct triple images consists of the following steps: 1. Storing three target images (f1, f2, f3) in QR codes (q1, q2, q3). 2. Generating two sparse POMs (p1s, p2s) from two images (g1, g2), which are used as reference for authentication and differ from the target images. 3. Numerical calculation of the two POMs P1 and P2 using the sparse POMs and the QR codes. g p To generate two sparse POMs, two secret images g1 and g2 need to be respectively encoded in two different POMs by using a modified Fresnel domain GSA11,12 with the two intensity constraints, i.e., a unit amplitude in the input plane and the image to be encoded in the output plane at a certain distance. Note that the two images used as references are not QR codes used for image encoding. The images g1 and g2 are independently encoded into POMs pm and ′ ′ pm in two iterative processes where the propagation distances are d1 and d2. Results O i l i Note that the subscripts m and m′ represent the number of iterations in iterative processes. When pm is illuminated with incident plane wave, an approximation of g1 can be observed in the object plane at distance d1, which can be written by ′( , ) =  ( , ). ( ) λ , g x y p x y FrT 2 d m 1 1 1 0 0 1 ( ) 2 Likewise, we can obtain an approximation of g2 in the output domain at the distance d2, which can be given by ′( , ) = \| ′ ( , ) \| λ , ′ g x y p x y FrT [ ] d m 1 2 2 0 0 2 . Likewise, we can obtain an approximation of g2 in the output domain at the distance d2, which can be given by ′( , ) = \| ′ ( , ) \| λ , ′ g x y p x y FrT [ ] d m 1 2 2 0 0 2 . f h d d b f ll d f f h λ , g y p y d m 1 2 0 0 2 Sparse representation of the encrypted data can be successfully used for information authentication in some DRPE-based security systems14,21–25. In this step, two sparse phase functions p1s and p2s that used for the calculation of P1 and P2 can be randomly extracted from the outcomes of the iterative processes, i.e., pm, ′ ′ pm . Then the following step is numerical calculation of the two authenticated POMs P1 and P2 by using the obtained sparse POMs together with the QR codes. A modified double-phase retrieval algorithm (MDPRA) is proposed to achieve this purpose, where the sparse POMs and the QR codes are used as support constraints. In the MDPRA, let functions ( ) P n 1 and ( ) P n 2 respectively denote the two esti- mates for P1 and P2, where the superscript n represents the nth iteration of the algorithm. In the initial stage, two random POMs can be used as ( ) P1 1 and ( ) P2 1 , respectively. Results O i l i Due to its fast readability, great storage capacity and high damage tolerance capability, storing data in QR codes during the processes of optical encryption27–29 and authentication21–23 holds many practical advantages. Once the reconstructed QR codes are scanned by smartphones or tablets, the target images can be successfully revealed. y In our simulations, the pixel dimensions and the illumination wavelength are set as 8μm × 8μm and λ = 633 nm, respectively. Figure 3(a,c) show two 500 × 500 pixels binary images used as references for authentication. The propagation distances are given by d1 = 6 cm and d2 = 8 cm, respectively. Figure 3(b,d) demonstrate two sparse phase functions p1s and p2s that are randomly extracted from the outcomes of the iterative processes, p50 and ′ p50, for which both the percentages of the extracted pixels with respect to the pixel size of their originally recovered phase images are 28%.h p g y p g The two sparse POMs are used as two constraints in the proposed MDPRA, together with the three QR codes. The correlation coefficient (CC) is applied to evaluate the similarity between the recovered images ′ qi and their original images qi, which is defined by =  −  ′ − ′   −   ′ − ′  , ( ) { } { } { } E q E q q E q E q E q E q E q CC [ ] [ ] 4 i i i i i i i i 2 2 ( ) 4 where E[] denotes the expected value operator. The CC values get the maximum value of 1 if ′ qi are perfectly correlated with qi. Figure 4(a) shows the relation between number of iterations and CC values (between qi and ′ qi ), where we set the parameters as z0 = 8 cm, z1 = 12 cm, z2 = 20 cm and z3 = 30 cm. It can be seen from Fig. 4(a) that the CC value increases as the number of iterations increases. At the beginning iterations, the three curves shown in Fig. 4(a) overlap almost completely, which implies that the three recovered QR codes have almost identical CC values at the first several iterations. After 100 iterations, the CC values increase very slightly. Results O i l i The QR codes, i.e., q1, q2 and q3, are the three amplitude constraints in the output planes with respect to different distances from the second POM ( ) P n 2 , i.e., z1, z2 and z3. 2 Note that the final solutions for P1 and P2 are ( + ) P N 1 1 and ( + ) P N 2 1 if the iterative stops after N iterations. The obtained two POMs P1 and P2 require authentication before being applied for optical image retrieval. For brevity, only the identifying of P1 is explained. The reconstructed signal from P1 given by ( , ) = \| ( , ) \| λ , g x y P x y FrT [ ] s d 1 1 1 1 0 0 1 will be compared with the original image g1, by nonlinear correlation in our proposal, where the two parameters d1 and λ can be used as keys for authentication. The authen- tication method is described as follows25,26: µ ν µ ν ( , ) = ( , ) ( , ) , ( ) ω− NC x y c c IFT[ ] 3 1 2 ( ) 3 Scientific Reports \| 5:15668 \| DOI: 10.1038/srep15668 2 www.nature.com/scientificreports/ Figure 2. Storing three target images in QR codes. (a) Girl, (b) Telescope, (c) input text information. (d–f) their respective QR codes. Photographs taken by author. Figure 2. Storing three target images in QR codes. (a) Girl, (b) Telescope, (c) input text information. (d–f) their respective QR codes. Photographs taken by author. where IFT[•] denotes inverse Fourier transform and ω defines the strength of the applied nonlinearity. Function c(μ, v) is given by µ ( , ) =  ( , )⋅  ( , ) ⁎ { } c v g x y g x y FT FT s 1 1 , where FT[•] denotes Fourier transform. Experimental Simulations and performance analyses. Figure 2(a,b) are two 500 × 500 color images, which can be respectively inserted into the QR codes shown in Fig. 2(d,e) via hyperlinks. When a user scans the QR code containing the hyperlink which automatically redirects the user to the image. Figure 2(c) shows input the input text information (NUS stands for National University of Singapore) and its respective QR code is presented in Fig. 2(f). All of the QR Codes have the size of 500 × 500. Results O i l i As expected, two authenticated POMs ( + ) P n 1 1 and ( + ) P n 2 1 can be generated after the nth iteration, which require authentication before being used for image recon- struction. Evaluation of the correlation outputs can be implemented by using peak-to-correlation (PCE)23, which is defined as the ratio between the maximum intensity peak value and the total energy of the output plane, usually indicates the sharpness and height of the output correlation peak. Figure 4 shows Scientific Reports \| 5:15668 \| DOI: 10.1038/srep15668 3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Secret images used as references for authentication and their respective sparse POMs. (a) g1; (b) phase distribution of p1s; (c) g2; (d) phase distribution of ′ p s 1 . Figure 3. Secret images used as references for authentication and their respective sparse POMs. (a) g1; (b) phase distribution of p1s; (c) g2; (d) phase distribution of ′ p s 1 . Figure 4. Performance of the proposed iterative algorithm. (a) Relation between CC and number of iterations and (b) the PCE curves versus the number of iterations. Figure 4. Performance of the proposed iterative algorithm. (a) Relation between CC and number of iterations and (b) the PCE curves versus the number of iterations. the two PCE curves obtained by using all correct authentic keys and the nonlinearity strength ω = 0.4. Different from the PCE curve corresponding to P2, the curve with respect to P1 rises rapidly during the first twenty iterations. After that, the PCE values increased slowly and then reaches a plateau (0.2846) at 145 iteration. However, the PCE values obtained with the second POM P2 moves up and down in a limited range. It reaches its maximum (0.0390) at 99 iterations. In order to obtain high-quality correla- tion peak intensity in authentication, the POMs generated after 99 iterations are chosen as the two POMs the two PCE curves obtained by using all correct authentic keys and the nonlinearity strength ω = 0.4. Different from the PCE curve corresponding to P2, the curve with respect to P1 rises rapidly during the first twenty iterations. After that, the PCE values increased slowly and then reaches a plateau (0.2846) at 145 iteration. However, the PCE values obtained with the second POM P2 moves up and down in a limited range. It reaches its maximum (0.0390) at 99 iterations. Scientific Reports \| 5:15668 \| DOI: 10.1038/srep15668 Results O i l i In order to obtain high-quality correla- tion peak intensity in authentication, the POMs generated after 99 iterations are chosen as the two POMs Scientific Reports \| 5:15668 \| DOI: 10.1038/srep15668 4 www.nature.com/scientificreports/ Figure 5. Two obtained POMs and their respective diffractive patterns. Phase distributions (a) P1 and (b) P2, and their respective Fresnel diffraction intensity patterns (c) at distance d1; (d) at distance d2. Figure 5. Two obtained POMs and their respective diffractive patterns. Phase distributions (a) P1 and (b) P2, and their respective Fresnel diffraction intensity patterns (c) at distance d1; (d) at distance d2. Figure 6. Authentication of the POMs based on nonlinear correlation. (a) Correlation outputs corresponding to P1 and (b) P2. Figure 6. Authentication of the POMs based on nonlinear correlation. (a) Correlation outputs corresponding to P1 and (b) P2. used for authentication and image retrieval since the CC values increase very slowly after 100 iterations.ht The two POMs P1 and P2 designed for triple-image reconstruction and obtained after iteration num- ber of 99 are respectively shown in Fig. 5(a,b). They are required for authentication before being used for image reconstruction. The diffraction patterns of P1 and P2 are shown in Fig. 5(c,d), at the distances of d1 and d2 respectively, from which no information about the two secret binary images can be observed. The two POMs P1 and P2 designed for triple-image reconstruction and obtained after iteration num- ber of 99 are respectively shown in Fig. 5(a,b). They are required for authentication before being used for image reconstruction. The diffraction patterns of P1 and P2 are shown in Fig. 5(c,d), at the distances of d1 and d2 respectively, from which no information about the two secret binary images can be observed. When those two visually unrecognizable images [Fig. 5(c,d)] are respectively compared with the ref- erences, i.e., Fig. 3(a,c), by nonlinear correlation, sharp correlation peaks could be obtained as shown in Fig. 6(a,b), which implies that the two POMs are successfully authenticated with correct parameters. The security performance of the two POMs is further investigated. When only one of the POMs is placed in the optical scheme shown in Fig. 1, the diffraction patterns in the three object planes are shown in Fig. 7, from which no valuable information about the QR codes could be observed. Note that Fig. Discussions We developed an algorithm for jointly designing two POMs that allow for the encryption and noise-free retrieval of triple images. Compared with previous works, the proposed algorithm based on sparsity constraints and QR codes has the following features: We developed an algorithm for jointly designing two POMs that allow for the encryption and noise-free retrieval of triple images. Compared with previous works, the proposed algorithm based on sparsity constraints and QR codes has the following features: • This approach allows users to authenticate the two POMs without visual observation of those images used as references for authentication. Since a huge number of differently encoded POMs can be sent out through communication channels14, the identifying and matching of the POMs in a simple and direct manner can help increase efficiency. The MPRA with sparsity constraints may expect to be used in the computation of digital holograms and meta-holograms30–32 for image display and information authentication.h • This approach allows users to authenticate the two POMs without visual observation of those images used as references for authentication. Since a huge number of differently encoded POMs can be sent out through communication channels14, the identifying and matching of the POMs in a simple and direct manner can help increase efficiency. The MPRA with sparsity constraints may expect to be used in the computation of digital holograms and meta-holograms30–32 for image display and information authentication.h g g g g y • There is no problem of information disclosure in the proposed method. No information can be vis- ually observed from the two POMs and their respective diffraction patterns. Our method can also be used for encryption of three-dimensional objects.h • There is no problem of information disclosure in the proposed method. No information can be vis- ually observed from the two POMs and their respective diffraction patterns. Our method can also be used for encryption of three-dimensional objects.h yp j • It allows user to friendly access and readout with mobile devices. The target images can be revealed without visible loss of information due to the property of high damage tolerance capability of QR codes. Optically secured information retrieval can be realized when the two designed POMs manu- factured by a number of techniques including embossing on plastic films and encoding on photopol- ymer are placed in the proposed scheme shown in Fig. 1. Results O i l i 8(d–f), the two target color images and the text information can be retrieved without visible loss of information. Results O i l i 7 only shows the intensity distributions within the area of 4 mm × 4 mm on the output display. The results When those two visually unrecognizable images [Fig. 5(c,d)] are respectively compared with the ref- erences, i.e., Fig. 3(a,c), by nonlinear correlation, sharp correlation peaks could be obtained as shown in Fig. 6(a,b), which implies that the two POMs are successfully authenticated with correct parameters.h The security performance of the two POMs is further investigated. When only one of the POMs is placed in the optical scheme shown in Fig. 1, the diffraction patterns in the three object planes are shown in Fig. 7, from which no valuable information about the QR codes could be observed. Note that Fig. 7 only shows the intensity distributions within the area of 4 mm × 4 mm on the output display. The results Scientific Reports \| 5:15668 \| DOI: 10.1038/srep15668 5 www.nature.com/scientificreports/ Figure 7. Images reconstructed from only one of the POMs. (a–c) are the three images reconstructed from P1 in the different object planes (located from near to far from P1); (d–f) are the three images reconstructed from P2 in the three different object planes. Figure 7. Images reconstructed from only one of the POMs. (a–c) are the three images reconstructed from P1 in the different object planes (located from near to far from P1); (d–f) are the three images reconstructed from P2 in the three different object planes. shown that neither of the two POMs has the problem of untended information disclosure. The proposed security system can be regard as an information sharing system. Each POM will be sent to different receivers through communication channels. Only when a matched pair of POMs are used for decryption, the primary images can be retrieved by scanning the decrypted QR codes.f p y g y g yp Results for the reconstructed images from the two POMs P1 and P2 in different object planes using all the correct physical and geometric parameters are shown in Fig. 8(a–c), which have worse quality than the original images due to the effect of energy loss. The energy of the above three recovered images respectively account for about 85%, 84% and 79% of the total energy in their corresponding object planes. Smartphone was used to display the decrypted images by scanning the reconstructed QR codes. As demonstrated in Fig. Scientific Reports \| 5:15668 \| DOI: 10.1038/srep15668 Methods Double-phase retrieval algorithm with sparsity constraints. Let functions ( ) P n 1 and ( ) P n 2 respec- tively denote the two solutions for P1 and P2, where the superscript n represents the nth iteration of the algorithm. In the initial stage, two random POMs can be used as ( ) P1 1 and ( ) P2 1 , respectively. The QR codes, i.e., q1, q2 and q3, are the three amplitude constraints in the output planes with respect to different distances from the second POM ( ) P n 2 , i.e., z1, z2 and z3. The process proceeds as follows:hi ( )i 2h (i) The first POM ( ) P n 1 illuminated with incident plane wave is first Fresnel-transformed at the prop- agation distance z0. The resultant wave function can be written as = ( ) λ ( ) , ( ) U P FrT [ ] 5 n z n 0 1 0 ( ) 5 which is then multiplied by the second POM ( ) P n 2 and propagates forward to the output plane through distances zi (i = 1, 2, 3) to obtain new wave functions = × , ( ) λ ( ) , ( ) ( ) U P U FrT [ ] 6 i n z n n 2 0 i ( ) 6 where the coordinates are omitted for simplicity.f (ii) Replace amplitude parts of the diffraction space wave functions ( ) Ui n with amplitude constraints qi. Then the modified functions in the three output planes simultaneously transform back to the plane where the second POM ( ) P n 2 locates to get a new wave function ( ) U n 4 . ∑ = ×    , ( ) λ ( ) = , ( ) { } U q U IFrT PR 7 n i z i i n 4 1 3 i ( ) 7 where IFrT[•] represent inverse Fresnel transform and PR[•] denotes phase reservation, retaining the hase of a complex function but truncating its amplitude part. ( ) ( ) ( ) ( ) (iii) Update the two input POMs ( ) P n 1 and ( ) P n 2 with ( + ) P n 1 1 and ( + ) P n 2 1 . Discussions • It allows user to friendly access and readout with mobile devices. The target images can be revealed without visible loss of information due to the property of high damage tolerance capability of QR codes. Optically secured information retrieval can be realized when the two designed POMs manu- factured by a number of techniques including embossing on plastic films and encoding on photopol- ymer are placed in the proposed scheme shown in Fig. 1. 6 www.nature.com/scientificreports/ Figure 8. The reconstructed QR codes from P1 and P2 at different object planes and their respective retrieved images using a smartphone. Figure 8. The reconstructed QR codes from P1 and P2 at different object planes and their respective retrieved images using a smartphone. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 9. Flowchart of the proposed double-phase retrieval algorithm. Figure 9. Flowchart of the proposed double-phase retrieval algorithm. Figure 9. Flowchart of the proposed double-phase retrieval algorithm. = × ⊕ , ( ) ( + ) ( ) ( ) ⁎ P U r p PR{ [ ] } 10 n n n s 2 1 4 2 2 ( ) 10 where the superscript * denotes conjugation. p p j g (iv) Repeat steps (i)–(iii) until the preset threshold value is satisfied. Suppose the iteration process stop at the Nth iteration. The convergence of the algorithm is demonstrated by the relation between the CC values [between qi and their approximates ′ qi obtained by substituting the two POMs computed with Eqs. (9) and (10) into Eq. (1)] and the iteration numbers.l p p j g (iv) Repeat steps (i)–(iii) until the preset threshold value is satisfied. Suppose the iteration process stop at the Nth iteration. The convergence of the algorithm is demonstrated by the relation between the CC values [between qi and their approximates ′ qi obtained by substituting the two POMs computed with Eqs. (9) and (10) into Eq. (1)] and the iteration numbers.l p p j g (iv) Repeat steps (i)–(iii) until the preset threshold value is satisfied. Suppose the iteration process stop at the Nth iteration. The convergence of the algorithm is demonstrated by the relation between the CC values [between qi and their approximates ′ qi obtained by substituting the two POMs computed with Eqs. (9) and (10) into Eq. (1)] and the iteration numbers.l p q q To sum up, a flowchart of the iterative phase retrieval algorithm is depicted in Fig. 9. So far, we have obtained the two POMs P1 and P2, which require authentication before being applied for optical image retrieval and can be respectively represented by function ( + ) P N 1 1 and ( + ) P N 2 1 . In general, the amplitude parts of function ( ) Ui n would be closer to the constraints qi with the increasing of iterations until stagna- tion. After N iterations, the estimates of qi are denoted by ( + ) Ui N 1 . From Eqs. www.nature.com/scientificreports/ (1), (5) and (6), we can readily obtain ′ = ( + ) q U i i N 1 , which implies that the finally recovered images ′ qi could be expected to be closer to their original images qi by increasing the number of iterations before reaching plateau values. It should be pointed out that the MDPRA is designed under the framework of nonlinear double random phase encoding. Different from previously proposed methods of single image encoding16,17, here we encode three QR codes into two POMs in Fresnel domain. Two sparse POMs calculated from two ref- erence images for authentication are used as constraints in the iteration process. Methods First, we obtain a POM function ( ) r n 1 and then update the input POM ( ) P n 1 with ( + ) P n 1 1 using ( ) r n 1 , which can be written as = \| \| × , ( ) λ ( ) , ( ) ( ) { } r U U PR IFrT { PR[ ]} 8 n z n n 1 4 0 0 = \| \| × , λ ( ) , ( ) ( ) { } r U U PR IFrT { PR[ ]} n z n n 1 4 0 0 ( ) 8 = ⊕ , ( ) ( + ) ( ) P r p 9 n n s 1 1 1 1 ( ) 9 where the symbol ⨁ denotes a particular way of data embedding. For clarity, the calculation of ( + ) P n 1 1 described by Eq. (9) is explained. We first extract the non-zero pixels of POM p1s as the data background for POM ( + ) P n 1 1 , and then embed the pixel values of ( ) r n 1 into ( + ) P n 1 1 pixel by pixel but keep the back- ground unchanged. where the symbol ⨁ denotes a particular way of data embedding. For clarity, the calculation of ( + ) P n 1 1 described by Eq. (9) is explained. We first extract the non-zero pixels of POM p1s as the data background for POM ( + ) P n 1 1 , and then embed the pixel values of ( ) r n 1 into ( + ) P n 1 1 pixel by pixel but keep the back- ground unchanged.i ( ) g g To generate another POM used for next iteration, we first compute a POM function ( ) r n 2 by using = λ ( ) , ( + ) r P PR{FrT [ ]} n z n 2 1 1 0 and then obtain POM ( + ) P n 2 1 by Scientific Reports \| 5:15668 \| DOI: 10.1038/srep15668 7 References 1. Gerchberg, R. W. & Saxton, W. O. A practical algorithm for the determination of phase from image and diffraction plane pictures. Optik 35, 237–246 (1972). 2. Fienup, J. R. Phase retrieval algorithms: a comparison, Appl. Opt. 21, 2758–2769 (1982). g pp p 3. Faulkner, H. M. L. & Rodenburg, J. M. Movable aperture lensless transmission microscopy: a novel phase retrieval algorithm. Phys. Rev. Lett. 93, 023903 (2004). h h h f b d ff l l y 4. Johnson, E. G. & Brasher, J. D. 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Commu 4, 2808 (2013). 2. Huang, Y. D. et al. Aluminum plasmonic multicolor meta-hologram, Nano Lett. 15, 3122–3127 (2015). Acknowledgements g This work was supported by the National Natural Science Foundation of China (Grant Nos. 61575178 and 61205006), Singapore MINDEF-NUS Joint Applied R&D Cooperation Programme (JPP) Project: MINDEF/NUS/JPP/14/01/02. g This work was supported by the National Natural Science Foundation of China (Grant Nos. 61575178 and 61205006), Singapore MINDEF-NUS Joint Applied R&D Cooperation Programme (JPP) Project: MINDEF/NUS/JPP/14/01/02. References g g p p transform domain. Opt & Laser Technol 44, 2238–2244 (2012 p ( ) 13. Rajput, S. K. & Nishchal, N. K. Fresnel domain nonlinear optical image encryption scheme based on Gerchberg–Saxton phase- retrieval algorithm. Appl. Opt. 53, 418–425 (2014). p 3. Rajput, S. K. & Nishchal, N. K. Fresnel domain nonlinear optical image encryption scheme based on Gerchberg–Saxton phase retrieval algorithm. Appl. 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Scientific Reports \| 5:15668 \| DOI: 10.1038/srep15668 8 www.nature.com/scientificreports/ 0. Fienup, J. R. Phase retrieval algorithms: a personal tour [Invited]. Appl. Opt. 52, 45–56 (2013).i Phase retrieval algorithms: a personal tour [Invited]. Appl. Opt. 5 1. Markman, A., Javidi, B. & Tehranipoor, M. Photon-counting security tagging and verification using optically encoded QR codes IEEE Photon. J. 6, 6800609 (2014).h 2. Markman, A., Wang, J. & Javidi, B. Three-dimensional integral imaging displays using a quick-response encoded elemental image array. Optica 1, 332–335 (2014). y p 3. Wang, X., Chen, W. & Chen, X. Optical information authentication using compressed double-random-phase-encoded image and quick-response codes. Opt. Express 23, 6239–6253 (2015). q p p p 4. Wang, X., Chen, W. & Chen, X. Optical Encryption and Authentication Based on Phase Retrieval and Sparse Constraints. IEEE Photon. J. 7, 7800310 (2015). 5. Pérez-Cabré, E., Cho, M. & Javidi, B. Information authentication using photon-counting double-random-phase encrypted images. Opt. Lett. 36, 22–24 (2011). g p 26. Sadjadi, F. & Javidi, B. Physics of Automatic Target Recognition (Springer, New York, 2007). Scientific Reports \| 5:15668 \| DOI: 10.1038/srep15668 Additional Informationi Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Wang, X. et al. Optically secured information retrieval using two authenticated phase-only masks. Sci. Rep. 5, 15668; doi: 10.1038/srep15668 (2015). How to cite this article: Wang, X. et al. Optically secured information retrieval using two authenticated phase-only masks. Sci. Rep. 5, 15668; doi: 10.1038/srep15668 (2015). 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/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/ by/4.0/ Scientific Reports \| 5:15668 \| DOI: 10.1038/srep15668 9
https://openalex.org/W2062763964	https://zenodo.org/records/1795925/files/article.pdf	English	null	ABDOMINAL CERVICAL CESAREAN SECTION.	Journal of the American Medical Association	1,908	public-domain	4,781	McCaskey, Fort Wayne, Ind.: The clinical im- portance of metabolism has increased greatly during the past few years, and the entire question of metabolism has assumed a very important place. One must look to the test-tube for a solution of many problems in connection with clinical medi- cine. I was particularly impressed by what Dr. Joslin stated about the four cases of severe diabetes in which there was a chlorid retention with a serous effusion ; whereas in the mild cases there was none. This seems to me to point to the existence of an impaired renal permeability, at least for salines. It has been shown by von Noorden that as diabetes continues on for a period of time the sugar in the blood must be increased considerably over 0.1 per cent, before glycosuria takes place. Normally when there is over 0.1 per cent, gly- cosuria ensues. Whether this is conservative on the part of Nature, or whether it is the result of the chronic irritation, there is diminished permeability of the renal epithelium and the sugars and ehlorids gradually increase in the blood. y y p On the other hand, those operators who have dealt with women previously subjected to the more or less frequent examination of midwives, or to attempts at delivery at the hands of the general practitioner, in spite of good asepsis, still report a mortality of 10 per cent. g g y Dr. Elliot P. Joslin, Boston: With the diminution in the excretion of urine there is a corresponding diminution in thirst. Recently Dr. Goodall and I published in the Boston Medical and Surgical Journal personal observations on the manner in which acidosis affects young and old diabetic pa- tients. We found that the latter succumbed to a degree of acidosis which young individuals could stand well. When an acidosis developed in a person of advanced years, or one past middle life, the prospect for life was less than when a corre- sponding degree of acidosis occurred in a young individual. Presumably it depended on the condition of the kidneys and their vulnerability. It is also interesting to know that it was only in the young persons that recovery from diabetic coma often occurred. The young person was capable of voiding large quantities of urine and so was able to wash the acids from the body. Read in the Section on Obstetrics and Diseases of Women of the American Medical Association, at the Fifty-ninth Annual Ses- sion, held at Chicago, June, 1908. In one case nine quarts of urine were voided during recovery. Frank's idea was to operate extraperitoneally. He was followed by Veit and Sellheim, who attempt to mod- ify his method in another direction. While Frank incised both the abdominal parietes and uterus trans- versely immediately above the symphysis, Veit planned his incision through the median line, and Sellheim adopted my transverse fascial incision through the ab- domen. All three operators direct their efforts to pro- tecting the peritoneal cavity from infectious material— especially from the amniotie fluid—by operating either entirely extraperitoneally or subperitoneally. They do this by reflecting the peritoneum from the bladder and cervix, entering the uterus below the plica vesico-uterina (Frank, Sellheim). Dr. J. M. Allen, Liberty. Mo.: Over thirty years ago I found chronic inflammation and ulcération of the duodenum on postmortem examination in two patients that died from dia- betes mellitus. Since then I have made postmortems in five additional cases, and in each found chronic inflammation and ulcération of the duodenum and no other pathologic condition, except the ordinary changes to be found in the kidney. In all I have treated twenty-seven eases: the clinical symptoms of each were the same as those of which I made postmortem examina- tions. Hence I conclude that diabetes mellitus is the result of faulty metabolism in the duodenum, caused by the pathologic condition mentioned. The practical abilities of the old mas- ters in our profession are nowhere indicated with more force than in the diet list given to diabetics by Watson, Wood, Flint and others, which mainly consisted in albuminoids, thereby requiring but little function of the duodenum, and lessening the possibility of physiologic hyperemia. I read a paper on this subject and along these lines before the Ameri- can Medical Association at its semi-centennial meeting at Philadelphia. Dr. J. M. Allen, Liberty. Mo.: Over thirty years ago I found chronic inflammation and ulcération of the duodenum on postmortem examination in two patients that died from dia- betes mellitus. Since then I have made postmortems in five additional cases, and in each found chronic inflammation and ulcération of the duodenum and no other pathologic condition, except the ordinary changes to be found in the kidney. In all I have treated twenty-seven eases: the clinical symptoms of each were the same as those of which I made postmortem examina- tions. Hence I conclude that diabetes mellitus is the result of faulty metabolism in the duodenum, caused by the pathologic condition mentioned. The practical abilities of the old mas- ters in our profession are nowhere indicated with more force than in the diet list given to diabetics by Watson, Wood, Flint and others, which mainly consisted in albuminoids, thereby requiring but little function of the duodenum, and lessening the possibility of physiologic hyperemia. I read a paper on this subject and along these lines before the Ameri- can Medical Association at its semi-centennial meeting at Philadelphia. obstetric methods. By this means the placental site is most surely avoided, and the method also admits of a more rapid hemostasis, be- cause the suture of the uterine wall is performed by lig- atures inserted vertical to the course of the blood vessels. L l h b d l h Lately an attempt has been made to enlarge the scope of the Cesarean section with the view of further safety to the child's life by the introduction of cervical Cesa- rean section through the abdomen. This new method is to be credited to Frank of Cologne. The fundamental idea was not the same as that involved in the practice of making an incision into the lower uterine segment in order to avoid the placental site, but is based on the effort to extend the indication for Cesarean section to infected cases. The results of Cesarean section in the last few years have only become so excellent after we had learned to avoid unclean cases and to confine our operations to such cases as presented a more or less complete guaranty of good asepsis; therefore, those clinics especially whose material consisted exclusively of waiting women show brilliant results, with a maternal mortality of only zero to 1 per cent. p Dr. G. W. McCaskey, Fort Wayne, Ind.: The clinical im- portance of metabolism has increased greatly during the past few years, and the entire question of metabolism has assumed a very important place. One must look to the test-tube for a solution of many problems in connection with clinical medi- cine. I was particularly impressed by what Dr. Joslin stated about the four cases of severe diabetes in which there was a chlorid retention with a serous effusion ; whereas in the mild cases there was none. This seems to me to point to the existence of an impaired renal permeability, at least for salines. It has been shown by von Noorden that as diabetes continues on for a period of time the sugar in the blood must be increased considerably over 0.1 per cent, before glycosuria takes place. Normally when there is over 0.1 per cent, gly- cosuria ensues. Whether this is conservative on the part of Nature, or whether it is the result of the chronic irritation, there is diminished permeability of the renal epithelium and the sugars and ehlorids gradually increase in the blood. h p Dr. G. W. This operation has shown good re- sults in cases in which the indication for rapid delivery, especially in eclampsia and in prolapse of the cord, necessitates such measures. Likewise the true Cesarean section, that is, delivery through incision of the uterus by the abdominal route, necessitated by narrowing of the bony pelvis, or because of stenosis of the vagina and cervix, has undergone changes in the last decade that are by no means inconsiderable. obstetric methods. This operation has shown good re- sults in cases in which the indication for rapid delivery, especially in eclampsia and in prolapse of the cord, necessitates such measures. Likewise the true Cesarean section, that is, delivery through incision of the uterus by the abdominal route, necessitated by narrowing of the bony pelvis, or because of stenosis of the vagina and cervix, has undergone changes in the last decade that are by no means inconsiderable. by Among these new methods Cesarean section, per- formed according to Fritsch, is of advantage because it consists of transverse incision through the fundus. By this means the placental site is most surely avoided, and the method also admits of a more rapid hemostasis, be- cause the suture of the uterine wall is performed by lig- atures inserted vertical to the course of the blood vessels. Lately an attempt has been made to enlarge the scope of the Cesarean section with the view of further safety to the child's life by the introduction of cervical Cesa- rean section through the abdomen. This new method is to be credited to Frank of Cologne. The fundamental idea was not the same as that involved in the practice of making an incision into the lower uterine segment in order to avoid the placental site, but is based on the effort to extend the indication for Cesarean section to infected cases. The results of Cesarean section in the last few years have only become so excellent after we had learned to avoid unclean cases and to confine our operations to such cases as presented a more or less complete guaranty of good asepsis; therefore, those clinics especially whose material consisted exclusively of waiting women show brilliant results, with a maternal mortality of only zero to 1 per cent. y Among these new methods Cesarean section, per- formed according to Fritsch, is of advantage because it consists of transverse incision through the fundus. Downloaded From: http://jama.jamanetwork.com/ by a University of Arizona Health Sciences Library User on 05/30/2015 ABDOMINAL CERVICAL CESAREAN SECTION. The drainage entails a further risk, as it favors the likelihood of abdominal hernia as well as the consequence of abnormal adhe- sions in the cervical region with the abdominal wall, by no means inconsequential to the future well-being of the patient. I i pIt is known that pregnant women examine them- selves, or allow themselves to be examined by other women, and thus infection can occur. The course of the infection is then puzzling to us, because the fact of its examination by unskilled hands has been concealed. I i h i h , by women, and thus infection can occur. The course of the infection is then puzzling to us, because the fact of its examination by unskilled hands has been concealed. It is always well when dealing with febrile patients, or even with those in whom no fever is present, to con- sider them as infected, or at least as suspicious, provid- ing we elicit the fact that the woman has been in labor for a long time or has been subjected to repeated exam- inations on the part of physicians or midwives whose asepsis is not reliable. If with these precautions we undertake Cesarean section, no matter by what method, there is no reason why a considerable percentage should not have a happy outcome, simply because no infection was present. On the other hand, it is equally true that in the fatal cases death might have occurred even if the delivery had been obtained through perforation instead of by Cesarean section, because we are dealing with a true, septic, progressive infection. But my standpoint is that it is not a matter of indifference whether the uterus of a febrile patient is left untouched or whether a complicated wound is made. The gravity of the prog- nosis is doubtless increased by the operation. h di i f i p It is questionable whether we are justified in exposing the mother to all the above dangers in order merely to save the life of the child, which, as we know, is ex- tremely jeopardized in the presence of infected amniotie fluid. ABDOMINAL CERVICAL CESAREAN SECTION. it is to distinguish between true infection and sapremic intoxication, or let us say for the sake of brevity, to distinguish dangerous from harmless microbie invasion sub partu, and, therefore, be in a position to form a correct prognosis. For a true infection may remain localized and consequently lead to a favorable outcome, while a sapremic intoxication may end fatally by pro- ducing general poisoning. The sharp distinction be- tween infection and intoxication is theoretic and re- quires revision. it is to distinguish between true infection and sapremic intoxication, or let us say for the sake of brevity, to distinguish dangerous from harmless microbie invasion sub partu, and, therefore, be in a position to form a correct prognosis. For a true infection may remain localized and consequently lead to a favorable outcome, while a sapremic intoxication may end fatally by pro- ducing general poisoning. The sharp distinction be- tween infection and intoxication is theoretic and re- quires revision. quires It is impossible in cases of fever sub partu to an- nounce with certainty that this case is one of septic infection and, therefore, extremely serious, or that case is sapremic and, therefore, harmless. It is true that the experienced diagnostician will be guided in some cases by the general impression obtained from seeing the febrile patient, but such impressions can be decep- tive. The simplest ease would be one in which we know how the pregnant woman has been treated in the last few days preceding labor. For instance, when one is dealing with waiting women, who for many days before labor have been kept scrupulously clean, and during labor have either not been examined at all or only under conditions of strictest asepsis. If in such cases a rise of temperature sub partu occurs one can assume that this in general is of harmless nature; but even then it is possible to he deceived, as I have seen from personal experience. Sellheim has frankly acknowledged that he made a mistake in one fatal case by omitting drainage, but experience has shown that in most cases such drainage is entirely unnecessary. The same difficulty which con- fronts us in deciding the question whether in a given febrile case infection is present or not confronts us in deciding whether the wound is to be left open or the pef- itoneal cavity completely closed. ABDOMINAL CERVICAL CESAREAN SECTION. I would like to answer this question in the neg- ative and to advise that in all cases in which infection may surely be assumed, or in which a distinct suspicion of it is justified, to sacrifice the child by perforation, or where the pelvis is too narrow, to resort to Cesarean section, after the method of Porro. I believe that our modern attempts to save the child's life shoot beyond the mark, and, therefore, at least for the present, I must refrain from endorsing the extra- peritoneal Cesarean section. But I am not prepared to discard Frank's idea completely in those cases in which the fever in a pregnant woman gives the general impres- sion that the rise of temperature is harmless. I would advise, however, not to operate subperitoneally, but transperitoneally. p y My experience in the realm of abdominal surgery has given me great confidence in the capacity of the undam- aged peritoneum to destroy bacteria. It is well known that the peritoneum possesses these faculties to a much higher degree than the widely opened connective tissue surfaces. y p In this connection it is not alone the direct infection of the peritoneum with bacteria that must be taken into consideration, but also the fact that stagnating blood in both the uterine wound and in the uterine cavity fur- nishes a nutritive medium for the growth of the bacteria. As important as it is to protect the peritoneal cavity from infected amniotic fluid, which is quite feasible by means of the above mentioned methods of Cesarean sec- tion, I am unwilling to acknowledge that this suffices to prevent fatal peritonitis. The same applies to the wound of the abdominal wall, which is the more imper- iled the more complicated this wound is. To these objections others must be added. The fundamental thought of Frank is to protect the peritoneal cavity against the infectious wound area. The question then arises, "Is this technically possible?" y p In this connection it is not alone the direct infection of the peritoneum with bacteria that must be taken into consideration, but also the fact that stagnating blood in both the uterine wound and in the uterine cavity fur- nishes a nutritive medium for the growth of the bacteria. 1. Reported from my clinic by Hoehne. ABDOMINAL CERVICAL CESAREAN SECTION. ( , ) The same result is readied by transperitoneal opera- tion, the parietal peritoneum being incised above the bladder. The peritoneum covering the cervix is then severed, and before the uterus is opened the visceral and the parietal peritoneal surfaces are united either by suture or forceps (Veit, also Frank and Sellheim). b f h d f h d J. PFANNENSTIEL, M.D. Professor of Obstetrics and Gynecology at the University of Kiel. KIEL, GERMANY. Obstetric therapy during the last twenty years has entered a new phase by the substitution of obstetric pubiotomy in the place of symphyseotomy. In the same way those methods of delivery which aim to extract the child by means of incision of the uterus have been markedly improved and enlarged. p ( ) A number of patients have recovered after this method of operation. Of course it is questionable whether these patients would not have recovered just as well if the classic Cesarean section had been employed; the results, however, have not appealed to me in favor of this idea. Baumm and Sellheim have already reported deaths due to sepsis. y p g The so-called vaginal Cesarean section of D\l=u"\hrssen, better termed hysterotomy, has opened a large field in sepsis. I do not doubt that other, as yet unpublished, case« had a fatal outcome. We are all well aware how difficult Complete subperitoneal operation, such as is recom- mended by Frank and Sellheim, is at times not feasible, as I have found by experience, and as is acknowledged by both these authors. They, therefore, advise in such cases—and Veit has adopted this in his longitu- dinal incision—cutting through the parietal peritoneum and the cervix, to undermine them, and to unite them by means of forceps. This produces a closed subperitoneal space, to which access to the cervix is obtained. Thus somewhat greater safety against infection of the peri- toneal cavity is secured. But this is not an absolute guaranty, as infectious bacteria are known to break through such artificial barriers. It is, therefore, logi- cal and right that Frank, who produces a broad skin and muscle wound, and, in addition, opens a large, sub- peritoneal, cellular area, should advocate open wound treatment and drainage. The abdominal wound is equally endangered if infection arises after my trans- verse fascial incision. oaded From: http://jama.jamanetwork.com/ by a University of Arizona Health Sciences Library User on 05/30/2015 ABDOMINAL CERVICAL CESAREAN SECTION. My operation excels that of Sellheim because of greater simplicity and the avoidance of all those objections previously discussed in criticising the extraperitoneal method of Frank and Veit. My operation also can be applied in cases in which the mother has an apparently harmless fever, and in which the life of the child must consequently receive due consideration. In these cases the oil injections are to be employed immediately after the extraction of the child. The application of the cervical Cesarean section is ex- tensive. First, in contracted pelvis, the indication for pubiotomy must, however, not be narrowed, as the excel- lent results recently obtained should be taken into con- sideration. Only in cases in which pubiotomy no longer promises a sure chance for the child is Cesarean section to be undertaken. Cases of stenosis due to scars or neo- plasms of the internal genitals stand on a similar plane as contracted pelvis, unless some radical tumor opera- tion (myoma) indicates a different technic. Further, cervical Cesarean section is applicable in cases of threat- ened rupture of the uterus with living child, unless an already existing infection is present as contraindica- tion. This is exclusive of the previously mentioned cases of contracted pelvis. Unlike Sellheim, I would like to emphasize the indications for vaginal Cesarean section, which are distinctly special. In fact, the results of this method have been good. g du g operation. Concerning the technic of the Cesarean section, I have rejected the extraperitoneal method of Frank, Veit and Sellheim; but the work of these operators has taught me that the classic Cesarean section, as well as the modification of Fritsch, can be improved by em- ploying the cervical incision. My technic which Î have reported2 resembles that of Sellheim, but differs from it basically in that I operate transperitoneal 1 v. bas ca y operate transperitoneal To recapitulate, my technic is as follows: A typical transverse incision, about 15 cm. long, is made at the level of the anterior superior iliac spines; the aponeu- rosis of the rectus muscle is reflected up to the symphy- sis and down to the umbilicus. The peritoneum is then incised longitudinally for a distance of about 10 cm. Then a careful incision of the cervix is made between the bladder reflection and the contraction ring. The peritoneum is carefully guarded against contamination of amniotic fluid by means of pads, etc. Read in the Section on Obstetrics and Diseases of Women of the American Medical Association, at the Fifty-ninth Annual Ses- sion, held at Chicago, June, 1908. 2. Zentralblatt f\l=u"\rGyn., 1908. No. 10. ABDOMINAL CERVICAL CESAREAN SECTION. A i i i th i l i g As important as it is to protect the peritoneal cavity from infected amniotic fluid, which is quite feasible by means of the above mentioned methods of Cesarean sec- tion, I am unwilling to acknowledge that this suffices to prevent fatal peritonitis. The same applies to the wound of the abdominal wall, which is the more imper- iled the more complicated this wound is. To these objections others must be added. The fundamental thought of Frank is to protect the peritoneal cavity against the infectious wound area. The question then arises, "Is this technically possible?" Attempts to protect the body against invasion of bac- teria within the abdominal cavity can be made by the injection of oil into the peritoneal cavity during the Cesarean section, as I have proposed, based on the ex- perimental work of Glimm. That this measure is capable of preventing fatal infection has been shown experimentally by Glimm as well as the fact that the injection of oil, at least into guinea-pigs, is harmless.1 My own clinical experience has as yet been too scanty to Downloaded From: http://jama.jamanetwork.com/ by a University of Arizona Health Sciences Librar permit of any final judgment, especially as I have been unable to apply the method in Cesarean section. So far as I am concerned, I would not hesitate to perform transperitoneal Cesarean section, combined with the injection of oil, in cases of apparently harmless fever and a living child. The proper dose consists of 200 c.c. of olive oil, which has been sterilized for half an hour on a water bath at a temperature of 100 C. (212 F.). This fluid is simply poured into the abdomen by means of an irrigator during the course of the operation. permit of any final judgment, especially as I have been unable to apply the method in Cesarean section. So far as I am concerned, I would not hesitate to perform transperitoneal Cesarean section, combined with the injection of oil, in cases of apparently harmless fever and a living child. The proper dose consists of 200 c.c. of olive oil, which has been sterilized for half an hour on a water bath at a temperature of 100 C. (212 F.). This fluid is simply poured into the abdomen by means of an irrigator during the course of the operation. said, more closely a physiologic process. ABDOMINAL CERVICAL CESAREAN SECTION. by pads, If one does not operate too early, that is, if one waits until the cervix has dilated and been partly absorbed, then the bladder rests merely against the vagina and no longer against the cervix. It is then unnecessary to separate the bladder from its underlying parts, as one has sufficient room to deliver the child by the direct cervical incision. g In conclusion I would say that the extraperitoneal method of Cesarean section should be discarded, because it is harmful to the mother and of uncertain value, at least in febrile cases, to the child. In contrast to this, transperitoneal cervical Cesarean section marks a dis- tinct progress. In some cases in which there is fever in the mother it is indicated, but must then be combined with a prophylactic injection of oil into the peritoneal cavity. Should the plica vesico-ttterina still be high in situa- tion, this .fold is incised, separated from the bladder and the bladder pushed down, exactly as in the abdom- inal operation for fibroid uterus. The child is now de- livered by means of lateral pressure or extracted by the feet. After a short pause, which is occupied with cleansing, changing pads, etc., the placenta is delivered either spontaneously or by means of pressure exerted on the fundus (through the abdominal wall) similar to its ordinary delivery. The cervical wound is sutured in two layers and a typical closure of the abdominal wall is made. I have employed this method so far in one case with recovery. Rubeska reports a similar case. The method has numerous advantages over the classical oper- ation of Saenger and that of Fritsch. oaded From: http://jama.jamanetwork.com/ by a University of Arizona Health Sciences Library User on 05/30/2015 ABDOMINAL CESAREAN SECTION, ABDOMINAL CESAREAN SECTION, AS PERFORMED AT THE SOCIETY OF THE LYING-IN HOS- PITAL OF THE CITY OF NEW YORK, WITH AN ANALYSIS OF 186 CASES. ROSS McPHERSON, M.D. Attending Surgeon. Lying-in Hospital. NEW YORK. AS PERFORMED AT THE SOCIETY OF THE LYING-IN HOS- PITAL OF THE CITY OF NEW YORK, WITH AN ANALYSIS OF 186 CASES. AS PERFORMED AT THE SOCIETY OF THE LYING-IN HOS- PITAL OF THE CITY OF NEW YORK, WITH AN ANALYSIS OF 186 CASES. ROSS McPHERSON, M.D. Attending Surgeon. Lying-in Hospital. NEW YORK. In presenting this paper for consideration, it is my intention to take up, so far as the time allowed me will permit, the question of abdominal Cesarean section, with regard to the indications for its employment, the choice of time for its use, the technic of the operation, and the results obtained. In a general paper of this description, it would be trying to the patience, as well as unsuitable, to give more than a passing consideration to the details of the subject, the main object being to show the results obtained from a large number of sec- tions, together with certain especial points in the technic of the operation. Saenger 1. All the advantages inherent in my transverse fascial incision, that is, (a) the morbidity, and with it the mortality, has diminished, because the uterus remains in "ila, and the rest of the peritoneal cavity is guarded from contamination from without; (b) the danger of a ventral hernia is eliminated; (c) the patient is able to leave her bed early (in normal cases from the third day on), which shortens the convalescence con- siderably. p The series of cases reported comprise the total num- ber done in the Lying-in Hospital during the eighteen years of its existence, and are, therefore, in no sense of the word, selected cases. It will be my endeavor to show some of the dangers, as well as the advantages of this method of delivery. y 2. Further advantages are due to the situation of the uterine wound. In most cases the hemorrhage is min- imal—with the exception of placenta prœvia. (a) No large vessels are situated within reach of the incision ; (b) detachment of the placenta is entirely left to Nature. The course of labor resembles, as Sellheim has justly
https://openalex.org/W2897498865	https://europepmc.org/articles/pmc6195607?pdf=render	English	null	The role of MHC supertypes in promoting trans-species polymorphism remains an open question	Nature communications	2,018	cc-by	4,678	1 Institute of Environmental Sciences, Jagiellonian University, ul. Gronostajowa 7, 30-387 Kraków, Poland. 2 Centre for Ecology and Evolution in Microbial model Systems – EEMiS, Linnaeus University, 39182 Kalmar, Sweden. 3 School of Biological, Earth & Environmental Sciences, University College Cork, Cork T23 N73K, Ireland. 4 Marine Institute, Furnace, Newport, Co. Mayo F28 PF65, Ireland. 5 Evolutionary Biology Group, Institute of Environmental Biology, Adam Mickiewicz University, ul. Umultowska 89, Poznan, Poland. Correspondence and requests for materials should be addressed to J.R. (email: jradwan@amu.edu.pl) CORRESPONDENCE CORRESPONDENCE The role of MHC supertypes in promoting trans-species polymorphism remains an open question DOI: 10.1038/s41467-018-06821-x OPEN OPEN Maciej J. Ejsmond1,2, Karl P. Phillips3,4, Wiesław Babik1 & Jacek Radwan 5 I I n a recently published paper on the evolution of the vertebrate major histocompatibility complex (MHC), Lighten et al.1; (henceforth ‘Lighten et al.’) set out to explain an apparent incompatibility between the dynamic nature of host-parasite coevolution, which could accelerate MHC allele turnover2,3, and the apparent long-term persistence of allelic lineages that may underpin MHC trans-species polymorphism (TSP4,5). TSP arises when multiple allelic lineages that originated in an ancestral species are maintained in descendant species. TSP is usually a transient phenomenon, but at the MHC, TSP is common and seemingly long-term, leading to profound discordance between genealogies of MHC alleles and species phylogenies4,5. Lighten et al. offer a scenario in which several functionally divergent MHC ‘supertypes’ (clusters of MHC alleles with similar physi- cochemical properties at their antigen-binding sites6) are main- tained by balancing selection, whereas functionally similar alleles within supertypes undergo fast turnover. The scenario is based on an empirical ﬁnding that population-genetic structure by super- types is signiﬁcantly lower than allele-based null expectations, and on simulations modelling MHC alleles as coordinates in paratope space. Here, we argue that the empirical patterns do not support a major role of supertypes in the maintenance of TSP, and that the theoretical arguments provided by Lighten et al. are based on disputable assumptions. supertype loss in either species. The tree Lighten et al. present in their Supplementary Fig. 2 is based on amino acid sequences at 15 codons under signiﬁcant positive selection. Although such tar- geted trees are useful in studying MHC evolution, longer sequences will provide a more rigorous test of a phylogenetic property such as TSP. We therefore constructed a phylogeny based on Lighten et al.’s full nucleotide sequences (Fig. 1b). We did not observe the predicted pattern: most supertypes were far from monophyletic, which suggests convergent origin rather than common ancestry of alleles within supertypes. Lighten et al. argue that gene conversion between alleles of different supertypes has broken down monophyly for all supertypes except ST9 (legend to Supplementary Fig. 2 in ref. 1), but do not explain how polyphyly of supertypes can be reconciled with their proposed role in TSP. Instead, we think that different allelic clades of the same super- type would readily ﬁx in different species, even under strong selection maintaining supertypes themselves, thus erasing TSP. NATURE COMMUNICATIONS \| (2018) 9:4362 \| DOI: 10.1038/s41467-018-06821-x \| www.nature.com/naturecommunications The role of MHC supertypes in promoting trans-species polymorphism remains an open question DOI: 10.1038/s41467-018-06821-x OPEN used to support balancing selection acting on supertypes, a deﬁcit of “supertype homo- zygotes”, is unconvincing because the authors could not assign alleles to loci (genotypes consisted of up to nine alleles, implying a minimum of ﬁve co-amplifying loci in some individuals), meaning that true zygosity is not known. Furthermore, the unique properties of ST9 also mean that the observed homo- zygosity deﬁcit needs to be treated cautiously (6.7% of individuals are ST homozygotes in the full dataset, but 29.1% if ST9 and all its alleles are removed). few alleles per supertype (Fig. 2a). With a mutation rate = 10−4, which is in line with the upper limit of reported per locus MHC mutation rates9,10, all supertype diversity was lost within a few thousand generations. Thus, under more realistic mutation parameters, the model of Lighten et al. does not generate realistic MHC diversity. Based on their simulations, the authors argue that TSP arises because supertypes are stable in paratope space, and show only slight “wobbles” in the position of their centroids over time. However, our versions of the simulations showed that the long- term stability of supertypes hinges on a crucial feature of the published simulation code that was not mentioned in the verbal model description: in addition to point mutations in pathogens, 1% new parasite genotypes, distributed randomly in epitope space, enter the population at each generation (i.e. ≈100 entirely new, typically very divergent haplotypes in a population of 10,000 individuals). When we used the original parameters of Lighten et al. but ‘switched off’ host-parasite coevolution by recruiting parasites to the next generation at random (i.e. no selection on parasite genotypes), this seeding alone was sufﬁcient to maintain supertype and allelic diversity similar to that reported by Lighten et al. (Fig. 2b). It thus appears that this easily overlooked feature of unclear biological context (it is debatable whether 100 entirely new, divergent parasite genotypes are likely to enter any popu- lation every generation), and not host-parasite coevolution, drove the dynamics of the original simulations. The theoretical argument of Lighten et al. is based on simu- lations suggesting that supertypes may persist nearly indeﬁnitely without much change in their antigen-binding properties, shown by stable positions of supertypes in paratope space. In contrast, alleles persist for very short times, being replaced by new mutant alleles within the same supertype (Fig. 4 of Lighten et al.). The role of MHC supertypes in promoting trans-species polymorphism remains an open question DOI: 10.1038/s41467-018-06821-x OPEN Our attention was drawn by ST9, the only supertype that forms a well-supported clade in the phylogenetic tree (Fig. 1b). Diver- gence within this clade is relatively shallow, indicating rapid coalescence, and a long branch separates ST9 clade from other MHC alleles. This pattern is strikingly different from the rest of the tree (Fig. 1b). Lighten et al. note a correlation between the number of ST9 alleles and the number of microsatellite alleles within populations and suggest that ST9 alleles are subject to drift. Thus, ST9 may represent a specialized MHC locus that evolves differently from other MHC supertypes. In support of this, alleles comprising ST9 map best to a different scaffold in the guppy genome (LG18: 22 Mb) than alleles of the other supertypes: (unplaced scaffold 55 kb or unplaced scaffold 1.5 kb). Impor- tantly, our analyses showed that the central observation of Lighten et al.—of weaker population-genetic structuring of supertypes compared to structuring of MHC alleles—appears to be entirely driven by ST9, as indicated by jack-knife removal of each supertype (Supplementary Figs 1, 2). The effect of ST9 does not appear to be solely due to its high frequency in the dataset TSP is a feature of gene genealogy and requires phylogenetic analysis to demonstrate it. In a phylogeny, TSP is detected as monophyletic groups of alleles with each group represented in multiple species, or by extensive paraphyly should some lineages be lost in one or more descendent species. Despite the com- plexities of molecular evolution (recombination, gene conversion) that often cause departures of the true genealogy from a bifur- cating tree, such TSP-diagnostic patterns are commonly detected in the MHC5,7. If TSP is caused by long-term stability of MHC supertypes, the phylogeny of MHC alleles from two sister species should be characterized by predominantly monophyletic super- types present in both species (Fig. 1a), barring occasional 1 NATURE COMMUNICATIONS \| (2018) 9:4362 \| DOI: 10.1038/s41467-018-06821-x \| www.nature.com/naturecommunications CORRESPONDENCE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06821-x 0.03 a b * 1 2 4 3 6 5 8 7 10 9 11 13 12 14 15 Supertype Fig. 1 Predictions and results of phylogenetic analysis. a The hypothetical pattern expected if balancing selection on supertypes maintains TSP, but alleles within supertypes experience rapid turnover. Hypothetical supertypes are marked with diamonds of the same colour, and the species-speciﬁc branches are in turquoise and deep blue. The role of MHC supertypes in promoting trans-species polymorphism remains an open question DOI: 10.1038/s41467-018-06821-x OPEN Supertype lineages should be shared between species, but sharing of identical alleles (dashed line) should be rare. b Neighbour- joining tree of 539 MHC class II exon 2 alleles reported by Lighten et al.1. The tree was constructed from a matrix of Jukes–Cantor distances calculated using all 209 nucleotide positions. Alleles from each supertype are marked with the same colour as in Lighten at al. Supertype 9 is marked yellow; the asterisk indicates that it is the only monophyletic supertype (bootstrap support 97%) b a 0.03 Fig. 1 Predictions and results of phylogenetic analysis. a The hypothetical pattern expected if balancing selection on supertypes maintains TSP, but alleles within supertypes experience rapid turnover. Hypothetical supertypes are marked with diamonds of the same colour, and the species-speciﬁc branches are in turquoise and deep blue. Supertype lineages should be shared between species, but sharing of identical alleles (dashed line) should be rare. b Neighbour- joining tree of 539 MHC class II exon 2 alleles reported by Lighten et al.1. The tree was constructed from a matrix of Jukes–Cantor distances calculated using all 209 nucleotide positions. Alleles from each supertype are marked with the same colour as in Lighten at al. Supertype 9 is marked yellow; the asterisk indicates that it is the only monophyletic supertype (bootstrap support 97%) (Supplementary Table 1), which further suggests that this supertype may be experiencing different evolutionary pressures from the other supertypes. The lower genetic structuring of supertypes is therefore not a general feature of the dataset. Another argument which Lighten et al. used to support balancing selection acting on supertypes, a deﬁcit of “supertype homo- zygotes”, is unconvincing because the authors could not assign alleles to loci (genotypes consisted of up to nine alleles, implying a minimum of ﬁve co-amplifying loci in some individuals), meaning that true zygosity is not known. Furthermore, the unique properties of ST9 also mean that the observed homo- zygosity deﬁcit needs to be treated cautiously (6.7% of individuals are ST homozygotes in the full dataset, but 29.1% if ST9 and all its alleles are removed). (Supplementary Table 1), which further suggests that this supertype may be experiencing different evolutionary pressures from the other supertypes. The lower genetic structuring of supertypes is therefore not a general feature of the dataset. Another argument which Lighten et al. NATURE COMMUNICATIONS \| (2018) 9:4362 \| DOI: 10.1038/s41467-018-06821-x \| www.nature.com/naturecommunications The role of MHC supertypes in promoting trans-species polymorphism remains an open question DOI: 10.1038/s41467-018-06821-x OPEN Parasite haplotypes (yellow) and host supertypes (black and other colours) are represented as coordinates in 1000 × 1000 grid, reﬂecting their functional properties (the closer a parasite is to the host, the more likely is a successful host immune response). Each panel shows how changes in speciﬁed parameters in the model, compared to the parameters used by Lighten et al. to produce their Fig. 4, affect the outcome (parameters other than stated in the description are as in Lighten et al.). a Mutation rate set to 10−2 for pathogens and 10−3 for hosts, population size increased to 100,000. In contrast to the results reported by Lighten et al. using much higher mutation rates, the effective number of alleles maintained in a population is small. b Selection on parasites (but not on hosts) ‘turned off’. This simulation gives the most similar outcome to that reported by Lighten et al., despite the lack of host-parasite coevolution (see Supplementary Fig. 6 for the scenario with no random pathogen genotypes added). c Simulations that do not seed each pathogen generation with ≈100 new genotypes, but mutation parameters as in Lighten et al. Host-parasite coevolution utilizing mutational variance alone does not maintain several stable supertypes, even though the simulations started from creating a set of random MHC alleles and pathogens in the same way as Lighten et al. d Parameters as in c, but 10 independent parasites simulated. Several supertypes are observed at any time point, but they are not stable through time. Effective number of alleles (#alleles) has been calculated for a sample of 100 individuals to allow comparison with the results of Lighten et al. The simulations were written in MatLab and the algorithm follows that described by Lighten et al. The MatLab code is provided in our Supplementary Data 1 maintained was very low compared to that observed in natural populations (Supplementary Fig. 5). and stable persistence of monophyletic supertypes in paratope space without the need for seeding random parasite genotypes. We simulated 10 independent parasite pools, and observed multiple supertypes maintained with realistic numbers of alleles (Fig. 2d). However, the dynamics of these simulations was very different from long-term stability; instead, some supertypes walked large distances in paratope space (implying change in supertype identity), some supertypes were lost, and others bran- ched into daughter supertypes (Fig. 2d). Adding more parasite species did not qualitatively change this conclusion (Supple- mentary Fig. 4). The role of MHC supertypes in promoting trans-species polymorphism remains an open question DOI: 10.1038/s41467-018-06821-x OPEN to produce their Fig. 4, affect the outcome (parameters other than stated in the description are as in Lighten et al.). a Mutation rate set to 10−2 for pathogens and 10−3 for hosts, population size increased to 100,000. In contrast to the results reported by Lighten et al. using much higher mutation rates, the effective number of alleles maintained in a population is small. b Selection on parasites (but not on hosts) ‘turned off’. This simulation gives the most similar outcome to that reported by Lighten et al., despite the lack of host-parasite coevolution (see Supplementary Fig. 6 for the scenario with no random pathogen genotypes added). c Simulations that do not seed each pathogen generation with ≈100 new genotypes, but mutation parameters as in Lighten et al. Host-parasite coevolution utilizing mutational variance alone does not maintain several stable supertypes, even though the simulations started from creating a set of random MHC alleles and pathogens in the same way as Lighten et al. d Parameters as in c, but 10 independent parasites simulated. Several supertypes are observed at any time point, but they are not stable through time. Effective number of alleles (#alleles) has been calculated for a sample of 00 individuals to allow comparison with the results of Lighten et al. The simulations were written in MatLab and the algorithm follows that described by Lighten et al. The MatLab code is provided in our Supplementary Data 1 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06821-x CORRESPONDENCE 1000 a 1000 5000 5000 10,000 10,000 15,000 15,000 Time Y-co-ordinate X-co-ordinate Time 500 500 150 100 0 50 # alleles 1 1 1000 b 1000 5000 10,000 15,000 Time Y-co-ordinate X-co-ordinate 5000 10,000 15,000 Time 500 500 150 100 0 50 # alleles 1 1 1000 b 1000 5000 10,000 15,000 Time Y-co-ordinate X-co-ordinate 500 500 1 1 b a X-co-ordinate e 5000 10,000 15,000 Time 150 100 0 50 # alleles 1000 c 1000 5000 10,000 15,000 Y-co-ordinate X-co-ordinate 5000 10,000 15,000 Time Time 500 500 150 100 0 50 # alleles 1 1 1000 1000 d 5000 10,000 15,000 Y-co-ordinate X-co-ordinate 5000 10,000 15,000 Time Time 500 500 150 100 0 50 # alleles 1 1 d c Fig. 2 Results of simulations based on the model proposed by Lighten et al. The role of MHC supertypes in promoting trans-species polymorphism remains an open question DOI: 10.1038/s41467-018-06821-x OPEN While the numbers of supertypes (~ 8) and alleles (~ 40 in a subsample of 100 individuals) in the simulated populations of Lighten et al. are comparable to those observed in natural populations, the MHC mutation rate assumed in the model was very high (~10−1). With simulated population sizes of the order of 103–104, this means that even under neutrality, the expected equilibrium het- erozygosity (H = 4Neµ/(1 + 4Neµ))8 would be ≈1. In order to investigate more realistic mutation rates, we ﬁrst reconstructed the model based on the description provided in the methods and supplementary materials of Lighten et al. and veriﬁed that we were able to recover their result (Supplementary Fig. 3). We then re-ran the simulations using host mutation rate = 10−3, but MHC polymorphism was not maintained. When we increased population size to 105 we observed several supertypes but very Without this repeated invasion of so many novel and highly divergent parasite genotypes each generation, we found that host- parasite coevolution led to strikingly different dynamics. All supertype diversity was lost, and there remained a single host supertype chasing a dominant parasite genotype through the whole epitope space (Fig. 2c). However, because Lighten et al. modelled only one parasite species, we investigated whether modelling multiple species could maintain MHC polymorphism NATURE COMMUNICATIONS \| (2018) 9:4362 \| DOI: 10.1038/s41467-018-06821-x \| www.nature.com/naturecommunications 2 CORRESPONDENCE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06821-x 1000 1000 1000 1000 1000 a b c d 1000 1000 1000 5000 5000 5000 5000 5000 10,000 10,000 10,000 10,000 10,000 15,000 15,000 15,000 15,000 15,000 Time Time Y-co-ordinate Y-co-ordinate Y-co-ordinate Y-co-ordinate X-co-ordinate X-co-ordinate X-co-ordinate X-co-ordinate Time 5000 10,000 15,000 Time 5000 10,000 15,000 Time 5000 10,000 15,000 Time Time Time 500 500 500 500 500 500 500 500 150 100 0 50 # alleles 150 100 0 50 # alleles 150 100 0 50 # alleles 150 100 0 50 # alleles 1 1 1 1 1 1 1 1 Fig. 2 Results of simulations based on the model proposed by Lighten et al. Parasite haplotypes (yellow) and host supertypes (black and other colours) are epresented as coordinates in 1000 × 1000 grid, reﬂecting their functional properties (the closer a parasite is to the host, the more likely is a successful host immune response). Each panel shows how changes in speciﬁed parameters in the model, compared to the parameters used by Lighten et al. TURE COMMUNICATIONS \| (2018) 9:4362 \| DOI: 10.1038/s41467-018-06821-x \| www.nature.com/naturecommunications References Phylogenetic analysis. The relationships between 539 MHC class II exon 2 alleles reported in the Supplementary Data 1 of Lighten et al.1 were reconstructed from f ll 209 b l id i h i hb J i i h d i MEGA 711 1. Lighten, J. et al. Evolutionary genetics of immunological supertypes reveals two faces of the Red Queen. Nat. Commun. 8, 1294 (2017). y g y p reported in the Supplementary Data 1 of Lighten et al.1 were reconstructed from full 209 bp nucleotide sequences using the neighbor Joining method in MEGA 711. The matrix of evolutionary distances was computed using the Jukes–Cantor method. The robustness of the relationships was assessed with 500 bootstrap replicates. 2. Takahata, N. & Nei, M. Allelic genealogy under overdominant and frequency- dependent selection and polymorphism of major histocompatibility complex loci. Genetics 124, 967–978 (1990). 3. Ejsmond, M. J. & Radwan, J. Red Queen processes drive positive selection on major histocompatibility complex (MHC) genes. PLoS Comput. Biol. 11, e1004627 (2015). Population genetics. We downloaded Lighten et al.’s population-genetic data and analysis scripts from Ben J. Ward’s GitHub repository for the paper (https://github. com/BenJWard/Supertypes_RedQueen_TSE) on 17/04/2017, and followed their annotated analysis to produce the data used for Supplementary Fig. 2 and the top- left panel of Supplementary Fig. 1. This analysis ﬁrst calculates the observed MHC DEST12 between all population pairs in the dataset, and uses each population’s mean pairwise DEST as the red, ‘observed’ dots in the plots. This is followed up by reassigning alleles to supertypes at random, keeping the number of alleles in each supertype constant, and then recalculating each population’s mean pairwise DEST. The means of 1000 repeats of this randomisation become the blue, ‘expected’ dots in the respective ﬁgures, with the standard deviations used for the blue dots’ error bars. Conceptually, the blue dots and their error bars represent a scenario in which there is no selection at the supertype level, i.e. supertype population genetics are a reduced-diversity reﬂection of allele-based population genetics. An observed supertype-based population structure stronger than this neutral expectation would imply diverging selection on supertypes, whereas lower population structure would imply balancing/stabilising selection. 4. Klein, J. Origin of Major Histocompatibility Complex polymorphism - the transspecies hypothesis. Hum. Immunol. 19, 155–162 (1987). 5. Klein, J., Sato, A. & Nikolaidis, N. MHC, TSP, and the origin of species: from immunogenetics to evolutionary genetics. Annu. Rev. Genet. References One population (Cumana, n = 6) was homozygous for ST13 and was removed from that supertype’s analysis. To 11. Kumar, S., Stecher, G. & Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874 (2016). 12. Jost, L. GST and its relatives do not measure differentiation. Mol. Ecol. 17, 4015–4026 (2008). compare the effects of removing each supertype, we calculated the mean and SD of the difference between the red and blue dots for each jack-knifed dataset (second column of Supplementary Table 1). We also calculated the Spearman correlation coefﬁcient between each jack-knifed dataset’s red dots and the red dots of the full dataset (third column of Supplementary Table 1). Acknowledgements We thank Mateusz Konczal, Magda Herdegen-Radwan and Joshka Kaufman for their comments on earlier versions of this manuscript. This research was partly supported by the Jagiellonian University (grants, DS/WB/INoS/756/2018, DS/WB/INoS/757/2018) and Adam Mickiewicz University in Poznan (DS/S/P-B/48). To assess the degree to which each supertype’s jack-knifed dataset might be inﬂuenced by supertype membership size (number of alleles) or supertype frequency, we performed three additional sets of randomised deletions for each supertype. In the ﬁrst, we repeatedly removed all occurrences of a random subset of unique nucleotide sequences from the dataset, corresponding in size to the number of sequences in the focal supertype (columns 7–8 of Supplementary Table 1). In the second, we made randomized deletions that matched the frequency of the focal supertype but without systematically removing any particular nucleotide sequences (columns 9–10 of Supplementary Table 1). The third was a spatially structured version of the second, matching the number of random deletions within each population to the frequency of the focal supertype within each population (columns 11–12 of Supplementary Table 1). The role of MHC supertypes in promoting trans-species polymorphism remains an open question DOI: 10.1038/s41467-018-06821-x OPEN Supertypes were more stable in paratope space at more realistic mutation rates, but the number of alleles The above analyses indicate that under more realistic para- meters of host-parasite coevolution, the model of Lighten et al. does not provide support for the scenario of long-term main- tenance of stable supertypes, proposed by the authors as an explanation of TSP. We conclude that the proposition of Lighten et al.—that supertypes maintain TSP—is not convincingly sup- ported by empirical evidence, and awaits ﬁrm theoretical foun- dation. What maintains TSP, despite constant pressure for MHC novelty under host-parasite coevolution3, thus remains an open question. NATURE COMMUNICATIONS \| (2018) 9:4362 \| DOI: 10.1038/s41467-018-06821-x \| www.nature.com/naturecommunications 3 3 Author contributions J.R. and M.E. designed the simulations and M.E. wrote the code and ran the simulations; W.B. carried out phylogenetic analyses; K.P.P. carried out population-genetic analyses; J. R. drafted the paper with all authors contributing to writing. Additional information Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 018-06821-x. Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 018-06821-x. Simulations. To analyze the theoretical results of Lighten et al. we ﬁrst reconstructed the simulation model of immune gene evolution presented in their work1; the detailed description of the model can be found in the Lighten et al. We then tested their model in an extended parameter space of pathogen mutation rate, host mutation rate and population size, as speciﬁed in our ﬁgures. Additionally, we ran simulations in three scenarios not considered by Lighten et al. In the ﬁrst, we removed the feature of Lighten et al.'s simulations that seeded pathogen population with 1% of random genotypes (‘random’ meaning randomly distributed in physicochemical parameter space) every host generation. The second was a neutral scenario in which parasites were sampled randomly every generation, i.e. without taking into account their ability to infect hosts, thereby preventing parasite adaptation. The third extended simulations of Lighten et al. by including more than one pathogen co-evolving with one host species. The algorithm of the simulation model used in our work can be found in Supplementary Data 1. Competing interests: The authors declare no competing interests. Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ reprintsandpermissions/ 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. Code availability. The code for our simulations is available in Supplementary Data 1. CORRESPONDENCE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06821-x References 41, 281–304 (2007). 6. Doytchinova, I. A. & Flower, D. R. In silico identiﬁcation of supertypes for class II MHCs. J. Immunol. 174, 7085–7095 (2005). p p p p p mean pairwise DEST as the red, ‘observed’ dots in the plots. This is followed up by reassigning alleles to supertypes at random, keeping the number of alleles in each supertype constant, and then recalculating each population’s mean pairwise DEST. The means of 1000 repeats of this randomisation become the blue, ‘expected’ dots in the respective ﬁgures, with the standard deviations used for the blue dots’ error bars. Conceptually, the blue dots and their error bars represent a scenario in which there is no selection at the supertype level, i.e. supertype population genetics are a d d di it ﬂ ti f ll l b d l ti ti A b d 7. Garrigan, D. & Hedrick, P. W. Perspective: Detecting adaptive molecular polymorphism, lessons from the MHC. Evolution 57, 1707–1722 (2003). 8. Kimura, M. The neutral theory of molecular evolution. (Cambridge University Press, 1983). 9. Melvold, R. W., Wang, K. & Kohn, H. I. Histocompatibility gene mutation rates in the mouse: a 25-year review. Immunogenetics 47, 44–54 (1997). supertype-based population structure stronger than this neutral expectation would imply diverging selection on supertypes, whereas lower population structure would imply balancing/stabilising selection. 10. Högstrand, K. & Böhme, J. A determination of the frequency of gene conversion in unmanipulated mouse sperm. Proc. Natl Acad. Sci. USA 91, 9921–9925 (1994). p y g g We then repeated these analyses removing each supertype in turn, i.e. removing all instances of all alleles in the focal supertype, and thereby any individuals that were homozygous for the focal supertype. One population (Cumana, n = 6) was homozygous for ST13 and was removed from that supertype’s analysis. To compare the effects of removing each supertype, we calculated the mean and SD of the difference between the red and blue dots for each jack-knifed dataset (second column of Supplementary Table 1). We also calculated the Spearman correlation coefﬁcient between each jack-knifed dataset’s red dots and the red dots of the full dataset (third column of Supplementary Table 1). We then repeated these analyses removing each supertype in turn, i.e. removing all instances of all alleles in the focal supertype, and thereby any individuals that were homozygous for the focal supertype. Data availability y The study used data published by Lighten et al.1—see information therein for data availability. Derived datasets and R codes used for the population genetics analyses are available from K.P.P. (karl.p.phillips@gmail.com). Received: 15 January 2018 Accepted: 27 September 2018 Received: 15 January 2018 Accepted: 27 September 2018 Received: 15 January 2018 Accepted: 27 September 2018 © The Author(s) 2018 © The Author(s) 2018 NATURE COMMUNICATIONS \| (2018) 9:4362 \| DOI: 10.1038/s41467-018-06821-x \| www.nature.com/naturecommunications 4
https://openalex.org/W4206121294	https://scholarlypublications.universiteitleiden.nl/access/item%3A3561412/view	English	null	A recurrent neural network architecture to model physical activity energy expenditure in older people	Data mining and knowledge discovery	2,022	cc-by	16,855	A recurrent neural network architecture to model physical activity energy expenditure in older people Paraschiakos, S.; Sa, C.R. de; Okai, J.; Slagboom, P.E.; Beekman, M.; Knobbe, A. Citation Paraschiakos, S., Sa, C. R. de, Okai, J., Slagboom, P. E., Beekman, M., & Knobbe, A. (2022). A recurrent neural network architecture to model physical activity energy expenditure in older people. Data Mining And Knowledge Discovery, 36, 477-512. doi:10.1007/s10618-021-00817-w Citation Paraschiakos, S., Sa, C. R. de, Okai, J., Slagboom, P. E., Beekman, M., & Knobbe, A. (2022). A recurrent neural network architecture to model physical activity energy expenditure in older people. Data Mining And Knowledge Discovery, 36, 477-512. doi:10.1007/s10618-021-00817-w Citation Paraschiakos, S., Sa, C. R. de, Okai, J., Slagboom, P. E., Beekman, M., & Knobbe, A. (2022). A recurrent neural network architecture to model physical activity energy expenditure in older people. Data Mining And Knowledge Discovery, 36, 477-512. doi:10.1007/s10618-021-00817-w Citation Version: Publisher's Version License: Creative Commons CC BY 4.0 license Downloaded from: https://hdl.handle.net/1887/3561411 Publisher's Version Creative Commons CC BY 4.0 license https://hdl.handle.net/1887/3561411 Publisher's Version Creative Commons CC BY 4.0 license https://hdl.handle.net/1887/3561411 Version: License: Note: To cite this publication please use the final published version (if applicable). Data Mining and Knowledge Discovery (2022) 36:477–512 https://doi.org/10.1007/s10618-021-00817-w Responsible editor: Panagiotis Papapetrou. A recurrent neural network architecture to model physical activity energy expenditure in older people Stylianos Paraschiakos1,2 · Cláudio Rebelo de Sá3 · Jeremiah Okai2 · P. Eline Slagboom1 · Marian Beekman1 · Arno Knobbe2 Received: 20 May 2020 / Accepted: 29 November 2021 / Published online: 10 January 2022 © The Author(s) 2022 Received: 20 May 2020 / Accepted: 29 November 2021 / Published online: 10 January 2022 © The Author(s) 2022 B Stylianos Paraschiakos s.paraschiakos@lumc.nl 1 Introduction At older age, the extension of health span and maintenance of mobility are of great importance for the quality of life. Regular physical activity (PA) of moderate intensity is known to offer positive effects on the reduction of disease incidence and mortality risk (Manini et al. 2006; Chen et al. 2012; Cicero et al. 2012; Petersen et al. 2012). To quantify and monitor the intensity of PA, estimation of energy expenditure dur- ing physical activity is an obvious necessity. By monitoring physical activity energy expenditure (PAEE), older people may better engage in physical activities, leading to better health and reduced (multi)morbidity and mortality risk (Manini et al. 2006). PAEE is one component of total energy expenditure (TEE), where TEE is the sum of PAEE, resting energy expenditure (REE or RMR) by a fasted individual, and thermic effect of food (TEF). One way to measure PAEE is using direct calorimetry and measurements of heat production, but expensive equipment is required. Also, the Doubly Labeled Water Technique (DLW) provides an accurate technique of TEE esti- mation from where PAEE can be estimated, however, similar to direct calorimetry, it requires sophisticated lab-based equipment to analyse urine samples. Therefore, indi- rect calorimetry (Leonard 2012) is commonly used, which involves the measurement of oxygen and carbon dioxide exchange by ventilated mask or hood. Because such forms of calorimetry cannot be performed under free-living condi- tions, methods to estimate PAEE from wearable accelerometers have been developed (Lyden et al. 2011; Staudenmayer et al. 2009; Ellis et al. 2014; Montoye et al. 2017a; Caron et al. 2020; O’Driscoll et al. 2020). This form of indirect calorimetry is estimated by accelerometer data and their combinations with physiological measurements such as heart rate, and individual-level data (demographic, anthropometric) using both lin- ear and non-linear methods (Liu et al. 2012). For example, linear or multiple regression methods can be used to estimate PAEE (Lyden et al. 2011), but also non-linear ensem- bles like random forest regressors (Gjoreski et al. 2013; Ellis et al. 2014; O’Driscoll et al. 2020) and deep learning method such as artiﬁcial neural networks (ANN) (Stau- denmayer et al. 2009; Montoye et al. 2017a) and convolutional neural networks (CNN) (Zhu et al. 2015) have been employed. Good estimates of PAEE can be derived from accelerometry data. Abstract Through the quantiﬁcation of physical activity energy expenditure (PAEE), health care monitoringhasthepotentialtostimulatevitalandhealthyageing,inducingbehavioural changes in older people and linking these to personal health gains. To be able to mea- sure PAEE in a health care perspective, methods from wearable accelerometers have been developed, however, mainly targeted towards younger people. Since elderly sub- jects differ in energy requirements and range of physical activities, the current models may not be suitable for estimating PAEE among the elderly. Furthermore, currently available methods seem to be either simple but non-generalizable or require elaborate (manual) feature construction steps. Because past activities inﬂuence present PAEE, we propose a modeling approach known for its ability to model sequential data, the recurrent neural network (RNN). To train the RNN for an elderly population, we used the growing old together validation (GOTOV) dataset with 34 healthy participants of 60 years and older (mean 65 years old), performing 16 different activities. We used accelerometers placed on wrist and ankle, and measurements of energy counts by means of indirect calorimetry. After optimization, we propose an architecture con- sisting of an RNN with 3 GRU layers and a feedforward network combining both accelerometer and participant-level data. Our efforts included switching mean to stan- dard deviation for down-sampling the input data and combining temporal and static data (person-speciﬁc details such as age, weight, BMI). The resulting architecture pro- duces accurate PAEE estimations while decreasing training input and time by a factor of 10. Subsequently, compared to the state-of-the-art, it is capable to integrate longer 123 478 S. Paraschiakos et al. activity data which lead to more accurate estimations of low intensity activities EE. It can thus be employed to investigate associations of PAEE with vitality parameters of older people related to metabolic and cognitive health and mental well-being. activity data which lead to more accurate estimations of low intensity activities EE. It can thus be employed to investigate associations of PAEE with vitality parameters of older people related to metabolic and cognitive health and mental well-being. Keywords Recurrent neural networks · Physical activity energy expenditure · Accelerometer · Wearables · Indirect calorimetry · Monitoring older adults 123 1 Introduction Since the majority of currently available methods to estimated PAEE from accelerometer data are mainly developed and tested on a young or middle-aged popu- lation (Montoye et al. 2017a; Caron et al. 2020), these models may not be suitable for estimating PAEE among the elderly. This is due to the fact that the elderly differ in energy requirements (Roberts and Dallal 2005; Hortobágyi et al. 2003), expenditure (Frisard et al. 2007; Knaggs et al. 2011), and range of physical activities (Jones et al. A recurrent neural network architecture to model physical... 479 2009; Martin et al. 2014) while it is also seen that the older the individuals tent to spend more time in sedentary activities (van Ballegooijen et al. 2019). 2009; Martin et al. 2014) while it is also seen that the older the individuals tent to spend more time in sedentary activities (van Ballegooijen et al. 2019). There are two main drawbacks in the currently available methods. First, while linear models are pretty simple to deploy and use, they are unable to ﬁt to all the activities (van Hees et al. 2009). Second, the non-linear can be quite elaborate and computationally intensive, since they require steps of features construction and selection in order to capture the temporal nature of the accelerometer signal. Thus, a PAEE modeling method that does not require any sophisticated or handcrafted pre-processing is called for, in addition to the development and testing of the model on older adults. p g Therefore, we propose a neural network modeling approach that is known for its ability to model sequential data, the Recurrent Neural Network (RNN). The RNN is a network architecture that can deal with raw sensor data or minimum feature extraction, and can model temporal data by sequential processing. The nature of the processing in RNNs provides the possibility to remember information from the near as well as distant past, which is an advantage in comparison to ANN or CNN. Because past activities inﬂuence present PAEE, RNN modeling seems to be an excellent ﬁt. To train the RNN for application on an elderly population, we used the Grow- ing Old Together Validation (GOTOV) dataset (Paraschiakos et al. 2020) with 34 healthy participants of 60 years and older (mean 65 years old), performing 16 differ- ent physical activities. 1. We propose an original GRU-based approach for modeling PAEE, and demonstrate its efﬁcacy in an elderly population. Once before, an RNN-based approach has been used for PAEE estimation (Mardini et al. 2020) combining LSTM and CNN layers. 1 Metabolic Equivalent of Task, where 1 MET at rest (e.g. sitting) equals 1 Kcal/kg/h. 1 Introduction We show that longer windows of prior sensor (up to 2min) lead to better PAEE estimation, and that GRU model based on standard deviation can deal with these longer windows efﬁciently. 3. We show that longer windows of prior sensor (up to 2min) lead to better PAEE estimation, and that GRU model based on standard deviation can deal with these longer windows efﬁciently. 4. We demonstrate how the addition of participant-level data (for example age and weight of a subject) can improve the sensor-based model. 4. We demonstrate how the addition of participant-level data (for example age and weight of a subject) can improve the sensor-based model. The rest of the paper is structured as following. Section 2 presents the related work, while Sect. 3 presents the dataset used for model development. Then, Sect. 4 discusses the methodological steps needed to model PAEE, such as model architecture, data preparation (including the predictors down-sampling steps), model evaluation and experimental pipeline. This is followed by the results section (Sect. 5) presenting the main ﬁndings of our analysis. Finally, our ﬁndings, modeling strengths and limitations, and our future work is discussed in Sect. 6. 1 Introduction This dataset is one of the ﬁrst datasets publicly available with a focus on physical activity modeling of the elderly, both for activity recognition and energy expenditure. It includes multiple sensors (accelerometry, indirect calorimetry, physiological measurements) placed at multiple body locations. In the current study, we used a combination of accelerometers placed on wrist and ankle (GENEActiv), because accelerometers combined on hand and foot can be good PAEE estimators (Dong et al. 2013; Ellis et al. 2014). Furthermore, Montoye (Montoye et al. 2017a,b) argues that both wrist and ankle separately produce the best PAEE estimations. Finally, the measurements of energy counts (per-breath calories) were collected by means of the medical-grade COSMED device (McLaughlin et al. 2001). Our proposed RNN architecture exploits Gated Recurrent Units (GRU) layers combined with a shallow ANN in order to make use of both accelerometer and participant-level data (age, gender, weight, height, BMI). This means that both tempo- ral data and attribute-value data are given as input to the model and it combines them to give estimates of PAEE. In more detail, the model takes as an input sequences of temporal data representing a time window of past accelerometer, and creates output- features that are combined with the participant-level data in order to produce a PAEE estimation. Summarising, the main contribution of this paper is the development of a novel PAEE modeling architecture without any sophisticated feature construction step focused on a population group that is often overlooked: adults over 60 years of age. The speciﬁc contributions of our work are the following: 1. We propose an original GRU-based approach for modeling PAEE, and demonstrate its efﬁcacy in an elderly population. Once before, an RNN-based approach has been used for PAEE estimation (Mardini et al. 2020) combining LSTM and CNN layers. 123 480 S. Paraschiakos et al. While we will demonstrate that LSTMs work equally well on our dataset, we have adopted GRUs for reasons of higher efﬁciency. While we will demonstrate that LSTMs work equally well on our dataset, we have adopted GRUs for reasons of higher efﬁciency. 2. We prove that using statistical dispersion metrics like standard deviation to down- sample the accelerometer data can signiﬁcantly improve the accuracy achieved, while reducing the training time by approximately 10 times while using 10 times less data, compared to averaging (mean). 3. 2 Related work In the past few years, multiple PAEE methods have been developed, ranging from simple linear regression and linear mixed models (Montoye et al. 2017a) to non-linear ones, based on machine learning (Montoye et al. 2017a; Ellis et al. 2014; Zhu et al. 2015). Here, we give a short introduction of these by examining their modeling aspects in detail. Table 1 displays the three publications explained in this sections and their modeling set-ups. Montoye et al. (2017a) already provided an interesting comparison of multiple PAEE methods. In this work, a linear regression model (LM) was compared to a linear mixed model (LMM), and a shallow artiﬁcial neural network (ANN). These models were developed in a dataset of N = 40 healthy participants (≈50% female) between ages 18 and 44 years (mean = 23.7). The dataset included recordings from 4 different accelerometers on the right hip, right thigh and both wrists, while a portable metabolic analyser on their backs connected to a breathing mask. The participants performed a 90-min semi-structured protocol of 13 activities of different intensity levels, such as lying down, sitting, household, climbing stairs, walking, jogging, stationary cycling and others in order and duration as determined by the participant. In order to train the different models, time-domain predictor features of 30 non- overlapping seconds were developed per device. The features were chosen based on previous work of the authors and included: mean, standard deviation, minimum, maximum, co-variance of adjustment windows, and 10th, 25th, 50th, 75th and 90th percentiles per acceleration axis (triaxial accelerometers were used). While, as a target variable, they used 30s of aggregated MET1 values, synchronous with the predictors. 123 12 A recurrent neural network architecture to model physical... 481 Table 1 State-of-the-art methods and their characteristics Publication Montoye et al. (2017a) Ellis et al. (2014) Zhu et al. (2015) N 40 40 30 Age 23.7 35.8 27.8 Body Location Hip, thigh, wrists Hips, wrist Waist Features 36 time domain 45 time and freq-domain – Window 30s 1min 5.12s Anthropometrics False False True Methods LM, LMM, ANN RF CNN Based on these, the different models (LM, LMM, ANN) were trained per device, where the two different ANNs developed were based on prior work of Staudenmayer et al. (2009). Based on these, the different models (LM, LMM, ANN) were trained per device, where the two different ANNs developed were based on prior work of Staudenmayer et al. (2009). 2 Related work The models were compared per body location using Pearson correlations, root mean squared errors (RMSE) and bias. The model correlations ranged from 0.82 to 0.89 and RMSE ranged from 1.07 to 1.31 MET for the four accelerometers with the ANN models, whereas the linear models (LM and LMM) from 0.71 to 0.88 and RMSE ranged from 1.11 to 1.61 MET. The differences between the ANN and the linear models (LM and LMM) were statistically signiﬁcant for the wrists while for the thigh there was no signiﬁcant difference for all models and for the hip only one of the ANNs had higher correlations and lower RMSE than the linear models. g Similar to Montoye et al. (2017a) and Ellis et al. (2014) developed a random for- est regressor (RFr) and compared it to the ANN approach of Staudenmayer et al. (2009). This time, a broader set of features is used, N= 45, with both time- and frequency-domain features computed from non-overlapping windows of 1 minute. The majority of these features were aggregations of signal vector magnitude (SVM = x2 + y2 + z2) and angular features that capture orientation information of the accelerometer. Adding to that, participants wore a heart rate (HR) monitor and an extra feature of HR is included. The dataset included recordings from N = 40 healthy participants (≈50% female) with mean age = 35.8 years, wearing two accelerom- eters (both hips and dominant wrist) and a portable indirect calorimeter. Participants performed 3 household activities (out of a set of 5) and 3 locomotion activities (slow walk, brisk walk, treadmill jog) for 6min each. The RFr model was developed by learning 500 regression trees with a minimum leaf size of 5 using MET values as a target. Then, the predictions were evaluated on the minute level using the leave-one-subject-out (LOSO) cross validation proce- dure by measuring the bias, standard error and the RMSE. During the experiments, Ellis et al. compared how the models perform for a single body location, but also by combining them, and by adding HR data. In terms of RMSE, the RFr approach outperforms the ANN ones with an RMSE= 1.00 versus an RMSE between 1.12 and 1.35. Furthermore, about the body-locations, placing an accelerometer on the right or left hip produces similar performance to the wrist. 2 Related work However, when wrist and right hip are combined, the performance improves signiﬁcantly compared to the single body 123 482 S. Paraschiakos et al. location. Finally, when HR data are included, both for one the body location set-up or for multiple, the performance further improved signiﬁcantly. location. Finally, when HR data are included, both for one the body location set-up or for multiple, the performance further improved signiﬁcantly. The above work exploits handcrafted features in order to estimate PAEE, while the recent advances in deep learning give us the advantage to automate this procedure and extract complex features of sensor data while training. Zhu et al. (2015), such a method is introduced where a convolution neural network (CNN) architecture is used on a dataset of N = 30 healthy subjects (≈33% female) with ages between 19 and 45 years (mean age = 27.8). The subjects performed a 30-min protocol of 6 activi- ties (walking, climbing stairs, running, static standing/sitting, riding elevator) inside and outside a regular hospital facility. During the data collection, the subjects were equipped with a smartphone with triaxial accelerometer, placed in a waist pouch, and a portable indirect calorimeter that also records HR. Additionally, anthropometric fea- tures (height, weight, age, gender, etc.) per participant were included in the modeling procedure. p Before training, the triaxial accelerometer data (50 Hz) were transformed into sequences of 256 samples representing a time window of 5.12s. As target data, the output of the indirect calorimeter (Kcal/min) was used, aggregated to the same rate as the accelerometer sequences. The trained CNN consisted of 2 convolution layers connected with one dense layer that takes as input the concatenation of the CNN fea- tures with the anthropometric data. The ﬁrst CNN layer employs 8 ﬁlters of kernel size 5 and a pooling factor of 2, while the second CNN layer has 4 ﬁlters of size 5 and the same pooling factor, and the dense layer had a size of 400 nodes. As activation functions, both CNN layers used tanh while for the dense layer no activation function is used (linear transformation layer). Unfortunately, other important hyperparameters like the number of epochs and batch sizes during training, were not reported. Their model was evaluated with LOSO cross-validation by measuring the RMSE and it was also compared to an activity-speciﬁc linear regression model and an ANN approach using handcrafted features. 2 Related work Overall, their CNN approach shows the lowest RMSE (≈1.12) while the activity-speciﬁc one follows with RMSE = 1.59 and the ANN with RMSE = 1.79. When the models are tested per clusters of activities still, the CNN clearly outperforms both models in every activity cluster. Concluding, different statistical or machine learning seem to estimate PAEE quite well. However, it is quite challenging to compare their reported performances since they are developed on (1) different datasets, (2) using data from accelerometers on different body locations (hip, thigh, waist, wrist), and (3) down-sampled these to different windows (from 5s to 1min). For this reason, in Sect. 5.5, we try to fairly compare all the above including our proposed method using a similar settings. In order to cope with the aforementioned challenges, we will develop all the methods using the same dataset [GOTOV (Paraschiakos et al. 2020)] with accelerometers on the ankle and wrist. 2 DOI link: https://doi.org/10.4121/12716081. 3 Body-Mass Index, the body mass divided by the square of the body height. 4 https://youtu.be/jvx5FGhqPxw. 3.1 Study population TheparticipantsintheGOTOVstudyrespondedtoadvertisementsonbulletinboardsin public spaces in the city of Leiden, the Netherlands. People were eligible to participate in the study if they: 1. were older than 60 years old. 1. were older than 60 years old. 2. had a healthy to overweight BMI3 between 23 and 35kg/m2. 3. had no restrictions in their movement caused by health conditions. 4. owned and had access to their own bicycle. A total of 35 individuals (14 female, 21 male) between the ages 60 and 85 years old (mean 65) and mean BMI 27 kg/m2 were recruited. Besides age, gender, height and weight, no additional clinical information was recorded on the participants. The GOTOV study was approved by the Medical Ethical Committee of LUMC (CCMO reference NL38332.058.11). 3 Dataset The dataset used for our experiment is part of the Growing Old Together Validation (GOTOV) study. The GOTOV dataset is designed to develop both activity recognition 123 A recurrent neural network architecture to model physical... 483 (Paraschiakos et al. 2020; Okai et al. 2019) and energy expenditure models that will serve multiple free-living ageing studies with similar population and devices (van de Rest et al. 2016; Westendorp et al. 2009; Wijsman et al. 2013). The dataset includes calorimetry measurements combined with the ankle and wrist accelerometer, among other data and since June 2020, is freely available in the 4TU data repository.2 3.2 Data collection protocol The 35 participants performed a set of 16 activities according to a speciﬁc protocol of approximately 90min. The 16 activities were performed successively for speciﬁc time windows and with short breaks of standing still in between (1 minute). A researcher monitored the activities duration without giving any instructions or illustrations of the activities and wrote down their starting and ending timestamp. The activity protocol took place at two locations; indoors and outdoors of the Leiden University Medi- cal Center (LUMC) facilities. The indoor activities consisted of lying down, sitting, standing, walking stairs and several household activities, such as dish washing, stak- ing shelves and vacuum cleaning. The indoor activities were performed in a room equipped with all the necessary instrumentation. The outdoor activities included dif- ferent types of walking slow, normal, fast, as well as cycling. A visual example of the procedure can be found in a recorded video.4 The detailed protocol of the activities performed is described in Table 2, have in mind that between every two activities there was a break of 60s standing, but in Table 2 this is represented only once, at the second row. Other than that, due to adverse weather conditions, only 25 out of 35 participants were able to perform the outdoor activities (walking, cycling). 123 3 S. Paraschiakos et al. 484 Table 2 Activities and their duration performed in the GOTOV protocol Activity (s) Description Light jumping (20s) Synchronising sensors Standing (60s) Get some rest between activities Steping (60s) A step test on a step (∼20 times) Sedentary activities (180s each) 1. Lying down (left and right) 2. Sitting on a sofa (legs on) 3. Sitting on a sofa (legs on the ground) 4. Sitting on a chair while working Walking stairs up (20s) Ascending two ﬂights of stairs Household activities (180s each) 1. Washing dishes (while standing) 2. Stacking shelves with books 3. Vacuum cleaning Walking outside (300s each) 1. Slow pace 2. Medium pace 3. Fast pace Cycling outside (900s) Participant’s pace in normal trafﬁc conditions Fig. 1 GOTOV study devices and their body location (Paraschiakos et al. 2020) Table 2 Activities and their duration performed in the GOTOV protocol Activity (s) Description Light jumping (20s) Synchronising sensors Standing (60s) Get some rest between activities Steping (60s) A step test on a step (∼20 times) Sedentary activities (180s each) 1. Lying down (left and right) 2. 3.2 Data collection protocol Sitting on a sofa (legs on) 3. Sitting on a sofa (legs on the ground) 4. Sitting on a chair while working Walking stairs up (20s) Ascending two ﬂights of stairs Household activities (180s each) 1. Washing dishes (while standing) 2. Stacking shelves with books 3. Vacuum cleaning Walking outside (300s each) 1. Slow pace 2. Medium pace 3. Fast pace Cycling outside (900s) Participant’s pace in normal trafﬁc conditions Fig. 1 GOTOV study devices and their body location (Paraschiakos et al. 2020) Fig. 1 GOTOV study devices and their body location (Paraschiakos et al. 2020) 123 12 A recurrent neural network architecture to model physical... 485 Time Fig. 2 Model input per device for the different activity groups Fig. 2 Model input per device for the different activity groups 3.3 Devices and body locations During the data collection, the participant used 4 different devices in 6 body locations (see Fig. 1). The set of devices included both accelerometers and sensors measuring physiological indicators, e.g. indirect calorimetry (VO2, VCO2), breathing rate (BR) and heart rate (HR). In this study, we focus on the data coming from accelerometers and indirect calorimetry. This is mainly motivated by the fact that the models will serve existing free-living studies using the same sensor setup. Accelerometry The GENEActiv accelerometers placed on ankle wrist (a and w in Fig. 1) were used in order to recognise and measure activity levels of the participants. The GENEActiv accelerometers provided triaxial (x,y,z) acceleration measurements (±8 g) with a sampling rate of 83 Hz. In order to create a recognisable pattern in data for synchronisation, the participants started the sequence of activities with a light jumping for 20s while waving arms. The recorded signal of ankle and wrist per axis is presented in Fig. 2. Indirect calorimetry The volume of oxygen (VO2) and carbon dioxide (VCO2) was measured per breath continuously during the activities, with a short break between the indoor and outdoor part of the protocol. The calorimetry measurements were obtained through the COSMED K4b2 (McLaughlin et al. 2001) device, with a portable unit on the torso and a ﬂexible mask covering the participant’s nose and mouth (K4 in Fig. 1). The mask is connected to the portable unit that contains O2 and CO2 analysers, a sampling pump, a barometric sensors and electronics. The gas analyser measures the exchange of oxygen and carbon oxygen (in mlkg−1) and outputs PAEE metrics such as energy expended per minute, EEm in Kcal per minute, or per hour, EEh in Kcal per hour or MET, where 1 MET at rest equals 1 Kcal/kg/h. Measurements in these three units can be straightforwardly translated between one another. The COSMED 123 123 S. Paraschiakos et al. 3.3 Devices and body locations 486 Table 3 Description of the ﬁnal study population and their average COSMED measurements Indoors (12 Activities) Outdoors* (4 Activities) Total (16 Activities) N (female %) 13 (27%) 18 (46%) 31 (35%) Age in years (SD) 66.8 (4.5) 64.9 (4.4) 65.7 (5.0) Height in cm (SD) 172.1 (8.3) 176.2 (7.4) 174.5 (7.9) Weight in kg (SD) 82.2 (13.5) 83.7 (10.2) 83.1 (11.5) BMI in kg/m2 (SD) 27.7 (3.5) 26.9 (2.0) 27.2 (2.7) EEm in Kcal (SD) 2.9 (0.4) 6.1 (0.9) 3.8 (1.1) BR in breaths per sec (SD) 0.30 (0.05) 0.39 (0.04) 0.31 (0.04) 4 out of 18 participants with outdoor activities did not perform cycling (1 female) Description of the ﬁnal study population and their average COSMED measurements 4 out of 18 participants with outdoor activities did not perform cycling (1 female) metrics are calculated per breath based on formula that combines VO2 and VCO2 measurements and is similar5 to the Weir formula (Weir 1949): Metabolic rate (calories per minute) or EEm = 3.94 V O2 + 1.11 VCO2 The output from this sensor in EEm, see Fig. 2, was used as our target for training and evaluating our PAEE estimation models. The sampling rate (SR) of the target is equal to the breathing rate of the participant and depends also from the activity at a speciﬁc moment. This results in an SR that is not stable, with a mean SR among all existing data being equal to 0.3 Hz. Before every individual started the sequence of activities, the system was manually calibrated according to the manufacturer instructions. If a device was severely limiting a participant’s movement (COSMED unit and battery weighs 1.5 kg), it was removed and the participant was excluded from our current analysis. 5 COSMED uses a slightly different estimation (unknown formula) giving an average overestimation of approx. 0.0098976 cal compared to Weir. 3.4 Resulting dataset – EE increases with height (Hills et al. 2014). g – EE increases with body composition (BMI) (Weinsier et al. 1992). – EE in males is on average higher compared to the female participants (Keys et al. 1973). 3.4 Resulting dataset There were 35 participants recruited in the GOTOV dataset, from whom 31 participants had both COSMED (indirect calorimetry) and GENEActiv (ankle, wrist accelerom- eter) data. Of those, there were 13 participants with only indoor activity data, so 12 out 16 activities. Finally, for all the other participants with both indoor and outdoor activities, there were 4 participants that did not perform the outdoor cycling activity. Table 3 presents the participant-level data of this study and the average measure- ments of COSMED. In detail, in the ﬁrst block it displays the number of female participants out of the total 31 participants, and the average (mean and SD) age, height, weight and BMI. Furthermore, we can see the average EEm measurements by COSMED and breathing rate (sampling rate) for indoors, outdoors and total. From that, it is observed that there is a clear difference between the indoors and outdoors measurement in terms of EEm, where the mean outdoor EEm measurement is a bit more than double that of the indoor. This is something expected since the outdoor mea- 123 12 A recurrent neural network architecture to model physical... 487 Fig. 3 Trend of indoor activities Energy Expenditure (y-axis) across Age, Height, BMI and Gender Fig. 3 Trend of indoor activities Energy Expenditure (y-axis) across Age, Height, BMI and Gender surements include high intensity activities such as walking and cycling with a bigger range of EEm values compared to the indoors that have a smaller range. Similarly, the breathing rate is higher for the outdoor activities, which implies more data inputs for the same window of time when compared to the indoors (outdoors EEm SR higher than indoors), again as expected. g In total, the data set includes 2.8 hours of sedentary activity (MET < 1.5), 5.4 hours of light activity (1.5 ≤MET < 4), 1.8 hours of moderate (4 ≤MET < 6) and 0.73 hours of vigorous activity (6 ≤MET). An initial view of the dataset is presented in Fig. 3, where the indoors energy expenditure measurements is plotted against gender, age, height and body composition per participant. We plotted the indoors EEm since all 31 participants had indoors COSMED data. From the plots, we see that the trends from the GOTOV dataset conﬁrm what is known from the literature. In detail: – EE decreases with age (Frisard et al. 2007; Roberts and Dallal 2005). 4 Methodology In this section, we explain the methodological contributions of the paper. In detail, we describe our model choice and its architecture. Following that, we analyse the steps 123 S. Paraschiakos et al. 488 Fig. 4 Proposed model architecture combining both temporal and static data. The grey layers are sufﬁcient when only temporal data are used (no static data) Fig. 4 Proposed model architecture combining both temporal and static data. The grey layers are sufﬁcient when only temporal data are used (no static data) of data preparation and their different combinations. Then, the training and evaluation process is explained. Finally, we summarise the experimental setup. of data preparation and their different combinations. Then, the training and evaluation process is explained. Finally, we summarise the experimental setup. 4.1 Modeling architecture A Recurrent neural network (RNN) is a type of artiﬁcial neural network that has the ability to ‘remember’ older information from sequences. In more detail, an RNN con- tains feedback loops within its hidden layers whose activation at each time depends on that of the previous layer (Chung et al. 2015). Consequently, RNNs have a mod- eling advantage when used on sequential or temporal data over traditional ANNs. RNNs have been used for a variety of tasks, both regression and classiﬁcation, such as natural language processing (Li and Xu 2018), speech recognition (Lee et al. 2018), in clinical application (Tomašev et al. 2019), and more recently, activity recognition from accelerometer data (Edel and Köppe 2016; Guan and Plötz 2017) and modeling of long-term human activity (Kim et al. 2017). Because PAEE is inﬂuenced by past activities (lag effect), RNNs could be a suitable modeling candidate for tackling the challenge of PAEE estimation. Traditional RNN networks are known to struggle with information from long sequences due to the so-called vanishing gradient problem (Hochreiter 1998). The most popular solution to this problem is introducing Long Short Term Memory (LSTM) or Gated Recurrent Unit (GRU) layers. LSTM and GRU layers contain cells that act either as memory or gates controlling the information ﬂow to the next layers. LSTMs contain 3 gates, namely, forget, input and output, while GRUs have only 2 gates, the reset and update gate. The reset gate controls which of the memory cell information needs to be forgotten. The update gate controls which information needs to be updated. This allows them to remember long sequences of information without losing relevant information. Adding to that, in a recent work of ours (Okai et al. 2019), we tested different RNN architectures with either LSTM or GRU layers for the task of activity recognition with predictors missing data. Based on these experiments we proved that RNNs with GRU layers and a proper architecture are robust for such a task. A recurrent neural network architecture to model physical... 4.2 Data preparation and choices In order to build PAEE estimation models using RNNs, there is a need for several transformations both in the predictors data (accelerometers, activities, participant- level data) and the target (COSMED EEm). As a ﬁrst step, the target and numeric predictor data were z-normalized to have zero mean and a standard deviation of 1. Additionally, in order to model discrete predictors, like gender or activity class, label encoding was used (with values in [0, n] with n being the number of values). 4.2.1 Indirect calorimetry as target data COSMED produces energy expenditure measurements per breath, meaning that the target doesn’t have a ﬁxed sampling rate. On average, the COSMED sampling rate is 0.3 Hz, which means one input approximately every 3.3s, see EEm signal in Fig. 2. In order to stabilize the sampling rate for the training, we down-sampled the COSMED signal to 0.1 Hz by taking the mean of every interval of 10s. This way, we avoid the creation of more training data in periods with higher breathing rate, but we also smooth the outlier EEm values occasionally produced by COSMED. Finally, we assign a sequence of predictors per EEm value which captures the movements that preceded the EEm measurement (see box C of Fig. 5). 4.1 Modeling architecture 489 Table 4 Proposed model architecture and training parameters Input 1: Sequential data Layer Nodes Kernel regularizer (L2) Dropout GRU1 32 0.0001 0.5 GRU2 256 0.0001 0.5 GRU3 32 0.0001 0.5 Layer Nodes Activation function Dropout layer Denseseq 32 ReLU – Input 2: static data Layer Nodes Activation function Dropout layer Densesta 32 ReLU – Concatenate inputs Layer Nodes Activation function Dropout layer Dense1 32 ReLU 0.2 Dense2 16 ReLU 0.2 Model output: linear Training parameters Batch size Epochs Optimizer Opimizing variable 512 50 Adam Mean squared error (MSE) As a result, our proposed RNN architecture is based on our recent work (Okai et al. 2019) and consists of an input layer followed by 3 GRU layers with 32, 256 and 32 nodes respectively, 2 dense layers with 32 and 16 nodes and an output layer (see Fig. 4 grey layers). Models are trained to minimize the mean squared error (MSE) using the optimization method Adam (Kingma and Ba 2015). To prevent over-ﬁtting, a dropout ratio of 0.5 (50%) is applied to all three GRU layers. In Table 4, we describe in detail the different parameters chosen. In order to test if participant-level data could improve PAEE estimation, we con- catenated the aforementioned RNN setup with a single feedforward network, into the ﬁnal architecture demonstrated in Fig. 4. The reason behind such an architecture is the need to model two types of data, temporal (sensor measurements, activities) and static data (participant-level). Therefore, we feed the accelerometer sequences to the GRU layers and at the same time, we feed the static data to a feedforward network. This feedforward network consists of an input layer and a hidden layer with 32 neurons. The output layers of both networks were concatenated and connected to 2 dense layers consisting of 32, and 16 neurons respectively, see Table 4. Finally, the output layer is made up of only a single neuron, which is used to predict the COSMED EEm values. 123 123 S. Paraschiakos et al. 490 4.2.2 Predictors data to sequences To train the RNN model, we need to build sequences, where each is associated with one EEm measurement. A sequence is deﬁned as a ﬁnite list of inputs arranged in a deﬁnite order (Volchan 2002). For our problem, the order of inputs is based on time. The sequences represent the predictors data in the time immediately before every EEm measurement in windows of time, with speciﬁc number of inputs and resolution (sampling rate). We have two types of inputs in our network. First, the activity data is of temporal nature, notably the accelerometer data include three numeric time series inputs for every device (see ankle, wrist in Fig. 2) and the activity labels (a discrete sequence). Second, wehavetheparticipant-level datathat includes demographicinformation(age, gender) and body composition information (height, weight, BMI), as static attribute- value data. Figure 5 shows the different elements and steps taken to transform this data to training sequences. In detail, I, II, III, and IV in Fig. 5 display the different types of input, while for the accelerometer data (I I), we display the extra steps needed in order to transform the signal to training sequences. In the following paragraphs, we explain the different sequence conﬁgurations developed and tested. Accelerometers In order to transform the accelerometer signal into training sequences, see light shading in box A, Fig. 5, we need to decide on the number of inputs to be used (sequence size of the RNN), the length of the window that those will represent (time interval) and the resolution (sampling rate). These 3 variables are inter-connected, as presented in the following equation: 123 12 A recurrent neural network architecture to model physical... 491 Fig. 5 Building sequences for temporal data SR = Sequence Size Window Size , Fig. 5 Building sequences for temporal data where Window Size is calculated in seconds. For example, if we want a sequence with a size of 480 inputs to represent a time window of 240 s (4min), we will need to down-sample the accelerometer data from 83 Hz (original SR) to 2 Hz, since the sampling rate (SR) depends on both sequence and window size. On the one hand, longer sequences allow for higher sampling rates in the accelerom- eter data, but they will produce longer training times. On the other hand, for a ﬁxed sequence size, a choice of longer time windows will result in lower sampling rate. 4.2.2 Predictors data to sequences Nevertheless, the choice of window length is crucial since long enough windows are needed in order to include any PAEE bias from activities performed further in the past. We experimented with different sequence sizes representing different intervals of time (time windows) and data resolutions. The down-sampling decisions are displayed in box B of Fig. 5. In order to adjust the predictors to the given sequence sizes and window lengths, we need to down-sample the accelerometer data to the desired SR (B, Fig. 5). For this down-sampling, which aggregates several values into a single one, we compared two different aggregation approaches, one that uses the mean function, and one that makes use of statistical dispersion functions (standard deviation, interquartile range, percentiles difference). Our motivation to use statistical dispersion measures comes from the fact that PAEE depends on the range of movement and therefore dispersion measures are more suitable for this task than the mean. As an example, see Fig. 6 where aggregated accelerometer values with mean and standard deviation (SD) are compared. Here, we can observe that during walking, the 3 axes of the ankle signal aggregated by SD (Ankle SD in ﬁgure) correlate nicely with the values of EEm compared to the Ankle Mean signal 123 123 492 S. Paraschiakos et al. Fig. 6 The ankle and wrist accelerometer signal down-sampled to 1 input every 2.4s (our optimal down- sampling) with mean and SD, compared to the recording of COSMED Fig. 6 The ankle and wrist accelerometer signal down-sampled to 1 input every 2.4s (our optimal dow sampling) with mean and SD, compared to the recording of COSMED which is represented in a more linear way. Similarly, during household activities, the Wrist SD correlates with EEm much more than Wrist Mean does. As a simple example, let’s consider 2 different movements represented in 2 windows of accelerometer data, W1 and W2. Let the windows also have different ranges of movement represented by only 2 values, x1 ∈{−2, 2} and x2 ∈{−4, 4}. As a result, the energy spent for the movement in W1 would be lower than in W2 since the effort needed to go from −2 g to 2 g is lower than the effort needed to go from −4 g to 4 g. However, the mean magnitude in both windows is equal (meanw1 = meanw2 = 0). 4.2.2 Predictors data to sequences On the other hand, the standard deviation of these ranges are different, SDw1 = 2 and SDw2 = 4, which correctly captures of the relative expected energy spent per window. Concluding, the signal aggregated with statistical dispersion functions is more likely to capture the variation of PAEE compared to the ones of mean. For this reason, in this work we want to test this hypothesis (see Sect. 5). Participant-level data Combined with the accelerometer data, we test whether participant-level data like demographics (age, gender) and anthropometric features (height, weight and BMI), see Table 3, could contribute to PAEE estimation (I V , Fig. 5). For this reason, we had to prepare such data input and combine it into the data sequences. The anthropometric data were z-normalized to have zero mean and A recurrent neural network architecture to model physical... A recurrent neural network architecture to model physical... 493 Table 5 Table of time spent and EEm (Kcal/min) per activity Activity Time (h) EEm (SD) Lying down 0.8 (7.4%) 2.4 (1.1) Sitting 1.6 (14.8%) 2.0 (1.0) Standing 2.5 (23.2%) 3.2 (1.7) Household 2.2 (20.4%) 3.5 (1.6) Walking 2.3 (21.4%) 5.2 (2.1) Cycling 1.3 (12.1%) 8.2 (3.0) Jumping 0.1 (0.6%) 3.1 (1.7) a standard deviation of 1 and the gender was hard encoded. This way the model will take as an input a sequence of accelerometer data and the details of the corresponding participant. a standard deviation of 1 and the gender was hard encoded. This way the model will take as an input a sequence of accelerometer data and the details of the corresponding participant. Activity classes data Finally, we would like to test whether adding symbolic data in the form of a label describing current or past activities can be beneﬁcial to estimate PAEE (IV, Fig. 5), when combined with either accelerometer data only, or with both accelerometer and participant-level data, as seen before in Bonomi et al. (2009) and Altini et al. (2015). In order to obtain such activity labels, we had to predict the activity types using learned activity recognition methods. We need to derive the labels from the acceleration data, since they will not be available in a free-living scenario either. For this goal, we used a previously developed and published method that was already tested with the GOTOV devices (Paraschiakos et al. 2020). 4.2.2 Predictors data to sequences This model can produce activity predictions per second with an accuracy of more than 90% based solely on ankle and wrist accelerometers for 7-class activity classiﬁcation. Through this model, we can predict the following 7 classes: lying down, sitting, standing, household, walking, cycling and jumping. Having the activity labels predicted per second, we encoded them and combined them with the accelerometer sequences as input to our model. Table 5 summarises the predicted classes and presents also some statistics about their EEm cost. 4.3 Training and evaluation We trained and tested our models using Leave One Subject Out cross validation (LOSO-CV). This means that we train using all subjects (participants), leaving the data of one subject out as a test set. We then iterate the process in order to test all subjects separately. The aim of this type of cross-validation is that we emulate the future situation where we would like to process as yet unseen subjects. The LOSO- CV process prevents training set leakage within a subject, as normal cross-validation procedures might allow. Additionally, during training, 2 participants were selected as validation set, one with only indoor activities and one with all activities. These 2 sets were randomly chosen per subject and were the same across the different model set- tings tested in order to have fair comparisons. All models were trained for 50 epochs with a batch size of 512. After testing 4 different batch sizes (64, 128, 256, 512) we 123 123 494 S. Paraschiakos et al. Fig. 7 Three examples of MSE error (left) and loss (right) during training for 50 epochs. The orange lines represent the evolution of MSE and loss for the training set and the blue for the validation set accordingly Fig. 7 Three examples of MSE error (left) and loss (right) during training for 50 epochs. The orange lines represent the evolution of MSE and loss for the training set and the blue for the validation set accordingly selected the max (512) since the accuracy gain for the smaller ones was too little com- pared to the cost of training time. Similarly, we tested three sizes of training epochs (50, 100, 200) and it was proven that, with our optimal set-up of SD-aggregated sig- nal, 50 epochs were enough for the model to converge while the cost of extra training epochs was not translated to signiﬁcant performance gains. This can also be seen in Fig. 7 where the mean squared error (MSE) and loss evolution during training of 3 LOSO examples is presented. We would also like to point out that the model’s validation and test sets during LOSO-CV are used with their original sampling rate (once per breath). This means that we trained our models using the smoothed EEm values with a stable SR (0.1 Hz), as indicated in the previous section, but we evaluate them per breath (COSMED recordings). 4.3 Training and evaluation This way, we can see which model can ﬁt better the input data since we evaluate our models by measuring their performance on the original EEm values, including the extreme COSMED measurements. 123 495 A recurrent neural network architecture to model physical... Hence, to get the overall performance of a model, we train 31 different models using LOSO-CVandwereporttheiraggregated(median)result,asRootMeanSquaredError (RMSE) and R-squared (R2). Additionally, we compute the RMSE and R2 separately forindoorandoutdoordata.Therearemultiplereasonsbehindthisdecision.First,since there are participants without outdoor data and there is a clear difference between EEm levels with the indoor ones (see Tables 3, 5), we can see how our models behave on low and high-intensity activities separately. Additionally, it is suggested in the literature (van Hees et al. 2009) that PAEE estimation of sedentary or low-intensity physical activities (typically performed indoors) is still a challenging task since their differences in acceleration magnitude are minor. Finally, our main focus is to estimate PAEE of older individuals and it is observed that this group of people spends signiﬁcantly more time in sedentary or low-intensity activities (van Ballegooijen et al. 2019). 6 To make our experiments, scripts and results accessible to other researchers (open-source) we share them on the following git repository, https://github.com/parastelios/GOTOV_PAEE_publication. 4.4 Experimental pipeline In this section, we explain the experiments performed, the motivation behind their set-up and their order.6 Optimise data input First, we tested the architecture with only accelerometer data (grey in Fig. 4) comparing the different accelerometer aggregation functions into time windows of different sequence size and resolution (SR). The aggregation functions tested were mean, standard deviation (SD), interquartile range (IQR), and difference between 5th and 95th percentile (PD), with: – sequence sizes of 10, 50, 160 and 480 inputs per sequence, and – sequence sizes of 10, 50, 160 and 480 inputs per sequence, and – window lengths, for each sequence size, of 1, 2, 4, and 8min. – window lengths, for each sequence size, of 1, 2, 4, and 8min. Here, in order to avoid training and testing 4 × 4 × 4 = 64 different combinations of sequence size, window length, and aggregation function, we optimize the search process by ﬁrst comparing all aggregation functions with the longest sequence size (480) representing the 4-min windows. Since we observed that the sequences built with statistical dispersion functions had very similar performance, we subsequently ﬁx our dispersion measure to SD, and compared this with Mean for the remaining combinations (2 functions ×9 combinations = 18 in total). In the end, when the optimal setting of window and sequence size is found for the SD and Mean, we tested them also for the IQR and PD aggregations. The training parameters used were a batch size of 512, for 50 epochs for all experiments since we observed that with this combination the model converged faster. Anthropometrics and activity classes data As a second analysis step, we tested whether the addition of participants-level data or the predicted activity classes improves the performance. In order to do that, we make use of the complete architecture and param- eters presented in Fig. 4 and Table 4 and the best combination of aggregation function (SD), window size (w = 2min) and sequence size (N = 50), as concluded from the step above. In the end, we compare the performance of this model set-up using: (1) 12 3 3 496 S. Paraschiakos et al. 4.4 Experimental pipeline Table 6 Table of architectures tested at ablation study Architecture GRU layers Dense layers Data 5xGRU_3xDense 1, 1, 2, 3, 3 Seq, sta, 1, 2 a, w 4xGRU_3xDense 1, 1, 2, 3 Seq, sta, 1, 2 a, w 3xGRU_3xDense 1, 2, 3 Seq, sta, 1, 2 a, w 3xGRU_2xDense 1, 2, 3 Seq, sta, 1 a, w 2xGRU_3xDense 1, 2 Seq, sta, 1, 2 a, w 2xGRU_2xDense 1, 2 Seq, sta, 1 a, w 1xGRU_2xDense 1 Seq, sta, 1 a, w 3xGRU_3xDense_a 1, 2, 3 Seq, sta, 1, 2 a 3xGRU_3xDense_w 1, 2, 3 Seq, sta, 1, 2 w 1xGRU_2xDense_a 1 Seq, sta, 1 a 1xGRU_2xDense_w 1 Seq, sta, 1 w The number and descriptions of the layers refer to the ones in Fig. 4 and Table 4. Data refers to ankle (a) and/or wrist (w) inputs used for training The number and descriptions of the layers refer to the ones in Fig. 4 and Table 4. Data refers to ankle (a) and/or wrist (w) inputs used for training accelerometer and participants level data (GRUI D) and (2) accelerometer, participant- level and activity classes data (GRUI D_AC). accelerometer and participants level data (GRUI D) and (2) accelerometer, participant- level and activity classes data (GRUI D_AC). Ablation study Subsequently, we performed an ablation study for our proposed archi- tecture, and we tested our model against different RNN layers. For these experiments we used the best combination of data resolution and data combination as found in the previous steps. For the ablation study, we tested 2 more coomplicated and 3 simpler architectures of our model by excluding different layers of our model. The different architectures are presented in Table 6. Additionally, we compared the performances of our proposed model (3xGRU_3xDense) to the minimal one (1xGRU_2xDense) using data (train and test) from ankle or wrist alone. The motivation behind this is to test how robust each architecture is to training data from only ankle or only wrist since in our previous work (Okai et al. 2019), the maximum (3xGRU_3xDense), was proven to be robust to missing data for a classiﬁcation task (activity recognition) and that is why it was selected. Furthermore, most of PAEE related work tests models that exploit data from single devices and we would like to compare our model to theirs (see Sect. 2). 4.4 Experimental pipeline Finally, we tested our maximal architecture with two different types of RNN layers, the long short term memory (LSTM) and simple RNN layers. For all experiments, we compared the performance using both R-squared and RMSE for all activities, and for indoor and outdoor ones separately. Compare to related work Following to ablation study, we compared our proposed model against state-of-the-art methods as presented in Sect. 2. We trained a linear model (LM), a linear mixed model (LMM), a random forest regressor (RFr), an arti- ﬁcial neural network (ANN) and a convolutional neural network (CNN) using our dataset. In detail, we tested the LM, LMM and ANN as presented in Montoye et al. (2017a) with the proposed set of 30 time-domain features for non-overlapping win- dows of 30s per device. In order to have comparable results, we used the same set of features to train an RFr similar to the one that Ellis et al. (2014) introduced but 123 A recurrent neural network architecture to model physical... 497 using 1000 trees (compared to 500) as O’Driscoll et al. (2020) recently presented to be more robust. Finally, we trained a CNN as presented in Zhu et al. (2015) using both the authors’ data resolution with a window of 5.12s for sequences of size 256 inputs, and our approach of down-sampled predictors with standard deviation for a window of 2min and a sequence size of 50 inputs. About that, since the authors did not include the number of epochs and batch size, we decided to train the CNN for 50 epochs and a batch size of 512, similar to our GRU approach. Here, we need to clarify that all of the above publications (Montoye et al. 2017a; Ellisetal.2014;Zhuetal.2015)recommendmodelswithdatafromasingleaccelerom- eter placed at different body locations per study. Even when multiple devices exist, only (Ellis et al. 2014) combined them and tested their performance against the single ones. In contrast, our purpose in this work is to predict PAEE by combining the data of ankle and wrist and not compare them separately. However, in order to compare our work to the above methods, we also trained our optimal architecture with only ankle or wrist data. Demonstrate the model’s use in the future Finally, we test our selected model’s per- formance over different EEm aggregation windows and activity types. The motivation behind this is twofold. 4.4 Experimental pipeline First, we wanted to test how our model performs in a free-living setting where breathing rate is not included, and second, in order to have comparable results to the literature where models are evaluated in aggregated windows of 30s or 1min. In particular, we report the performance across different EEm windows from the original COSMED sampling rate (breath by breath), to 10, 30s and 1, 5, 60min aggregations and per activity type. This way, we can have an idea of how our approach can be used to estimate PAEE of longer windows. 5 Results and discussion In this section, we present and discuss the results of the experiments performed. First, we discuss the training input optimisation (Sects. 5.1, 5.2), followed by the results of the ablation study (Sects. 5.3, 5.4) and the comparison of our results to related work (Sect. 5.5). Finally, we demonstrate the results of our suggested model for different windows of PAEE aggregations and activities (Sect. 5.6). 5.1 Standard deviation as optimal aggregation function Here, we present the performance of the different input data setups. Since it is not feasible to display all 64 combinations tested, Table 7 displays the best setup per aggregation function. In the ﬁrst column, the aggregation function is displayed, fol- lowed by its resulting best data setup (sequence size, window length, sampling rate distribution). Then we compare their R2 and RMSE in total, indoor, and outdoor activ- ities. Additionally, the last column indicates the signiﬁcance of the difference between mean and the rest functions in terms of R2, using a paired t-test. Examining Table 7, we observe that models built with statistical dispersion func- tions outperform signiﬁcantly the one using the mean, for α = 0.05, all p-values are 123 S. Paraschiakos et al. 498 Table 7 Comparing the performance of different data setups Model R2 inR2 outR2 RMSE inRMSE outRMSE p-Value Mean SeqSize = 480 WinSize = 4min 0.38 0.23 0.38 1.46 1.24 2.32 – SR = 2Hz SD SeqSize = 50 WinSize = 2min 0.45 0.31 0.33 1.35 1.16 2.03 0.01* SR = 0.42Hz IQR SeqSize = 50 WinSize = 2min 0.41 0.29 0.33 1.39 1.18 2.15 0.02* SR = 0.42 Hz PD SeqSize = 50 WinSize = 2 min 0.43 0.30 0.28 1.36 1.17 2.03 0.01* SR = 0.42 Hz The p-value corresponds to the paired t-test of mean with SD, IQR, in terms of R2, with * pointing out when p < 0.05 Table 7 Comparing the performance of different data setups signiﬁcant. Additionally, when we feed our model with SD-aggregated accelerometer data instead of averaging, not only is the model’s performance signiﬁcantly improved, but this improvement is achieved by using approximately 10 times less input data (sequences of 50 versus 480 inputs) and double the window size (4 versus 2min), leading to approximately 10 times lower training time. This is because statistical dis- persion metrics can represent the original signal in a more characteristic way compared to averaging with mean (Lyden et al. 2011). In detail, for the estimation of PAEE using the developed RNN, a two-minute win- dow can be represented by statistical dispersion functions using only 50 inputs per sequence without signiﬁcant loss of accuracy compared to windows down-sampled by averaging. This is of importance when applying the RNN model to data sets with large sample size, e.g. 5.1 Standard deviation as optimal aggregation function intervention studies (hundreds of participants or more) or epidemio- logical studies like UK Biobank (thousands) (Sudlow et al. 2015). Accurate estimation of PAEE in such studies will be relevant so that personal advise can be given to older persons with respect to the most effective and still achievable beneﬁcial changes in lifestyle. This sums up to a computationally efﬁcient and accurate method to estimate PAEE in older adults. Furthermore, comparing the models built with SD, IQR and PD, there is no clear performance difference. That is because all three measures are similar in behaviour, and their main differences are in the magnitude of training values. Based on that, if we have to choose one of them, we believe that the SD model seems to be slightly better than the others, both in terms of R2 and RMSE. Adding to that, SD is more intuitive as a metric compared to IQR and PD. Therefore, for the rest of our analysis, we will focus on the model built with the following settings for accelerometer data: (1) a sequence A recurrent neural network architecture to model physical... 499 Table 8 Comparing models with participant-level data and/or activity classes R2 inR2 outR2 RMSE inRMSE outRMSE p-Value GRU 0.45 0.31 0.33 1.35 1.16 2.03 – GRUI D 0.55 0.41 0.36 1.25 1.09 2.05 0.02* GRUAC 0.42 0.32 0.35 1.33 1.14 1.96 0.38 GRUI D_AC 0.50 0.33 0.40 1.29 1.13 2.00 0.30 The p-value corresponds to the paired t-test of GRU (only accelerometry data) with any data addition, GRUI D (accelerometer and participant-level data), GRUAC (accelerometer and activity class data), and GRUI D_AC (accelerometer, participant-level and activity classes), in terms of R2, with * indicating when p < 0.05 Table 8 Comparing models with participant-level data and/or activity classes The p-value corresponds to the paired t-test of GRU (only accelerometry data) with any data addition, GRUI D (accelerometer and participant-level data), GRUAC (accelerometer and activity class data), and GRUI D_AC (accelerometer, participant-level and activity classes), in terms of R2, with * indicating when p < 0.05 size of 50 inputs, (2) representing a time window of 2min, (3) down-sampled to a resolution of SR = 0.42 Hz with SD. 5.2 Adding participant-level data results in better PAEE estimation Table 8 demonstrates the effect of participant-level data and activity classes in our RNN model to estimate PAEE (ﬁrst row marked GRU). The addition of participant level data (such us age, sex, height, weight, and BMI) improves the results, both in terms of R2 and RMSE error (GRUI D model). In more detail, the model’s performance improves signiﬁcantly (p = 0.02) from 0.45 (GRU) to 0.55 (GRUI D) for R2, while for RMSE the error decreased from 1.35 Kcal/min to 1.25. This development is mainly a result of the improved performance in the lower intensity activities (indoor activities), where RMSE decreased from 1.16 to 1.09 Kcal/min and R2 from 0.31 to 0.41. This is quite an important observation since it is mentioned in the literature that estimating PAEE of lower intensity activities is challenging (van Hees et al. 2009) and it seems that anthropometric data can help in this respect. On the other hand, adding activity labels doesn’t seem to improve the results (see GRUAC in Table 8), where the R2 drops (not signiﬁcantly though), and RMSE increases. Interestingly, the addition of (predicted) activity classes, even when com- bined with participant-level and accelerometer data (model GRUI D_AC), did not produce any signiﬁcant improvement to our model (R2 = 0.50, (p = 0.3)). This is a notable observation for our architecture since it contradicts with what is shown in previous work (Bonomi et al. 2009; Altini et al. 2015). It seems that the way RNNs model the input sequences and its ability to ‘remember’ past information, the exact activity labels are not needed for efﬁcient PAEE estimations. Therefore, when the objective is only PAEE estimation and not its association with speciﬁc activities, there is no need for applying activity recognition algorithms beforehand. Still, we need to mention that our dataset might not be ideal in order to prove this point, since activity windows are not equal and the breaks between each activity are not long enough to avoid the PAEE lag effect. 123 123 500 S. Paraschiakos et al. 5.2 Adding participant-level data results in better PAEE estimation Table 9 Comparing the different architectures of the ablation study, where t is the average training time per run in seconds Architecture R2 inR2 outR2 RMSE inRMSE outRMSE t (s) 5xGRU_3xDense 0.51 0.38 0.41 1.38 1.12 1.87 732.8 4xGRU_3xDense 0.49 0.40 0.45 1.41 1.13 1.92 656.5 3xGRU_3xDense 0.55 0.41 0.36 1.25 1.06 2.05 607.4 3xGRU_2xDense 0.5 0.39 0.42 1.28 1.09 1.97 589.8 2xGRU_3xDense 0.47 0.35 0.39 1.41 1.15 1.92 237.2 2xGRU_2xDense 0.52 0.37 0.44 1.30 1.08 2.00 191.6 1xGRU_2xDense 0.52 0.39 0.46 1.32 1.09 1.99 98.2 3xGRU_3xDense_a 0.50 0.33 0.35 1.32 1.09 2.00 377.1 3xGRU_3xDense_w 0.41 0.23 0.29 1.37 1.25 2.17 352.1 1xGRU_2xDense_a 0.45 0.27 0.42 1.38 1.16 2.03 60.8 1xGRU_2xDense_w 0.25 -0.01 0.02 1.57 1.35 2.60 56.6 ble 9 Comparing the different architectures of the ablation study, where t is the average training time r run in seconds 5.3 Ablation study For the ablation study, we are comparing our proposed architecture against simpliﬁed architectures to determine whether the increased complexity is warranted. As Table 9 demonstrates, in terms of R2 and RMSE, the proposed architecture is optimal (R2 = 0.55, RMSE = 1.25), although reasonably similar results could be obtained with archi- tectures with fewer layers, e.g. 2 GRU layers and 2 dense layers (2xGRU_2xDense). In more detail, separating indoor and outdoor activities, we note that simpler architectures with fewer GRU layers might perform better on outdoor activities, with the best results obtained by the simplest architecture tested 1xGRU_2xDense with an R2 = 0.46. This probably has to do with the fact that smaller networks are more robust against overﬁt- ting. On the other hand, for the challenging task of estimating PAEE of lower intensity activities (indoor activities), an extra GRU layer, as in the proposed model, is still the best option with an R2 = 0.41. Still, adding 2 further GRU layers has no beneﬁcial effect estimating indoor activities PAEE, see 5xGRU_3xDense. Subsequently, we study the beneﬁts of two devices versus only a single device on the ankle or wrist. The results demonstrate that a moderate price is paid for remov- ing one device from the proposed architecture [from R2 = 0.55 to R2 = 0.50 for 3xGRU_3xDense_a (−9%), and R2 = 0.41 for 3xGRU_3xDense_w (−25)%], whereas for 1xGRU_2xDense, removing a device incurs a large penalty: from R2 = 0.52 to R2 = 0.45 for 1xGRU_2xDense_a (−13.5)%, and R2 = 0.25 for 1xGRU_2xDense_w (−52%). We see the same phenomenon in RMSE and within the indoor and outdoor activities, showing that the proposed architecture is more robust against removal of a device. All in all, we observe that our proposed architecture (bold in Table 9) has a small advantage over the others, so if optimal accuracy is a priority, this architecture is the model of choice. However, if efﬁciency is important, quite reasonable results can still be obtained with smaller architectures. We feel the decent accuracy across the board 123 A recurrent neural network architecture to model physical... 501 Table 10 Comparing the different RNN layers RNN layer R2 inR2 outR2 RMSE inRMSE outRMSE GRU 0.55 0.41 0.36 1.25 1.06 2.05 LSTM 0.48 0.40 0.41 1.29 1.07 1.98 simpleRNN 0.13 −0.35 −0.05 2.02 1.66 2.62 is due to the rich representation of data (SD, 2-minute windows and anthropometric data). 5.3 Ablation study There appears to be no added beneﬁt for larger networks than the proposed one, although larger models still work reasonably well. Moving on to the bottom half of Table 9, where data from a single device is used, the proposed architecture (top two rows) handles the loss of data better than a smaller architecture (bottom two rows). This advantage was expected since we selected this architecture (3xGRU_3xDense) based on previous work of ours (Okai et al. 2019), where it was proven to be robust for the task of activity recognition under missing data. Concluding, if training speed is the priority, the simpler architectures are still reasonably accurate, but if there is a need for a model to be robust to data loss and lower intensity activities (indoor activities), the proposed one is preferred. 5.4 Comparing different RNN layers Adding to the ablation study, we replaced the GRU layers on our proposed architec- ture with 2 other RNN layers, the LSTM and simple RNN. The results in Table 10 demonstrate that interchanging the GRU layers with LSTM ones will not cost much of performance (R2 = 0.49) compared to the ones of simple RNN that did not manage to ﬁt at all (R2 = 0.12). However, training the same model with LSTM layers compared to GRU is more costly since every epoch takes almost 3 times longer than with the GRUs. Based on that, using GRU layers, we manage to have a better performance with a lower computational cost. 5.5 Our proposed model versus the state-of-the-art In order to fairly compare our best model (GRUI D) to the ones presented in related work (see Sect. 2), we performed our analysis in two steps. First, we compared our architecture to the convolution architecture proposed by Zhu et al. (2015) and then to the models proposed by Montoye et al. (2017a) and Ellis et al. (2014) (LM, LMM, RFr, ANN). We divided our analysis this way since the CNNs are tested with the original EEm (breath rate) while the rest of the methods are tested with 30-s aggregations of the target. In detail, we tested the CNN using two different data settings, one with windows of 5.12s for a sequences of 256 inputs (CNN5 sec), as suggested by the authors, and one with our best data set-up of 2-min windows aggregated with SD and sequence size = 50 (CNN2 min). Table 11 demonstrates in terms of R2 the performance per model and per devices. It is clear that the CNN developed with the short window of 5s 123 502 S. Paraschiakos et al. Table 11 Comparing R2 score of our proposed model (GRUI D) to CNN5sec for windows of 5s (Zhu et a 2015 set-up) and CNN2min with 2-min window Ankle & wrist Ankle Wrist Method R2 inR2 outR2 R2 inR2 outR2 R2 inR2 outR2 GRUI D 0.55 0.41 0.36 0.50 0.33 0.35 0.41 0.23 0.30 CNN5 sec 0.29 −0.12 0.21 0.20 −0.13 0.19 −0.05 −0.45 −0.08 CNN2 min 0.49 0.28 0.36 0.37 0.23 0.24 0.29 −0.19 −0.05 Table 12 Comparing R2 score of our proposed model (GRUI D) with RFr-random forest regressor (Ellis et al. 2014), LMM-linear mixed model, ANN-artiﬁcial neural network and LM-linear regression (Montoye et al. 2017a), using 30-s features Ankle & wrist Ankle Wrist Method R2 inR2 outR2 R2 inR2 outR2 R2 inR2 outR2 GRUI D 0.72 0.60 0.56 0.65 0.53 0.62 0.55 0.40 0.49 RFr 0.55 0.30 0.43 0.57 0.23 0.43 0.33 0.03 0.17 LMM 0.43 −0.07 0.47 0.29 −0.26 0.41 0.24 −0.19 −0.22 ANN 0.37 0.06 0.27 0.50 0.12 0.45 0.20 −0.26 0.24 LM 0.35 −0.12 0.45 0.24 −0.37 0.41 0.22 −0.37 −0.12 (CNN5 sec) fails to explain enough of the EEm variation for all set-ups. On the other hand, the CNN2 min has a somewhat comparable performance to our set-up (GRUI D) for ankle and wrist data combined, with an R2 = 0.49 versus R2 = 0.55. 5.5 Our proposed model versus the state-of-the-art However, when we compare per single device, the GRUI D set-up clearly outperforms the CNN with R2 = 0.50 for ankle and R2 = 0.41 for wrist, compared to the CNN with R2 = 0.37 and R2 = 0.29, for ankle and wrist respectively. (CNN5 sec) fails to explain enough of the EEm variation for all set-ups. On the other hand, the CNN2 min has a somewhat comparable performance to our set-up (GRUI D) for ankle and wrist data combined, with an R2 = 0.49 versus R2 = 0.55. However, when we compare per single device, the GRUI D set-up clearly outperforms the CNN with R2 = 0.50 for ankle and R2 = 0.41 for wrist, compared to the CNN with R2 = 0.37 and R2 = 0.29, for ankle and wrist respectively. To conclude, when our approach is compared to another deep learning approach, we see that the performance of our model outperforms the one of the CNNs both in combined ankle and wrist, as well as when compared to single-device settings. Remarkably, we can observe here too the effect that our robust representation of data (similar to the ablation study) has on the performance of the tested CNN model. Furthermore, it is clear that using longer windows of training data, down-sampled with SD, gives a big advantage in explaining PAEE variance. Next, Table 12 demonstrates the performance of the proposed model and the meth- ods trained with handcrafted features and both tested with 30-s EEm aggregations. Here, we observe that our method clearly outperforms all others discussed above. In detail, the GRUI D model manages to explain almost 72% of the total EEm variation (aggregated EEm) when both ankle and wrist predictors are used, while the second best (RFr) explains only 55%. When data from indoor activities only is used for test- ing, our model captures 60% of EEm variation which is double the second method (RFr) in performance. Similar to that, when data from ankle or wrist alone are used, our model explains more than 50% of aggregated EEm variation, with R2 = 0.65 for ankle and R2 = 0.55 for wrist. 123 A recurrent neural network architecture to model physical... 503 Fig. 8 Scatter plot of mean true over mean predicted EEm, per participant (left) and activity (right) Fig. 5.5 Our proposed model versus the state-of-the-art 8 Scatter plot of mean true over mean predicted EEm, per participant (left) and activity (right) When comparing the rest of the methods, the random forest regressor (RFr) clearly outperforms LMM, ANN and LM with an R2 = 0.55 for the ankle and wrist combined predictors and R2 = 0.57 for the ankle only. Interestingly, ANN performs pretty well when only ankle predictors are used for training with an R2 = 0.50, however this per- formance is mainly based to the higher intensity activities (outdoor walking, cycling) since inR2 = 0.12 while outR2 = 0.45. Still, when wrist and ankle are combined, ANN’s performance drops to R2 = 0.27. On the contrary, the LMM manages to cap- ture quite well the variation of the outdoor EEm for both ankle and wrist predictors combined (R2 = 0.47) and for ankle alone (R2 = 0.41). Finally, we observe for all models that using wrist features alone is not enough to explain enough of the EEm variation. Summarizing,comparingourmodeltomethodsthatuseasetofhandcraftedfeatures aspredictors,itisclearthatourapproachoutperformstherestofthemodelingmethods. This is probably due to our approach being able to incorporate longer windows of predictors data. This way, EEm bias from a longer activity time is taken into account. In order to fairly compare our model with the other modeling choices, we had to test our model in aggregated windows of the target (30-s aggregations). Such aggregation has the effect of smoothing the per-breath PAEE measurements, creating a less noisy target. As a result, the over- or under-estimations are also smoothed out and the model performance seem improved. This model application is close to how we intend to use our model with free-living accelerometer data in the future. Therefore, in next section, Table 12 we also present the performance of our approach with similar scenarios of target aggregation. 5.6 Demonstration of GRUID model estimating PAEE 505 Table 13 Comparing true and predicted EEm over different aggregations Aggregation R2 inR2 outR2 RMSE inRMSE outRMSE Per breath 0.55 0.41 0.36 1.25 1.09 2.04 10s 0.65 0.51 0.50 0.95 0.89 1.58 30s 0.72 0.60 0.56 0.82 0.74 1.40 1min 0.78 0.67 0.62 0.76 0.64 1.34 5min 0.82 0.78 0.63 0.62 0.48 0.97 60min 0.86 – – 0.40 – – Table 13 Comparing true and predicted EEm over different aggregations Adding to that, in Fig. 9, we plotted the predicted over true PAEE (COSMED) values (recordings per-breath) for 3 participants that performed both indoor and outdoor activities and have the worst, median and best ﬁt, R2 = 0.35, R2 = 0.55 and R2 = 0.80 respectively. Here, we see that the model overall captures nicely the trend of the true EEm,astheblackline(predictedEEm)followsthelonger-termchangesofthegreyline (true EEm). However, the short-term behaviour of the target is not captured sufﬁciently, except for the sudden changes. We need to point out here that, while our models are tested on the real data (per breath), they were trained with averaged EEm values of 10s both for smoothing out any non-generalizable noise (high peaks in grey in Fig. 9) of the data and for training with a stable sampling rate, since COSMED produces data per breath. As a result of this choice, we can see that our predicted EEm values do not capture the high-frequency ﬂuctuations, but follow the average trend on a 10-second scale. Finally, the gap in the middle of the plots is the transition in the protocol from indoor to outdoor activities, where no COSMED measurements took place. Subsequently, in Table 13, we present the performance of the GRUI D model by different target aggregations. We aggregated the original and predicted EEm values at 10 and 30, and at 1, 5 and 60min. This is really useful, since in a free-living setting, the model will be used to estimate PAEE for aggregated windows. From Table 13, it is observed that even with the shorter window of aggregation (10s) the model’s performance improves substantially both for R2, from 0.55 to 0.65, and RMSE, from 1.25 to 0.95 Kcal/min. Additionally, for commonly used time frames of 30-second and 1-minute windows (Staudenmayer et al. 2009; Montoye et al. 2017a; Ellis et al. 2014; O’Driscoll et al. 5.6 Demonstration of GRUID model estimating PAEE To appreciate the general performance of the GRUI D model, consider Figs. 8 and 9. In Fig. 8, we see two scatter plots of the average true over average predicted EEm value per participant (left) and per activity class (right). In the left plot, the dots (in blue) 123 504 S. Paraschiakos et al. Fig. 9 True versus Predicted EEm per breath for participants with lowest (top), median (middle) and higher (bottom) R2 examples, when indoor and outdoor activities are included Fig. 9 True versus Predicted EEm per breath for participants with lowest (top), median (middle) and higher (bottom) R2 examples, when indoor and outdoor activities are included display the participants with both indoor and outdoor activities, while the diamonds (in green) represent participants with only indoors data. Adding to that, the trend line (in red) is used to compare the predictions to the main diagonal (blue, representing x = y) which is the ground truth. From that, we observe that our model has on average a good ﬁt. However, it slightly overestimates the lowest EEm values (red line above main diagonal) and underestimates the highest ones (red line below main diagonal). In the right plot of Fig. 8, we can observe that our model captures really well the average EEm per activity since the average PAEE estimated for all the activities is either on or really close to the ground truth line. However, evaluating the performance over one activity class averages out a lot of the EEm signal. display the participants with both indoor and outdoor activities, while the diamonds (in green) represent participants with only indoors data. Adding to that, the trend line (in red) is used to compare the predictions to the main diagonal (blue, representing x = y) which is the ground truth. From that, we observe that our model has on average a good ﬁt. However, it slightly overestimates the lowest EEm values (red line above main diagonal) and underestimates the highest ones (red line below main diagonal). In the right plot of Fig. 8, we can observe that our model captures really well the average EEm per activity since the average PAEE estimated for all the activities is either on or really close to the ground truth line. However, evaluating the performance over one activity class averages out a lot of the EEm signal. A recurrent neural network architecture to model physical... 5.6 Demonstration of GRUID model estimating PAEE 2020), the model has an RMSE of only 0.86 and 0.76 Kcal/min respectively with the predictions explaining more than 70% of the original EEm signal variation for both aggregations, with R2 = 0.78 for the 1min. Finally, we can observe that our model has different performance for indoor and outdoor activities. In detail, when comparing windows of 30s, for indoor activities the RMSE is equal or less that 1.0 Kcal/min, while for outdoor activities, RMSE is much higher (walking RMSE = 1.20; cycling RMSE = 1.50), see Table 13. Characteris- tically, when we compare our model’s performance per participant, we see a slight overestimation of EEm for those with only indoor activities (low-intensity activities) and a slight underestimation for those with high average EEm, see Fig. 8. Especially, when ordering by R2 (see Fig. 10), we can clearly see that for participants with lower median EEm, the model explains less of its variance (lower R2), while for participants 123 S. Paraschiakos et al. 506 Fig. 10 Box plots of mean True EEm per participant ordered by R2 Fig. 10 Box plots of mean True EEm per participant ordered by R2 with longer range of EEm values, the model does capture the variation. This may be due to the fact that for low EEm values, even a small RMSE error can lead to lower R2. We indeed observe lower RMSE for indoor activities, while they produce lower R2 values. We conclude that the proposed RNN model on average performs reasonably well for both high and low intensity activities (see Fig. 8). Because estimating PAEE from sedentary or low-intensity activities is considered challenging (van Hees et al. 2009), our proposed RNN model makes an important contribution. 6 Conclusion and future work In this paper, we developed and tested a recurrent neural network architecture based on an efﬁcient down-sampling method that incorporates standard deviation for down- sampling the input data to estimate physical activity energy expenditure within an elderly population. This approach is based on accelerometers at two body locations (wrist and ankle) and is able to take advantage of long time windows of predictor data (2min to predict reasonably accurately the PAEE of older individuals. Moreover, the inclusion of participant-level data like age, gender and body composition further improved the accuracy of PAEE estimation, especially in activities with lower intensity ranges. In summary, the results of this study demonstrate that RNNs incorporating GRU layers can solve the challenge of PAEE estimation. While they do not require any com- plex feature construction steps and can be trained with lower-resolution accelerometer data, if this is down-sampled with statistical dispersion metrics, RNNs produce PAEE estimations similar or better than competing methods. Because RNNs take into account longer windows in activity history without increasing the size and dimensionality of A recurrent neural network architecture to model physical... 507 their input, we believe that such modeling techniques are attractive when applied to free-living accelerometer data that is collected in a continuous way. Subsequently, our proposed down-sampling using statistical dispersion metrics (like standard deviation) proves to be really efﬁcient since we achieved better results using ten times less data compared to averaging. Additionally, this strategy gives us the advantage of incorpo- rating longer windows of prior sensor data with lower computational cost which also lead to better PAEE estimation. Finally, adding participant-level data (age, weight, height) when training our model can improve PAEE estimation signiﬁcantly. Duringthedevelopmentofourmodels,werealizedthattheGOTOVdatasetinvolves some data collection limitations. Indirect calorimetry was collected in a continuous way with only small breaks in between (max 1 minute). The rather small breaks between activities might make it difﬁcult to estimate the EEm outcome per speciﬁc activityduetotheenergyexpenditure’slageffect.Indetail,withoutlongdiscriminating breaks between activities, it is likely that past activities inﬂuence the EEm records of future ones. Additionally, we did not randomise the order of activities which might have introduced a slight bias in our training. For these reasons, it would have been interesting to test our ﬁndings in other similar labeled datasets with older individuals. However, to the best of our knowledge, GOTOV is as yet the only publicly available PAEE dataset with a focus on older individuals. OpenAccess ThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If 6 Conclusion and future work 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/. 6 Conclusion and future work Summarising, if there is no need to predict PAEE per speciﬁc activity, such as in our setting, this data collection can be used nicely to represent free-living conditions. The RNN modeling advantage enabled taking into account preceding activity infor- mation by incorporating data of longer windows and letting the model decide on which information to emphasize on. The great advantage of the GOTOV dataset is that there are a satisfactory number of participants and that this data set is dedicated to people over 60 years of age. Because PAEE monitoring within the elderly might help stimulate vital and healthy ageing, the GOTOV dataset is perfectly suited for the development of activity recognition and PAEE estimation models. Applying such a model to free-living data collections was one of the motivations of our study. In our future work, we intent to apply our modeling technique to physical activity and lifestyle improvement intervention studies on older individuals. From such an application, we envision that better insights in energy expenditure of older people will contribute to better physical behaviour guidelines for them to stay healthy, and potentially further stimulate vital and healthy ageing. In order to achieve this, we aim to build characteristic features of PAEE levels and PA types of long time periods (weeks, months) and relate them with parameters of metabolic health, general health and well-being. These relations between life style and health can then be turned into distinct recommendations for effectively maintaining mobility among older adults and a continuous monitoring system to track the adherence and improvement of metabolic health. OpenAccess ThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If 12 3 3 508 S. Paraschiakos et al. 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/. A Table of Abbreviations across the paper Table 14 Abbreviations across the paper in alphabetical order Abbreviations a GENEActiv ankle ANN Artiﬁcial neural network BMI Body mass index CNN Convolutional neural network CNN2min CNN trained with 2min window of data CNN5sec CNN trained with 5s window of data DLW Doubly labeled water technique EE Energy expenditure EEh Energy expenditure per hour EEm Energy expenditure per minute GOTOV Growing old together Validation study GRU Gated recurrent unit GRUAC GRU trained with accelerometer and activity classes data GRUI D GRU trained with accelerometer and participant-level data GRUI D_AC GRU with accelerometer, participant-level and activity classes data HR Heart rate inR2 Indoors R-squared inRMSE Indoors root mean squared error IQR Interquartile range K4 COSMED K4b2 LM Linear model LMM Linear mixed model LOSO-CV Leave one subject out cross validation LSTM Long short-term memory LUMC Leiden University medical center MET Metabolic equivalent of task MSE Mean squared error outR2 Outdoors R-squared Table 14 Abbreviations across the paper in alphabetical order 123 12 A recurrent neural network architecture to model physical... 509 Table 14 continued Abbreviations outRMSE Outdoors root mean squared error PA Physical activity PAEE Physical activity energy expenditure PD Percentile difference REE Resting energy expenditure ReLU Rectiﬁed linear unit RF Random forest RFr Random forest regressor RMSE Root mean squared error RNN Recurrent neural network SD Standard deviation SR Sampling rate SVM Signal vector magnitude t Average training time TEE Total energy expenditure TEF Thermic effect of food VCO2 Volume of carbon dioxide VO2 Volume of oxygen w GENEActiv wrist B Open-source code To make our experiments, scripts and results accessible to other researchers (open- source) we share them on the following git repository, https://github.com/parastelios/ GOTOV_PAEE_publication. 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https://openalex.org/W2893545572	https://europepmc.org/articles/pmc6023591?pdf=render	English	null	Intake of Different Dietary Proteins and Risk of Heart Failure in Men	Circulation. Heart failure	2,018	cc-by	9,392	Circulation: Heart Failure Circulation: Heart Failure Circulation: Heart Failure ORIGINAL ARTICLE Key Words: calcium ◼ dietary proteins ◼ heart failure ◼ men ◼ prospective studies © 2018 The Authors. Circulation: Heart Failure is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. 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 that the original work is properly cited. http://circheartfailure.ahajournals.org The Kuopio Ischaemic Heart Disease Risk Factor Study BACKGROUND: Animal and plant protein intakes have indicated opposite associations with cardiovascular mortality risk. Whether dietary proteins are associated with risk of heart failure (HF) is unclear. Thus, we examined the associations of proteins from different food sources with risk of HF. Heli E.K. Virtanen, MSc Sari Voutilainen, PhD Timo T. Koskinen, MSc Jaakko Mursu, PhD Tomi-Pekka Tuomainen, MD, PhD Jyrki K. Virtanen, PhD Heli E.K. Virtanen, MSc Sari Voutilainen, PhD Timo T. Koskinen, MSc Jaakko Mursu, PhD Tomi-Pekka Tuomainen, MD, PhD Jyrki K. Virtanen, PhD METHODS AND RESULTS: The study included 2441 men aged 42 to 60 years at the baseline examinations in 1984 to 1989 in the Kuopio Ischaemic Heart Disease Risk Factor Study. Protein intakes at baseline were assessed with 4-day dietary records. Data on incident HF cases were obtained from national registers. HF risk according to protein intake was estimated by Cox proportional hazard ratios. During the mean follow- up of 22.2 years, 334 incident HF cases occurred. Higher intake of total protein indicated a trend toward increased risk of HF (multivariable- adjusted hazard ratio in the highest versus lowest quartile=1.33; 95% confidence interval: 0.95–1.85; P-trend=0.05). The associations between specific types and sources of protein with incident HF were consistent with this overall finding although not all associations reached statistical significance. For example, the hazard ratio in the highest versus lowest quartile was 1.43 (95% confidence interval: 1.00–2.03; P-trend=0.07) for total animal protein and 1.17 (95% confidence interval: 0.72–1.91; P-trend=0.35) for total plant protein. CONCLUSIONS: In middle-aged men, higher protein intake was marginally associated with increased risk of HF. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT03221127 CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT03221127 Key Words: calcium ◼ dietary proteins ◼ heart failure ◼ men ◼ prospective studies METHODS The data, analytic methods, and study materials will not be made available to other researchers for purposes of reproduc- ing the results or replicating the procedure. Circ Heart Fail. 2018;11:e004531. DOI: 10.1161/CIRCHEARTFAILURE.117.004531 Circ Heart Fail. 2018;11:e004531. DOI: 10.1161/CIRCHEARTFAILURE.117.004531 1 June 2018 Virtanen et al; Dietary Proteins and Risk of Heart Failure Virtanen et al; Dietary Proteins and Risk of Heart Failure To elucidate the role of dietary proteins in risk of HF, we examined whether total, animal, or plant pro- tein intakes are related to HF risk in a population of middle-aged and older Finnish men. We also compared the associations of proteins from more specific animal and plant sources. In the secondary analyses, we inves- tigated the associations of intakes of the main dietary protein sources with HF risk. Baseline Measurements One factor contributing to cardiac function and onset of HF may be dietary protein. Substituting either plant or animal protein for carbohydrates has lowered blood pressure in experimental studies.10,11 Because high blood pressure is a major risk factor for HF,1 pro- tein intake could also play a beneficial role in the patho- genesis of HF. Benefits are suggested also by other studies: sufficient protein intake (>0.7 g/kg ideal body weight) was associated with lower cardiovascular and all-cause mortality in hypertensive patients,12 and ami- no acid supplementation prevented cardiac dysfunction in patients with type 2 diabetes mellitus.13,14 Fasting venous blood samples were collected between 8 am and 10 am. Subjects were instructed to abstain from ingest- ing alcohol for 3 days and from smoking and eating for 12 hours before providing the sample. Detailed descriptions of the determination of serum lipids,19 lipoproteins,19 and magnesium20 and the assessment of medical history and medications,19 family history of diseases,19 smoking,19 alco- hol consumption,19 physical activity,21 and blood pressure19 have been published. Education years, annual income, mari- tal status, and dietary supplement use were obtained from self-administered questionnaires. Family history of coronary heart disease (CHD) was defined as positive if a first-degree relative of the participant had a CHD history. Body mass index (BMI) was computed as the ratio of weight in kilograms to the square of height in meters. Serum creatinine was mea- sured with the clinical chemistry analyzer Kone Specific (KONE Instruments Oy, Espoo, Finland) using Jaffe reaction, and esti- mated glomerular filtration rate was calculated by the Chronic Kidney Disease Epidemiology Collaboration formula.22 Instead of considering only the total protein intake, prospective studies have indicated that proteins from different dietary sources may be differentially related to health. For example, higher intake of animal pro- tein has been associated with increased cardiovascu- lar mortality in some observational studies15,16 while plant protein intake has had an inverse association.15,17 Whether different proteins with specific amino acid compositions have distinct roles also in relation to HF risk remains to be established. Study Population • This study suggests that high protein intake may not be the optimal dietary strategy in the preven- tion of heart failure. The KIHD (Kuopio Ischaemic Heart Disease Risk Factor Study) was designed to investigate risk factors for cardiovascular dis- ease (CVD), atherosclerosis, and related outcomes in a popu- lation based, randomly selected sample of men from eastern Finland.18 The baseline examinations were performed in 1984 to 1989. A total of 2682 men who were 42, 48, 54, or 60 years old at baseline (83% of those eligible) were recruited in 2 cohorts (Figure I in the Data Supplement). Re-examinations were conducted 4, 11, and 20 years after the baseline. The baseline characteristics of the entire study population have been described previously.18 The Research Ethics Committee of the University of Kuopio has approved the KIHD study, and dietary compound of KIHD has been registered at Clinicaltrials. gov. All subjects gave written informed consent. Subjects with diagnosed HF at baseline (n=194), unknown HF status (n=7), and those with missing data on dietary intakes (n=40) were excluded, leaving 2441 men for analyses. D espite the achieved improvements in heart fail- ure (HF) management, the disease dramatically shortens life expectancy,1 highlighting the need for efficient prevention. Investigations on dietary ap- proaches concentrating on HF prevention are scarce. Prospective studies have suggested that greater intake of fish or marine omega-3 polyunsaturated fatty acids2,3 and whole grain products4,5 may associate with lower HF risk. In contrast, greater intake of total2 or red meat6 and especially processed red meat7,8 or frequent intake of eggs9 may associate with increased HF risk. Results from previous studies are, nevertheless, inconclusive, and it is unclear which factors explain the differential relations of the protein sources with HF risk. D WHAT IS NEW? • Despite the increasing popularity of high-protein diets, the role of dietary protein in relation to heart failure risk has not been previously evaluated. • Despite the increasing popularity of high-protein diets, the role of dietary protein in relation to heart failure risk has not been previously evaluated. y • We observed that high total protein intake was marginally associated with increased risk of heart failure. • The associations with proteins from different sources were in accordance with the overall find- ing, although not all associations reached statisti- cal significance. • The associations with proteins from different sources were in accordance with the overall find- ing, although not all associations reached statisti- cal significance. Circ Heart Fail. 2018;11:e004531. DOI: 10.1161/CIRCHEARTFAILURE.117.004531 Dietary Assessment Baseline food consumption was assessed with a food record of 4 days, one of which was a weekend day, by using Circ Heart Fail. 2018;11:e004531. DOI: 10.1161/CIRCHEARTFAILURE.117.004531 June 2018 2 Virtanen et al; Dietary Proteins and Risk of Heart Failure monounsaturated, polyunsaturated, and trans fatty acids and fiber. In addition, the disease status was updated throughout the follow-up, and the diseases and medications mentioned above were included as time-dependent covariates. Model 2 was also mutually adjusted for other proteins. Models that include protein and fat but not carbohydrates can be inter- preted as replacement of carbohydrates with the protein in interest. All quantitative variables were entered in the models as continuous variables. The cohort mean was used to replace missing values in covariates (<2.4%). Tests of linear trend were conducted by assigning the median values for each category of exposure variable and treating those as a single continuous variable. Marital status, use of dietary supple- ments, serum total cholesterol/high-density lipoprotein cho- lesterol ratio, serum triglycerides, serum magnesium, baseline estimated glomerular filtration rate, history of atrial fibrilla- tion, cardiomyopathy, stroke, valvular defect, myocarditis or chronic obstructive pulmonary disease, or intake of fruits and vegetables were not included in the models because they did not appreciably affect the associations (change in estimates <5%). Potential nonlinear associations were assessed semi- parametrically using restricted cubic splines with 3 knots. To assess replacement of proteins with each other, all proteins were simultaneously added into the models, and the differ- ence of regression coefficients of 2 proteins of interest, their variance, and covariance were used for calculating HRs and 95% confidence intervals (CIs). household measures. A picture book of common foods and dishes was used to help in the estimation of portion sizes. To further improve accuracy, instructions were given, and completed records were checked by a nutritionist together with the participant. Nutrient intakes were estimated with NUTRICA 2.5 software (Social Insurance Institution, Turku, Finland). The software’s databank is mainly based on Finnish nutrient composition values of foods. We calculated protein coming from different sources (Table I in the Data Supplement). Total meat included red meat, white meat, and offal. Processed red meat included industrially processed red meat. For the analyses with major plant protein sources, we combined the most protein-rich plant foods, that is, grain products, legumes, nuts, and seeds. Dietary calcium included calcium from food sources but not from supplements. Diseases Incident HF cases between study entry and December 31, 2014, were obtained by computer linkage to the National Hospital Discharge Register (maintained by the National Institute for Health and Welfare in Finland). The International Classification of Disease, Tenth Revision codes I11.0 and I50.0-I50.9 were used for diagnostic classification of HF cases. There were no losses to follow-up. In the secondary analyses, we investigated the associa- tions of the protein sources with risk of HF. Identical list of covariates were used as in the main analysis. However, the possible effect mediators, that is, intakes of saturated, mono- unsaturated, polyunsaturated, and trans fatty acids and fiber, were not added to model 2, but instead an additional model 3 with these factors was created. Data on other incident diseases or medications during the follow-up were obtained by record linkage to the National Hospital Discharge Register and to the Social Insurance Institution of Finland register for reimbursement of medicine expenses or by diagnosis at the re-examination rounds. Dietary Assessment Each nutrient was energy adjusted by the residual method.23 Statistical Analysis The statistical significance of the interactions with baseline disease status (CHD or use of cardiac medications, diabetes mellitus, or hypertension), CHD status, BMI (below versus above median), and smoking status was assessed by likeli- hood ratio tests with the use of a cross-product term. All P values were 2-tailed (α=0.05). Data were analyzed with SPSS 21.0 for Windows (IBM Corp, Armonk, NY) and Stata 13.1 (Stata Corp, College Station, TX; for spline analysis). The univariate relations of total, animal, and plant protein intakes with baseline characteristics were assessed by means and linear regression (continuous, normally distributed vari- ables), by medians and Jonckheere–Terpstra test (continu- ous, skewed variables), or by χ2 tests (categorical variables). Correlations between intakes of different proteins were esti- mated by Spearman correlation coefficients. Person-years of follow-up were calculated from the base- line to the date of HF diagnosis, death, or the end of follow- up, whichever came first. Cox proportional hazards regression models were used to estimate hazard ratios (HRs) in expo- sure quartiles, with the lowest category as the reference. Schoenfeld residuals did not indicate significant evidence of violation of the proportional hazards assumption. Absolute risk (AR) change was calculated by multiplying the AR in the reference group by the multivariable-adjusted HR change in the comparison group. Baseline Characteristics The mean protein intake was 93.2 g/d (15.8% of total energy intake), of which 70.0% was from animal sourc- es and 27.7% from plant sources (Table II in the Data Supplement). Of the total protein intake, 2.3% was from mixed sources and was not included into either animal or plant protein. Dairy (28.8 g/d), meat (24.7 g/d), and fish (8.1 g/d) were the major animal protein sources whereas most of the plant protein came from grain products (20.5 g/d) and potatoes (2.4 g/d). Covariate selection was based on literature of identified and potential risk factors for HF or on associations with expo- sures and outcomes in the present analysis. Model 1 included age, examination year, and energy intake. The multivariable model (model 2) included the variables in model 1 plus edu- cation years, income, pack-years of smoking, alcohol intake, leisure-time physical activity, BMI, family history of CHD, baseline disease status (CHD or use of cardiac medications, diabetes mellitus or hypertension), and intakes of saturated, Table 1 shows the baseline characteristics of the study population. Men with greater total protein intake were younger, more likely to be married, had longer educa- tion, and higher income than those with lower protein June 2018 Circ Heart Fail. 2018;11:e004531. DOI: 10.1161/CIRCHEARTFAILURE.117.004531 3 1:e004531. DOI: 10.1161/CIRCHEARTFAILURE.117.00453 Virtanen et al; Dietary Proteins and Risk of Heart Failure Table 1. Baseline Characteristics According to Total, Animal, and Plant Protein Intake Among 2441 Men From the Kuopio Ischemic Heart Disease Risk Factor Study* istics According to Total, Animal, and Plant Protein Intake Among 2441 Men From the Kuopio Ischemic Heart Disease Table 1. Baseline Characteristics Baseline Characteristics According to Total, Animal, and Plant Protein Intake Among 2441 Men From the Kuopio Ischemic Heart Disease Risk Factor Study* Characteristic Total Protein Animal Protein Plant Protein Quartile 1 Median Intake 78.4 g/d Quartile 4 Median Intake 109.1 g/d Quartile 1 Median Intake 49.2 g/d Quartile 4 Median Intake 82.2 g/d Quartile 1 Median Intake 19.6 g/d Quartile 4 Median Intake 32.3 g/d Subjects, n 610 610 610 610 610 610 Demographic and lifestyle factors Age, y 53.5±4.7 52.4±5.7† 53.7±4.7 52.3±5.8† 52.3±5.3 53.2±5.2† Education, y 8.2±3.1 9.1±3.7† 8.3±3.2 9.0±3.6† 8.3±3.1 8.8±3.6† Income, 1000 € 10.4 (8.2) 12.6 (9.8)† 10.8 (8.7) 12.4 (10.1)† 11.4 (9.8) 12.1 (8.7)† Married, % 83.1 89.5† 83.6 87.7† 84.4 88.2 Current smoker, % 34.6 30.0 28.7 35.6† 47.9 17.7† Regular use of dietary supplements, % 6.1 8.2 5.7 7.5 4.9 9.0† Alcohol intake, g/wk 29 (108) 37 (89) 19 (72) 48 (108)† 88 (171) 12 (36)† Leisure-time physical activity, kcal/d 72 (150) 95 (153)† 81 (167) 80 (150) 65 (139) 106 (179)† Body mass index, kg/m2 26.3±3.3 27.5±3.8† 26.2±3.1 27.6±3.8† 27.0±3.7 26.3±3.4† Health and disease status Serum total cholesterol to HDL ratio 4.49 (1.80) 4.58 (1.83) 4.43 (1.81) 4.53 (1.77) 4.52 (1.92) 4.51 (1.88) Serum triglycerides, mmol/L 1.07 (0.65) 1.10 (0.78) 1.09 (0.70) 1.08 (0.82) 1.03 (0.74) 1.12 (0.76) Serum magnesium, mg/L 19.8±1.6 19.7±1.5 19.8±1.6 19.7±1.5 19.9±1.6 19.8±1.5 Systolic blood pressure, mm Hg 134±17 134±17 134±17 135±18 134±18 133±16 Diastolic blood pressure, mm Hg 89±10 89±11 88±10 89±11† 89±11 88±10† Estimated glomerular filtration rate, mL/min 85.2±13.0 85.9±13.1 84.8±12.6 86.5±13.1† 87.0±12.6 84.9±12.6† Family history of CHD, % 46.4 53.3† 47.4 51.0 43.3 50.3† CHD at baseline, % 23.3 21.8 23.9 22.6 23.4 20.0 CHD during follow-up, % 19.2 20.0 19.5 19.8 19.7 19.7 Cardiac medication at baseline, % 2.6 3.6 4.9 2.8† 1.3 6.1† Cardiac medication during follow-up, % 62.5 65.1 62.3 63.6 62.0 63.1 Diabetes mellitus at baseline, % 3.6 7.0† 4.3 7.2† 4.3 5.7 Diabetes mellitus during follow-up, % 18.9 22.3 17.0 22.3† 22.6 17.5† Hypertension at baseline, % 59.5 60.0 59.7 59.2 57.5 58.5 Hypertension during follow-up, % 24.4 25.6 24.4 26.2 27.2 26.2 Atrial fibrillation at baseline, % 0.2 0.8 0.5 0.8 1.0 1.0 Atrial fibrillation during follow-up, % 15.7 14.3 16.6 13.8 13.4 17.9 Cardiomyopathy at baseline, % 1.3 1.0 1.5 1.3 2.2 1.7 Cardiomyopathy during follow-up, % 0.3 1.1 0.5 1.0 0.8 1.0 History of stroke at baseline, % 3.0 2.3 3.6 2.6 2.5 1.6 Stroke during follow-up, % 10.8 10.7 11.6 10.5 9.3 10.7 Valvular defect during follow-up, % 4.1 3.4 3.9 3.6 3.3 5.2 Myocarditis during follow-up, % 0.2 0.2 0.2 0.2 0.5 0.0† Chronic obstructive pulmonary disease at baseline, % 11.0 8.9 10.8 9.5 8.5 6.6 Chronic obstructive pulmonary disease during follow-up, % 4.1 2.8 3.8 2.5 4.6 3.0 Dietary factors Energy, kcal/d 2546±666 2544±636 2541±678 2544±647 2551±657 2534±627 Protein, g/d 76.3±7.4 111.8±9.8† 78.0±8.8 110.5±11.0† 93.0±16.1 93.5±14.5 Protein, E% 12.9±1.1 18.8±2.0† 13.1±1.3 18.5±2.2† 15.6±2.6 15.7±2.4 (continued ) Circ Heart Fail. Baseline Characteristics 2018;11:e004531. DOI: 10.1161/CIRCHEARTFAILURE.117.004531 June 2018 4 Circ Heart Fail. 2018;11:e004531. DOI: 10.1161/CIRCHEARTFAILURE.117.004531 Virtanen et al; Dietary Proteins and Risk of Heart Failure Animal protein, g/d 48.9±9.0 83.7±11.8† 47.0±7.6 85.1±10.3† 72.2±16.2 57.8±14.9† Animal protein, E% 8.2±1.4 14.0±2.2† 7.9±1.2 14.3±2.0† 12.1±2.6 9.7±2.4† Plant protein, g/d 25.2±6.4 26.0±6.3 28.7±6.7 23.4±5.6† 18.8±3.1 33.5±4.2† Plant protein, E% 4.2±1.0 4.3±1.0 4.8±1.1 3.9±0.9† 3.2±0.4 5.6±0.7† Fat, E% 39.9±6.5 37.5±5.9† 38.1±6.2 38.9±6.0† 41.9±6.2 35.3±5.2† SFAs, E% 19.4±4.4 17.0±3.8† 18.2±4.3 17.9±4.0 20.2±4.3 16.2±3.7† PUFAs, E% 4.2±1.5 4.7±1.3† 4.3±1.4 4.7±1.4† 4.5±1.5 4.5±1.3 MUFAs, E% 11.7±2.4 11.7±2.3 11.2±2.2 12.1±2.3† 12.7±2.4 10.7±2.0† Trans fatty acids, E% 1.1±0.4 1.0±0.4† 1.1±0.4 1.0±0.4† 1.1±0.4 1.0±0.4† Carbohydrates, E% 43.7±7.1 41.2±6.3† 46.1±6.5 39.4±5.9† 37.4±5.8 47.8±5.2† Fiber, g/d 24.4±7.7 26.0±7.8† 27.5±8.2 23.3±7.0† 18.5±4.4 32.3±7.1† Calcium, mg/d 1035±291 1526±387† 1032±288 1530±390† 1357±404 1241±376† Fruits, berries, and vegetables,‡ g/d 208 (202) 258 (195)† 230 (196) 234 (198) 179 (183) 276 (201)† Whole grain products, g/d 149 (92) 163 (96)† 170 (108) 146 (88)† 113 (64) 211 (101)† Unprocessed red meat, g/d 55 (47) 90 (78)† 53 (46) 93 (79)† 73 (69) 61 (64)† Processed red meat, g/d 58 (74) 65 (81)† 47 (65) 73 (90)† 84 (92) 44 (59)† Fish, g/d 18 (43) 60 (89)† 17 (44) 60 (91)† 35 (70) 27 (58)† Nonfermented dairy, g/d 485±308 563±371† 457±290 592±385† 610±384 477±301† Fermented dairy, g/d 51 (167) 185 (360)† 56 (176) 171 (350)† 106 (265) 104 (260) CHD indicates coronary heart disease; E%, percentage of energy intake; HDL, high-density lipoprotein; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; and SFA, saturated fatty acid. Values are mean±SD for normally distributed variables, median (interquartile range) for skewed variables, and percentages for categorical variables. †P for trend across quartiles <0.05. P-trend was assessed with linear regression (normally distributed variables), Jonckheere–Terpstra test (skewed variables), or with χ2 test (categorical variables). ‡Excluding potatoes. Table 1. Continued Characteristic Total Protein Animal Protein Plant Protein Quartile 1 Median Intake 78.4 g/d Quartile 4 Median Intake 109.1 g/d Quartile 1 Median Intake 49.2 g/d Quartile 4 Median Intake 82.2 g/d Quartile 1 Median Intake 19.6 g/d Quartile 4 Median Intake 32.3 g/d earity in the association (Figure). Those in the highest versus the lowest quartile of total animal protein had 43% increased risk (95% CI: 0%–103%; P-trend=0.07); AR=12.3% in the lowest quartile, with a 5.3% increase in the highest quartile to AR=17.6%; model 2 in Table 2. intake. Baseline Characteristics On the contrary, they had higher BMI and were more likely to have diabetes mellitus. They had higher intake of fiber, polyunsaturated fatty acids, fruits, berries and vegetables, and processed red meat. High animal protein intake was also associated with more favorable socioeconomic factors, but with higher BMI, higher probability of being smoker and having diabetes mel- litus. Those with higher animal protein intake had lower intake of fiber but higher intake of polyunsaturated fat- ty acids. High plant protein intake was generally associ- ated with healthier lifestyle and dietary factors (Table 1). Those in the highest versus lowest quartile of dairy protein intake had multivariable-adjusted 49% (6%– 109%; P-trend=0.03) higher risk of HF (model 2 in Table 2; AR=12.0% in the lowest quartile, AR=17.9% in the highest quartile). However, there was evidence for nonlinearity (P-nonlinearity=0.03; Figure II in the Data Supplement). The association was stronger for protein from fermented dairy, with 70% (95% CI: 21%–140%; P-trend <0.001; P-nonlinearity=0.09) higher risk in the highest quartile as protein from nonfermented dairy indicated nonsignificant association (Table 2). Because of these findings, we explored the baseline character- istics according to the fermented dairy protein intake but found that higher intake of fermented dairy pro- tein was generally associated with healthier lifestyle and dietary factors (Table III in the Data Supplement). In more detailed analyses, protein from other ferment- ed dairy products (98.4% contained ≤2.5% fat) had a Associations of Dietary Proteins With Risk of HF During the mean follow-up of 22.2 years, 334 cases of HF were recorded. Total protein intake was not associ- ated with risk of HF in the age, examination year, and energy intake adjusted model (model 1; Table 2). After multivariable adjustments, those in the highest versus the lowest quartile of total protein had a borderline significant 33% (95% CI: −5% to 85%; P-trend=0.05) increased risk of HF. There was no evidence for nonlin- June 2018 5 Circ Heart Fail. 2018;11:e004531. DOI: 10.1161/CIRCHEARTFAILURE.117.004531 1:e004531. DOI: 10.1161/CIRCHEARTFAILURE.117.00453 Virtanen et al; Dietary Proteins and Risk of Heart Failure Table 2. Risk of Incident Heart Failure According to Protein Intake Among 2441 Men From the Kuopio Ischemic Heart Disease Risk Factor Study Intake Quartile 1 (n=610) 2 (n=610) 3 (n=611) 4 (n=610) P-Trend Per 5 g Increase Total protein Median intake, g/d 78.4 88.0 96.5 109.1 No. of events, incidence rate/1000 PY 81, 6.04 80, 5.82 92, 6.74 81, 6.03 Model 1 1 0.98 (0.72–1.33) 1.19 (0.88–1.61) 1.21 (0.89–1.65) 0.13 1.03 (0.99–1.07) Model 2 1 1.08 (0.79–1.49) 1.33 (0.97–1.81) 1.33 (0.95–1.85) 0.05 1.05 (1.01–1.09) Animal protein Median intake, g/d 49.2 59.8 68.8 82.2 No. of events, incidence rate/1000 PY 75, 5.54 95, 6.82 79, 5.83 85, 6.42 Model 1 1 1.23 (0.91–1.67) 1.14 (0.83–1.56) 1.44 (1.05–1.96) 0.04 1.04 (1.00–1.08) Model 2 1 1.20 (0.88–1.66) 1.17 (0.84–1.64) 1.43 (1.00–2.03) 0.07 1.05 (1.00–1.09) Protein from total meat† Median intake, g/d 12.4 20.0 27.1 37.7 No. of events, incidence rate/1000 PY 90, 6.82 88, 6.42 87, 6.41 69, 5.02 Model 1 1 0.95 (0.71–1.27) 1.07 (0.80–1.44) 0.97 (0.71–1.34) 0.95 1.00 (0.95–1.05) Model 2 1 0.99 (0.72–1.35) 1.12 (0.80–1.57) 1.11 (0.75–1.66) 0.50 1.03 (0.96–1.10) Protein from red meat Median intake, g/d 10.5 17.9 24.6 34.4 No. of events, incidence rate/1000 PY 83, 6.32 96, 6.96 86, 6.35 69, 5.00 Model 1 1 1.06 (0.79–1.42) 1.09 (0.81–1.48) 1.01 (0.73–1.40) 0.89 1.01 (0.96–1.06) Model 2 1 1.12 (0.82–1.53) 1.15 (0.81–1.63) 1.14 (0.76–1.71) 0.55 1.03 (0.96–1.10) Protein from processed red meat Median intake, g/d 1.9 6.0 10.1 17.0 No. of events, incidence rate/1000 PY 75, 5.58 100, 7.52 82, 5.95 77, 5.61 Model 1 1 1.39 (1.03–1.89) 1.16 (0.85–1.59) 1.28 (0.93–1.77) 0.32 1.06 (0.98–1.14) Model 2 1 1.44 (1.06–1.97) 1.02 (0.72–1.43) 1.09 (0.72–1.64) 0.85 1.02 (0.91–1.14) Protein from unprocessed red meat Median intake, g/d 3.9 9.3 14.4 23.2 No. Associations of Dietary Proteins With Risk of HF of events, incidence rate/1000 PY 88, 6.59 94, 6.90 76, 5.69 76, 5.47 Model 1 1 1.01 (0.76–1.35) 0.86 (0.63–1.17) 0.92 (0.68–1.26) 0.46 0.98 (0.91–1.04) Model 2 1 1.04 (0.77–1.41) 0.93 (0.67–1.28) 1.16 (0.82–1.65) 0.62 1.03 (0.95–1.11) Protein from fish Median intake, g/d 0 3.3 8.1 17.6 No. of events, incidence rate/1000 PY 83, 6.04 78, 5.69 84, 6.11 89, 6.83 Model 1 1 0.81 (0.59–1.11) 0.86 (0.63–1.17) 1.02 (0.75–1.38) 0.49 1.02 (0.97–1.08) Model 2 1 0.77 (0.56–1.06) 0.90 (0.65–1.23) 1.00 (0.73–1.39) 0.48 1.02 (0.96–1.09) Protein from egg Median intake, g/d 1.1 2.3 3.8 6.5 No. of events, incidence rate/1000 PY 82, 6.45 88, 6.48 86, 6.09 78, 5.64 Model 1 1 0.88 (0.65–1.19) 0.80 (0.59–1.08) 0.79 (0.58–1.07) 0.14 0.95 (0.77–1.18) Model 2 1 1.08 (0.79–1.47) 1.06 (0.77–1.45) 0.98 (0.71–1.36) 0.80 1.06 (0.86–1.30) Protein from dairy Median intake, g/d 17.1 25.2 31.6 40.8 No. of events, incidence rate/1000 PY 73, 5.31 85, 6.22 73, 5.40 103, 7.74 Model 1 1 1.05 (0.77–1.44) 0.90 (0.65–1.24) 1.45 (1.07–1.95) 0.03 1.06 (1.00–1.11) Model 2 1 1.06 (0.77–1.46) 0.92 (0.65–1.30) 1.49 (1.06–2.09) 0.03 1.08 (1.02–1.14) (continued ) Circ Heart Fail. 2018;11:e004531. DOI: 10.1161/CIRCHEARTFAILURE.117.004531 June 2018 6 Circ Heart Fail. 2018;11:e004531. DOI: 10.1161/CIRCHEARTFAILURE.117.004531 Circ Heart Fail. 2018;11:e004531. DOI: 10.1161/CIRCHEARTFAILURE.117.004531 Virtanen et al; Dietary Proteins and Risk of Heart Failure Protein from nonfermented dairy Median intake, g/d 6.6 13.5 19.9 29.2 No. of events, incidence rate/1000 PY 70, 4.96 83, 6.13 94, 6.89 87, 6.72 Model 1 1 1.09 (0.79–1.50) 1.18 (0.86–1.61) 1.25 (0.91–1.71) 0.15 1.04 (0.99–1.10) Model 2 1 1.10 (0.79–1.54) 1.19 (0.85–1.68) 1.23 (0.84–1.81) 0.27 1.06 (0.99–1.14) Protein from milk Median intake, g/d 5.8 12.8 19.1 28.8 No. of events, incidence rate/1000 PY 66, 4.64 85, 6.28 99, 7.30 84, 6.50 Model 1 1 1.18 (0.85–1.63) 1.34 (0.98–1.84) 1.30 (0.94–1.79) 0.10 1.05 (0.99–1.11) Model 2 1 1.14 (0.81–1.60) 1.30 (0.92–1.83) 1.27 (0.86–1.87) 0.21 1.06 (0.99–1.14) Protein from fermented dairy Median intake, g/d 1.4 6.9 12.8 22.7 No. of events, incidence rate/1000 PY 75, 5.75 76, 5.46 91, 6.67 92, 6.76 Model 1 1 0.84 (0.61–1.15) 1.10 (0.81–1.50) 1.21 (0.89–1.64) 0.07 1.03 (0.97–1.09) Model 2 1 0.97 (0.70–1.35) 1.52 (1.10–2.10) 1.70 (1.21–2.40) <0.001 1.09 (1.03–1.17) Protein from cheese Median intake, g/d −0.1 2.1 5.8 13.1 No. of events, incidence rate/1000 PY 88, 6.85 94, 7.06 80, 5.72 72, 5.11 Model 1 1 0.89 (0.66–1.21) 0.76 (0.56–1.03) 0.84 (0.61–1.16) 0.29 0.94 (0.86–1.04) Model 2 1 1.02 (0.75–1.38) 0.98 (0.72–1.35) 1.26 (0.90–1.77) 0.16 1.06 (0.97–1.17) Protein from other fermented dairy‡ Median intake, g/d −0.4 1.3 5.4 14.0 No. of events, incidence rate/1000 PY 71, 5.21 79, 5.85 72, 5.16 112, 8.52 Model 1 1 0.79 (0.55–1.12) 0.72 (0.51–1.02) 1.26 (0.93–1.72) 0.004 1.08 (1.01–1.16) Model 2 1 0.91 (0.64–1.30) 0.84 (0.59–1.20) 1.54 (1.10–2.16) 0.001 1.12 (1.03–1.21) Plant protein Median intake, g/d 19.6 23.8 27.3 32.3 No. of events, incidence rate/1000 PY 84, 6.53 77, 5.65 87, 6.32 86, 6.15 Model 1 1 0.73 (0.53–0.99) 0.78 (0.58–1.06) 0.80 (0.59–1.09) 0.27 0.93 (0.84–1.03) Model 2 1 0.82 (0.58–1.15) 1.03 (0.70–1.53) 1.17 (0.72–1.91) 0.35 1.03 (0.85–1.24) Protein from grain products Median intake, g/d 14.5 18.6 21.8 26.7 No. of events, incidence rate/1000 PY 77, 5.95 86, 6.37 90, 6.49 81, 5.82 Model 1 1 0.97 (0.71–1.32) 0.89 (0.65–1.21) 0.86 (0.63–1.18) 0.29 0.93 (0.83–1.03) Model 2 1 1.04 (0.74–1.44) 1.05 (0.72–1.54) 1.14 (0.71–1.84) 0.59 1.00 (0.83–1.22) Protein from nongrain plant protein sources Median intake, g/d 3.0 4.4 5.7 7.8 No. Sensitivity Analyses Dairy, fermented dairy, or any dairy proteins did not indicate statistically significant interactions by baseline disease history (CHD or use of cardiac medications, dia- betes mellitus or hypertension; P-interactions >0.32), CHD history (P-interactions >0.32), BMI (P-interactions >0.47), or smoking status (P-interactions >0.62). How- ever, in the stratified analyses based on disease his- tory, higher intake of the major plant protein sources was associated with increased risk of HF among those without disease history (n=832; 64 cases; HR per 100 g intake, 1.46; 95% CI: 1.00–2.12; model 2) but not among those with disease history (n=1609; 270 cas- es; HR=0.91; 95% CI: 0.76–1.10; P-interaction=0.04). We did not find evidence for interaction with intake of plant protein (P-interaction=0.13). For other proteins or protein sources, interactions by disease history (P-inter- actions >0.07), CHD history (P-interactions >0.08), BMI (P-interactions >0.06), or smoking status (P-interactions >0.15) were not statistically significant. stronger association with HF than protein from cheese (Table 2). Proteins from total meat and from meat sub- types, milk, and plant sources had nonsignificant asso- ciations toward increased HF risk (Table 2), without evi- dence for nonlinear associations (P-nonlinearity >0.10). Proteins from fish and eggs were not associated with HF risk. In the substitution models, replacing protein from one source with protein from another source did not reveal any statistically significant associations (P>0.05). Circ Heart Fail. 2018;11:e004531. DOI: 10.1161/CIRCHEARTFAILURE.117.004531 The model was adjusted for age (y), examination year, energy intake (kcal/d), education (y), income (euros/y), pack-years of smoking (packs smoked per day×years smoked), alcohol intake (g/wk), leisure-time physical activity (kcal/d), body mass index (kg/m2), family history of coronary heart disease (yes/no), diseases (coronary heart diseases or use of cardiac medications, diabetes mel- litus, or hypertension) at the baseline and during the follow-up, and intakes of saturated (g/d), monounsaturated (g/d), polyunsaturated (g/d), and trans fatty acids (g/d) and fiber (g/d). The solid lines represent the central risk estimates, and the shades are the 95% confidence interval, relative to the reference level (12.5th percentile). The dotted vertical lines correspond to 10th, 25th, 50th, 75th, and 90th percentile of the total protein intake. the multivariable-adjusted models (Table IV in the Data Supplement). Figure. Multivariable-adjusted hazard ratios of total protein intake with risk of heart failure in 2441 men, evaluated by restricted cubic splines from Cox proportional hazards models. The model was adjusted for age (y), examination year, energy intake (kcal/d), education (y) income (euros/y) pack years of smoking (packs smoked per the multivariable-adjusted models (Table IV in the Data Supplement). Circ Heart Fail. 2018;11:e004531. DOI: 10.1161/CIRCHEARTFAILURE.117.004531 of events, incidence rate/1000 PY 79, 5.97 91, 6.73 85, 6.17 79, 5.76 Model 1 1 1.04 (0.77–1.40) 0.96 (0.71–1.31) 0.96 (0.70–1.32) 0.70 1.00 (0.78–1.27) Model 2 1 1.29 (0.94–1.78) 1.28 (0.90–1.82) 1.30 (0.88–1.93) 0.29 1.20 (0.90–1.60) Model 1 adjusted for age (y), examination year, and energy intake (kcal/d). Model 2 adjusted for model 1 and education (y), income (euros/y), pack-years of smoking (packs smoked per day×years smoked), alcohol intake (g/wk), leisure-time physical activity (kcal/d), body mass index (kg/m2), family history of coronary heart Table 2. Continued Intake Quartile 1 (n=610) 2 (n=610) 3 (n=611) 4 (n=610) P-Trend Per 5 g Increase Model 1 adjusted for age (y), examination year, and energy intake (kcal/d). Model 2 adjusted for model 1 and education (y), income (euros/y), pack-years of smoking (packs smoked per day×years smoked), alcohol intake (g/wk), leisure-time physical activity (kcal/d), body mass index (kg/m2), family history of coronary heart disease (yes/no), diseases (coronary heart diseases or use of cardiac medications, diabetes mellitus, or hypertension) at the baseline and during the follow-up, and intakes of saturated (g/d), monounsaturated (g/d), polyunsaturated (g/d), and trans fatty acids (g/d) and fiber (g/d). Model 2 was also mutually adjusted for other proteins. PY indicates person-years. fi f Model 1 adjusted for age (y), examination year, and energy intake (kcal/d). Model 2 adjusted for model 1 and education (y), income (euros/y), pack-years of smoking (packs smoked per day×years smoked), alcohol intake (g/wk), leisure-time physical activity (kcal/d), body mass index (kg/m2), family history of coronary heart disease (yes/no), diseases (coronary heart diseases or use of cardiac medications, diabetes mellitus, or hypertension) at the baseline and during the follow-up, and intakes of saturated (g/d), monounsaturated (g/d), polyunsaturated (g/d), and trans fatty acids (g/d) and fiber (g/d). Model 2 was also mutually adjusted for other proteins. PY indicates person-years. Circ Heart Fail. 2018;11:e004531. DOI: 10.1161/CIRCHEARTFAILURE.117.004531 June 2018 7 June 2018 Virtanen et al; Dietary Proteins and Risk of Heart Failure Figure. Multivariable-adjusted hazard ratios of total protein intake with risk of heart failure in 2441 men, evaluated by restricted cubic splines from Cox proportional hazards models. Association of Calcium Intake With Risk of HF Because of the findings with dairy, in the post hoc anal- yses, we investigated the associations of dietary calcium with HF risk. After multivariable adjustments, those in the highest calcium intake quartile had 39% (95% CI: 1%–91%; P-trend=0.07) higher risk of HF when com- pared with the lowest quartile (model 2; Table V in the Data Supplement). The increased risk was only present in the highest calcium intake quartile (>1528 mg/d). Figure. Multivariable-adjusted hazard ratios of total protein intake with risk of heart failure in 2441 men, evaluated by restricted cubic splines from Cox proportional hazards models. We also tested whether adjustment for calcium intake attenuates the associations observed with fer- mented dairy protein. Adjusting model 2 further for calcium intake, the HRs (95% CIs) in the highest quar- tile for proteins from fermented dairy and fermented dairy excluding cheese were 1.86 (1.00, 3.55) and 1.46 (0.87, 2.47), respectively. The model was adjusted for age (y), examination year, energy intake (kcal/d), education (y), income (euros/y), pack-years of smoking (packs smoked per day×years smoked), alcohol intake (g/wk), leisure-time physical activity (kcal/d), body mass index (kg/m2), family history of coronary heart disease (yes/no), diseases (coronary heart diseases or use of cardiac medications, diabetes mel- litus, or hypertension) at the baseline and during the follow-up, and intakes of saturated (g/d), monounsaturated (g/d), polyunsaturated (g/d), and trans fatty acids (g/d) and fiber (g/d). The solid lines represent the central risk estimates, and the shades are the 95% confidence interval, relative to the reference level (12.5th percentile). The dotted vertical lines correspond to 10th, 25th, 50th, 75th, and 90th percentile of the total protein intake. The model was adjusted for age (y), examination year, energy intake (kcal/d), education (y), income (euros/y), pack-years of smoking (packs smoked per day×years smoked), alcohol intake (g/wk), leisure-time physical activity (kcal/d), body mass index (kg/m2), family history of coronary heart disease (yes/no), diseases (coronary heart diseases or use of cardiac medications, diabetes mel- litus, or hypertension) at the baseline and during the follow-up, and intakes of saturated (g/d), monounsaturated (g/d), polyunsaturated (g/d), and trans fatty acids (g/d) and fiber (g/d). The solid lines represent the central risk estimates, and the shades are the 95% confidence interval, relative to the reference level (12.5th percentile). The dotted vertical lines correspond to 10th, 25th, 50th, 75th, and 90th percentile of the total protein intake. DISCUSSION In this population-based cohort study in middle-aged and older men from eastern Finland, high protein intake was marginally associated with increased risk of HF. Most proteins from different animal and plant sources were associated with a higher risk although not all associations were statistically significant. Similar findings were observed with the protein sources. i g p Previously, high-protein–low-carbohydrate diets have been related to increased risk of type 2 diabetes mellitus and all-cause mortality,17 and high animal pro- tein intake with increased cardiovascular mortality,15,16 but studies have not examined protein intake in rela- tion to HF risk. Nevertheless, trials in humans have sug- gested that supplementation with a mixture of amino acids may prevent cardiac dysfunction in patients with type 2 diabetes mellitus.13,14 Of various amino acids, branched chain amino acids (BCAAs) that are abundant in dairy but also in other animal protein sources24 are of special interest as BCAA catabolism is impaired in a failing heart.25,26 The unanswered question is whether the dietary intake of BCAAs affects the development of HF.26 Human trials are lacking, and BCAA supple- mentation in animals has had both beneficial27,28 and harmful effects on cardiac function.29 Thus, the poten- tial effects of proteins and amino acids on HF risk need further clarification. In the post hoc analyses, a high intake of dietary calcium (>1528 mg/d) was related to increased HF risk. In contrast, calcium supplementation (1000 mg/d) with vitamin D (400 µg/d) did not affect HF risk in the over- all cohort of postmenopausal women but was ben- eficial for those with low HF risk.41 However, a meta- analysis observed a U-shaped association between total calcium intake and cardiovascular mortality: the risk gradually increased at intakes >800 mg/d.42 Stud- ies, however, suggest that it is mainly the excessive intake of supplemental calcium that associates with increased cardiovascular risk whereas dietary calcium is generally considered safe.43–45 The more pronounced increase in serum calcium after calcium supplementa- tion is hypothesized to promote aortic calcification and In our analyses, total dairy and fermented dairy were associated with increased HF risk. As we observed comparable associations for dairy and dairy proteins, it is hard to disentangle whether the results are because of the protein per se or because of some other fac- tors in dairy. Associations of Dietary Protein Sources With Risk of HF Intake of total dairy had a trend toward increased risk of HF in multivariable-adjusted model 2, with a 36% (95% CI: −5% to 94%; P-trend=0.07) higher risk in the highest versus the lowest quartile. Adjusting for nutri- ent intakes strengthened the association (model 3; Table IV in the Data Supplement). Those in the highest intake quartile of fermented dairy products had multivariable- adjusted 62% (95% CI: 20%–119%; P-trend=0.001) higher risk of HF when compared with the lowest quar- tile (model 2; Table IV in the Data Supplement). Fur- ther adjustment for nutrients had little impact on the associations (model 3). Nonfermented dairy intake had no association with HF risk (Table IV in the Data Sup- plement). Intakes of total meat or any meat subtype, fish, eggs, or main vegetable protein sources did not have statistically significant associations with HF risk in Because HF is typically an end-stage heart disease, some of the CVD subjects may die before developing HF. Thus, we considered the competing risk of CVD mortality by using a composite outcome of HF or CVD mortality (n=1640; cases=379 after excluding the par- ticipants with CVD at baseline). In these analyses, most associations were attenuated (eg, HR=1.04; 95% CI: 1.00–1.08 per 5 g higher total protein intake) whereas the association for nonfermented dairy was strength- ened (Table VI in the Data Supplement). Because the long follow-up may attenuate asso- ciations with the exposures that were assessed only at baseline, we tested the associations with 10 years June 2018 June 2018 Circ Heart Fail. 2018;11:e004531. DOI: 10.1161/CIRCHEARTFAILURE.117.004531 8 1:e004531. DOI: 10.1161/CIRCHEARTFAILURE.117.00453 Virtanen et al; Dietary Proteins and Risk of Heart Failure associated with decreased risk of fatal and nonfa- tal CHD.35 Dairy protein and major dairy amino acids have also induced beneficial metabolic effects in trials, such as lowered blood pressure and improved glyce- mic control.11,36–38 Interestingly, though a meta-anal- ysis observed an increased risk of all-cause mortality with high dairy intakes (>750 mL/d),39 indicating that high intakes may not be favorable. Accordingly, we observed higher HF risk mainly among those with the highest intake of total (>927 g/d) and fermented dairy (>281 g/d). Associations of Dietary Protein Sources With Risk of HF It is worth noticing that the median intakes of total dairy (682 g/d) and fermented dairy (103 g/d) in our study are higher than in many other cohorts, where the corresponding intakes have been 111 to 400 g/d and 40 to 100 g/d, respectively.34 shorter follow-up (n=96 cases). The associations were generally similar although not all of them reached sta- tistical significance. For example, HRs (95% CIs) per 5 g intake of total, animal, dairy, and fermented dairy protein were 1.06 (0.98–1.15), 1.06 (0.97, 1.14), 1.10 (0.99, 1.22), and 1.13 (1.01, 1.27), respectively (model 2; other data not shown). The association of cheese intake with risk of HF was stronger with the shorter fol- low-up (model 2 HR=1.58; 95% CI: 1.03–2.41 per 50 g intake of cheese), and the association remained statisti- cally significant after adjustment for nutrient intakes. We also tested the exclusion of HF cases that occurred during the first 2 years of follow-up (n=3) but this did not change the results. What could explain the stronger association of fer- mented compared with nonfermented dairy with HF risk in our study? A major source of fermented dairy was sour milk whereas nonfermented dairy was pre- dominantly milk. These products have fairly similar nutrient content (eg, BCAAs, other amino acids, cal- cium), indicating that these nutrients unlikely account for the stronger association of fermented dairy. More- over, as adjustment for different dietary fatty acids strengthened the direct association between fermented dairy and risk of HF, the fat quality is not a probable explanation, either, especially because the association was stronger with protein coming predominantly from reduced or low-fat fermented dairy products. Sour milk also includes probiotics, but probiotics may—based on animal studies—actually have beneficial effects on car- diac function,40 thus providing no clarification for our observation. Finally, as higher fermented dairy protein intake was associated with a healthier lifestyle, other lifestyle factors unlikely explain its association with higher HF risk. To sum up, we did not find a plausi- ble explanation for why fermented dairy or its protein would be more detrimental than nonfermented dairy. Thus, considering that we included many analyses in the present study, we cannot exclude the possibility of a chance finding explaining the result. DISCUSSION In one previous study, intake of high-fat dairy was related to increased HF risk.5 However, total2 or fermented dairy30,31 intake has not been related to HF risk in other studies. Furthermore, studies have reported beneficial or neutral associations of dairy and fermented dairy intakes with risk of type 2 diabetes mellitus and CVD.32–34 Also we have observed in this same cohort that high intake of fermented dairy was June 2018 June 2018 Circ Heart Fail. 2018;11:e004531. DOI: 10.1161/CIRCHEARTFAILURE.117.004531 9 Virtanen et al; Dietary Proteins and Risk of Heart Failure blood coagulation.44 Because we did not have data about calcium supplement use in our cohort, we were not able to compare dietary and supplemental calcium. However, higher dietary calcium intake compared with most studies42 may possibly explain its association with increased HF risk. excluded, and it may potentially explain the asso- ciations between dairy and HF. We also did not have information on calcium supplement use. Some misclas- sification in the outcome measure is possible although the use of national registers for ascertaining HF cases reduces false diagnoses compared with self-reporting. Finally, we were not able to separate the HF cases with preserved or reduced ejection fraction that have differ- ential pathogenesis1 and cannot, therefore, conclude whether the associations of protein intake differ with different subtypes of HF. In agreement with one previous meta-analysis,46 we did not observe protein from fish or fish intake to associate with HF risk. In contrast, another meta- analysis found that intake of fish and marine ome- ga-3 fatty acids is related to decreased HF risk.3 Preparation methods could explain the discrepancy in results; baked or broiled fish has been associated with decreased HF risk whereas fried fish has had an association with increased risk.47,48 Intakes of meat protein, total meat, or any meat subtype were not associated with HF risk, either. This result complies with one previous study, in which intake of red meat was not associated with HF risk.5 Yet, many studies have found a higher HF risk with greater intakes of total meat,2 red meat,6 and especially of processed red meat.7,8 Because consumption of total and pro- cessed meat in our study is comparable or higher than in other cohorts,2,8 the meat consumption patterns unlikely explain the difference between the studies. Differences in preparation methods of meat and dif- ficulties in categorizing meat as unprocessed or pro- cessed49 may, however, affect the results. Affiliation Institute of Public Health and Clinical Nutrition, University of Eastern Finland, Kuopio. i Our finding that egg protein or egg intake had no association with HF risk contradicts with a meta-anal- ysis that revealed a direct association between high egg intake and HF risk.9 Egg contains high quality protein and several bioactive compounds but is also a major source of dietary cholesterol and hence still has a controversial role on heart health and mortal- ity.39,50 Lifestyle factors related to egg intake may also affect its associations with health.50 More research on effects of egg intake in diverse populations is thus needed.50 Acknowledgments We thank Ari Voutilainen, PhD, for assistance with statistical analysis. Sources of Funding This study was supported by Finnish Cultural Foundation North Savo Regional fund (H.E.K. Virtanen), Päivikki and Sakari Sohlberg Foundation (H.E.K. Vir- tanen), Paavo Nurmi Foundation (H.E.K. Virtanen), and the Finnish Association of Academic Agronomists (H.E.K. Virtanen). ARTICLE INFORMATION Received August 29, 2017; accepted April 11, 2018. The Data Supplement is available at http://circheartfailure.ahajournals.org/ lookup/suppl/doi:10.1161/CIRCHEARTFAILURE.117.004531/-/DC1. Correspondence Jyrki K. Virtanen, PhD, Institute of Public Health and Clinical Nutrition, Univer- sity of Eastern Finland, Kuopio Campus, Yliopistonranta 1 C, PO Box 1627, 70211 Kuopio, Finland. E-mail jyrki.virtanen@uef.fi CONCLUSIONS Our results suggest that higher protein intake may be associated with a higher risk of HF in middle-aged and older men. Further studies in diverse study populations are needed to elucidate the role of protein intake in the pathogenesis of HF. DISCUSSION There is no apparent explanation for why a high consumption of major plant sources was associated with a higher HF risk among those without disease history. Because of low number of events in this group, the association might be a chance finding. Disclosures None. None. The strengths of our study are the prospective pop- ulation-based setting, no losses during the follow-up, and extensive measurement of diet and possible con- founding factors. Our study also has limitations. The single baseline measurement of diet and other lifestyle factors and the inability of a 4-day food recording to accurately assess typical intakes of occasionally con- sumed and seasonally varying foods could introduce random error and thus attenuate associations. How- ever, the associations were comparable with a shorter follow-up. Despite considering various covariates in our models, residual confounding cannot be completely Virtanen et al; Dietary Proteins and Risk of Heart Failure Association of animal and plant protein intake with all-cause and cause-specific mortality. JAMA Intern Med. 2016;176:1453–1463. doi: 10.1001/jamainternmed.2016.4182. 34. Gijsbers L, Ding EL, Malik VS, de Goede J, Geleijnse JM, Soedamah-Muthu SS. Consumption of dairy foods and diabetes incidence: a dose-response meta-analysis of observational studies. Am J Clin Nutr. 2016;103:1111– 1124. doi: 10.3945/ajcn.115.123216. 16. Hernández-Alonso P, Salas-Salvadó J, Ruiz-Canela M, Corella D, Estruch R, Fitó M, Arós F, Gómez-Gracia E, Fiol M, Lapetra J, Basora J, Serra-Majem L, Muñoz MÁ, Buil-Cosiales P, Saiz C, Bulló M. High dietary protein intake is associated with an increased body weight and total death risk. Clin Nutr. 2016;35:496–506. doi: 10.1016/j.clnu.2015.03.016. 35. 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https://openalex.org/W831705892	https://pdf.blucher.com.br/designproceedings/15ergodesign/02-E117.pdf	Portuguese	null	A OBSERVAÇÃO DO COMPORTAMENTO DO USUÁRIO PARA O WAYFINDING NO AMBIENTE CONSTRUÍDO	null	2,015	cc-by	5,208	RESUMO Este estudo apresenta uma sistematização da observação do comportamento do usuário perante a tarefa de se deslocar no ambiente construído. A estrutura da observação se pauta no “o quê” e no “como” observar. Apresenta revisão bibliográfica acerca do comportamento do usuário no wayfinding como base para a categorização de comportamentos observáveis. Para a observação/ registro de comportamento propõe-se a técnica do mapeamento do comportamento espacial. Tal técnica configura a maneira de observar o comportamento do usuário durante sua navegação e catalogar dados sobre o desempenho da informação do ambiente. Palavras-chave: Ergonomia do ambiente construídot; wayfinding, usuário Keywords: Ergonomics of the built environment; wayfinding, user A OBSERVAÇÃO DO COMPORTAMENTO DO USUÁRIO PARA O WAYFINDING NO AMBIENTE CONSTRUÍDO THE USER BEHAVIOR OBSERVATION FOR WAYFINDING IN THE BUILT ENVIRONMENT RANGEL, Márcia Moreira (1); Mont’Alvão, Cláudia (2) (1) IF Sudeste MG/ Câmpus Juiz de Fora, Mestre em Design e-mail:marcia.rangel@ifsudestemg.edu.br (2) Pontifícia Universidade Católica do Rio de Janeiro, Doutora em Engenharia de Transportes e-mail:cmontalvao@puc-rio.br RANGEL, Márcia Moreira (1); Mont’Alvão, Cláudia (2) (1) IF Sudeste MG/ Câmpus Juiz de Fora, Mestre em Design e-mail:marcia.rangel@ifsudestemg.edu.br (2) Pontifícia Universidade Católica do Rio de Janeiro, Doutora em Engenharia de Transportes il t l @ i b ia Universidade Católica do Rio de Janeiro, Doutora em Engenharia de Transport Universidade Católica do Rio de Janeiro, Doutora em Engenharia de Transportes 1. INTRODUÇÃO A pesquisa em Ergonomia visa estudar o homem e suas habilidades, a tarefa a ser realizada e o sistema no qual essa ocorre, sempre convergindo os métodos investigativos para a captação de dados no decorrer da tarefa. A tecnologia humano-sistema, é apontada por Hendrick (1993 apud MORAES.ORG. 2001), como a única tecnologia pertinente à ergonomia. A ergonomia moderna, que se caracteriza por ser centrada no indivíduo, indica ser este o controlador do sistema e capaz de alterá-lo conforme suas habilidades e vontade. Dessa forma, para sua efetividade, o sistema deve ser projetado sob o ponto de vista do usuário. Tal enfoque considera a interação humano/sistema como “controlada e conduzida” pelo usuário, contudo, visa um ambiente de interação adaptado às habilidades e necessidades desse usuário (MORAES E MONT’ALVÃO, 2003, p.27). Na realização de sua pesquisa, a ergonomia se utiliza de ferramentas metodológicas para abarcar conhecimentos acerca de “capacidades, limites e outras características do desempenho humano, à medida que elas se relacionam com o projeto de interfaces e outros componentes do sistema” (MORAES.ORG, 2001, p.9). O ambiente construído configura-se em um dos sistemas abordados pela pesquisa ergonômica, e suas ferramentas metodológicas visam compreender e identificar os elementos envolvidos na interação humano/ambiente. Entendendo-se aqui o ambiente construído como um cenário social de naturezas e interfaces diversas, que propiciam múltiplas interações aos usuários. Dentre as interfaces humano/ambiente, o deslocamento de um ponto ao outro para realizar determinada atividade é uma tarefa, que envolve o processamento da informação advinda dos diversos elementos que compõe o ambiente, a decisão da melhor rota, o monitoramento da rota para confirmar o caminho certo e o reconhecimento do destino. São questões cognitivas que abarcam a orientação espacial no ambiente construído, e trazem custos humanos quando o usuário, ao acionar seu mapa mental do ambiente, não consegue cumprir todas as etapas da tarefa de chegar/reconhecer o destino, a partir das informações captadas. Com enfoque no desempenho do usuário frente à sua tarefa de deslocar-se em determinada rota para realizar sua atividade, este artigo apresenta questões pertinentes à coleta de dados acerca da observação do comportamento espacial do usuário no ambiente construído. A abordagem é pautada nos fundamentos do wayfinding, uma vez que visa captar as soluções para navegação dos usuários em determinado ambiente, em seu processo de orientação espacial. O artigo primeiramente a aborda o usuário e seu comportamento elencados ao wayfinding. ABSTRACT This study introduces a systematic overview of user behavior observation when confronted with the user´s task of moving around in a built environment. The observation framework is based on "what" and "how" to observe. It includes a literature review about user wayfinding behavior as the basis for the categorization of observable behaviors. Spatial behavior mapping technique is proposed for behavior observation/registration. This technique sets the way to observe user behavior during his/her navigation and to log data on the performance of environmental information. Keywords: Ergonomics of the built environment; wayfinding, user 2. WAYFINDING E O USUÁRIO 2. Estar orientado é saber onde está e para onde ir sem a ajuda de terceiros, esse é o princípio da autonomia na navegação. É uma necessidade do ser humano que remonta ao seu instinto de sobrevivência. Na sociedade contemporânea a necessidade de orientação está elencada à segurança no ambiente, a não perder tempo e dinheiro, evitar frustração, estresse e crises de ansiedade. Essas são questões importantes para o indivíduo, portanto devem ser de interesse daqueles que administram e os que desenvolvem projetos para o ambiente construído. (ARTHUR E PASSINI, 1992; CARPMAN, 2000; CARPMAN E GRANT, 2008; RIBEIRO, 2009). A interface Ergonomia do Ambiente Construído (EAC)/wayfinding está na relação do ambiente construído e o comportamento do usuário, uma vez ser, segundo Ribeiro (2009), do interesse da EAC aperfeiçoar os espaços no sentido de torna-los cada vez mais adequados para a promoção do bem-estar e da segurança de quem neles interage. Esses são aspectos relevantes para o wayfinding. As contínuas transformações sociais plasmam ambientes com arranjo espacial complexo que impede e/ou limita a apreensão do espaço, dificultando ao usuário localizar-se e planejar seu deslocamento (CARPMAN, 2000; RIBEIRO, 2009). Ribeiro (2009) ressalta que as diversas pesquisas que apontam a desorientação como causa de insatisfação do usuário, revelam diversos problemas do ambiente em relação a sinalizar e a orientar o usuário. É, portanto, nesta lacuna entre o ambiente construído e o usuário que o wayfiniding se situa. O wayfinding aborda a relação sujeito/ambiente, considerando que estão envolvidos aspectos relativos a ambos. Quanto ao ser humano serão consideradas suas habilidades individuais, que irão caracterizar sua percepção e cognição frente ao ambiente construído em sua tarefa de deslocar-se. O ambiente construído será o provedor das informações para a orientação espacial. O wayfinding possui dois vieses complementares de abordagem. Para Arthur e Passini (1992) é um processo que se desenvolve gradualmente da relação sujeito/ambiente. Esse processo que envolve o processamento da informação, a tomada de decisão e a execução da decisão, tem como princípio norteador a relação dinâmica (de troca) entre o indivíduo e o ambiente. Para Carpman e Grant (2002) é um sistema conformado por três subsistemas comportamento + design + operacional, cujas ações desenvolvidas irão conduzir o indivíduo a saber onde está, encontrar um caminho para o seu destino e retornar ao local de onde partiu. 1. INTRODUÇÃO Depois, traz questões acerca da observação/registros de comportamento e apresenta uma sistemática para essa observação. Apresenta, ainda, as planilhas de observação com comportamentos típicos a serem observados, levantados da literatura. Cumpre asseverar que o estudo tem delineamento teórico/propositivo, pois é uma proposta ainda a ser aplicada. Contudo, apresenta conteúdo e relevância propícios à discussão. 2. WAYFINDING E O USUÁRIO Na relação sujeito/ambiente, sob a perspectiva do sujeito-usuário, “encontrar o caminho” é uma ação que deveria ser intuitiva e com o menor esforço cognitivo e interpretativo. Mediante essa assertiva, cabe ao ambiente fornecer as informações necessárias à orientabilidade dos indivíduos contemplando suas habilidades, ou mesmo, suas restrições (BINS ELY, IN MORAES.ORG. 2004). No processo de orientação espacial a relação sujeito/ambiente pode ser melhor compreendida a partir da modelagem do processamento da informação apresentada na figura 1. Essa modelagem, que envolve questões perceptivas e cognitivas, foi desenvolvida por Rangel (2011) a partir de estudos realizados por diversos autores. Figura 1 – Modelagem do processo perceptivo na interação humano-ambiente Fonte – Rangel, 2011, p.73 Figura 1 – Modelagem do processo perceptivo na interação humano-ambiente Fonte – Rangel, 2011, p.73 A tomada de decisão decorre do desenvolvimento de um plano de ação derivado do processamento da informação, em que entra em jogo a percepção dos elementos do ambiente, captadas pelos sentidos. A cognição decorre da atenção e da compreensão das informações captadas. Esses dois processos dependem das características próprias do sujeito-usuário e de suas habilidades individuais para interpretar a informação. A execução da decisão é um processo que demanda uma ação observável, é um comportamento que pressupõe o movimento do corpo em busca do destino. A orientação, portanto, ocorre a partir de uma interação dialogada – passo-a-passo, nó-a-nó, na qual surgem contínuas perguntas para o ambiente e respostas do indivíduo, conformadas nas ações interpretadas. Dessa forma, o comportamento espacial configura-se a partir de: 1) Habilidades e características próprias, que são as habilidades intrínsecas ao indivíduo; 2) Reação ao ambiente, que são as ações derivadas da interpretação dos estímulos de orientação e das tomadas de decisões. 3. O ser humano possui mecanismos naturais de navegação, decorrentes de um longo processo evolutivo, no qual suas habilidades para encontrar um local estão definitivamente ligadas ao seu instinto de sobrevivência. O comportamento espacial, dessa forma, é adaptável, pois nossos mecanismos de investigação e de fuga foram moldados, ao longo de nossa evolução, de maneira relativamente independente do conhecimento geométrico exato do ambiente. Podemos formatar um comportamento espacial a partir de modelos mentais que servirão como matrizes adaptadas para cada espaço a ser navegado (SEBEOK, 2001; ZINGALE, 2010). A partir dessa teoria podemos entender porque as pessoas tendem a encontrar um destino, mesmo em ambientes desconhecidos e com pouca informação para sua orientação. Primeiramente, os mecanismos interiores de busca irão investigar o ambiente para encontrar os meios necessários para se situar e se deslocar, e ao se sentir perdido, os mecanismos de defesa serão acionados colocando em alerta todos os canais sensórios para captar informações para a navegação. Isso também demonstra que o indivíduo tem predisposição a encontrar seu destino e busca por vários meios realizar sua tarefa de deslocar-se. O fato de perder-se, de demorar a encontrar o destino, de ter de pedir auxílio a outras pessoas é gerador de estresse, pois, se não impede, limita o ser humano a navegar com autonomia, seguindo seus próprios instintos e habilidades para captar a informação do ambiente. limita o ser humano a navegar com autonomia, seguindo seus próprios instintos e habilidades para captar a informação do ambiente. limita o ser humano a navegar com autonomia, seguindo seus próprios instintos e habilidades para captar a informação do ambiente. Zingale (2010) pontua que a orientação, a exploração e a navegação, configuram-se em três modalidades de comportamento espacial. Essas três modalidades são pertinentes às quatro fases de wayfinding – orientação, decisão da rota, monitoramento da rota e reconhecimento do destino – apontadas por Atkins et. al. (2008). A compilação e interpretação dos estudos dos autores são apresentadas a seguir. O comportamento de orientação decorre da pergunta “onde estou?”. O indivíduo precisa ter consciência de sua localização geográfica no espaço e ter conhecimento de sua posição dentro do mesmo. Visa interpretar o espaço e seus eventos a partir de conexões de reciprocidade, ou seja, estabelecer relações, dimensões, orientação e posicionamentos entre ele e o ambiente e os elementos entre si. A pergunta elencada ao comportamento de exploração é “para onde vou?”. O comportamento é investigativo. As pesquisas em ergonomia realizam de uma forma geral, duas modalidades de observação – a observação assistemática e a observação sistemática. Embora não seja o único e nem o determinante instrumento de coleta de dados, a importância da observação é apontada por Moraes e Mont’Alvão (2003, p.7), quando explicitam ser esta uma fase determinante para a pesquisa, por ser a base que configura problemas e hipóteses, como também, é sustentação para os demais métodos e técnicas. A observação sistemática é o enfoque desse estudo. Tal observação tem caráter estruturador, onde os propósitos – “o quê” e “como” observar – para se obter respostas acerca de questões da pesquisa, são definidos anteriormente. Utiliza usualmente como suporte, os dados preparados a partir da observação assistemática. Aqui, os dados coletados em pesquisa documental nas plantas arquitetônicas, são também apoio para a sistematização e o preparo dos documentos da observação. Para a eficiência da observação sistemática é necessário (1) delimitar a área da realidade a ser observada; (2) indicar a população (o que ou quem), as circunstâncias (quando) e o local (onde) a ser observado; (3) preparar material de apoio à observação – planilhas de registro, fichas de entrevista, equipamentos de fotografia e filmagem etc. Esses são alguns pontos pertinentes e fundamentais à observação (MORAES E MONT’ALVÃO, 2003, p.39). Os registros de comportamento estão elencados à observação sistemática, e compõe o estudo do comportamento do usuário frente à sua tarefa (MORAES E MONT’ALVÃO, 2003, p.39). Tal registro objetiva levantar e anotar os dados observados acerca das ações assumidas pelo usuário. Para Fagundes (2006, p.59), registrar os comportamentos é também importante na medida em que facilita “a análise posterior, dificultando a ação do esquecimento”. Fagundes (2006) explicita que para a observação, os comportamentos devem ser definidos para se eliminar possíveis contradições de interpretações. É necessário apresentar definições explícitas e completas, por meio de linguagem científica (objetiva, clara, exata, concisa e direta), e com denominações de rápida associação com o que está sendo designado. O resultado da observação deve apresentar somente o que foi percebido pelos sentidos, não devendo o observador acrescentar suas interpretações. Para facilitar a observação do comportamento, Fagundes (2006) recomenda ao observador, antes de começar o registro, se ambiente à situação para que o sujeito não estranhe a sua presença. 3. Visa compor o modelo mental do espaço real a partir de similaridades com mapas mentais padrões. É também interpretativo e de reconhecimento das qualidades sensoriais da configuração do ambiente, seus objetos e eventos, e ainda é associativo, uma vez que o indivíduo busca semelhanças para associá-las a ideias e “imagens evocativas”. A orientação e a exploração irão propiciar ao indivíduo decidir a rota para chegar ao seu destino. A Navegação responde à seguinte questão: “como prossigo?”. É o movimento em direção ao destino. É a competência para se deslocar e utilizar o mapa mental. Para navegar é necessário ao sujeito-usuário determinar direções, calcular a distância do objetivo, ser capaz de identificar os nós, de reconhecer marcos referenciais, etc. A navegação envolve o deslocamento e o monitoramento da rota para não perder a noção de lugar (onde está) em nenhum ponto do trajeto. A orientação, portanto, é uma constante em todo o processo. É interessante observar que o monitoramento abarca a memorização dos locais por onde passa, para ser capaz de refazer o percurso tanto em direção ao destino quanto para retornar ao local de partida, quantas vezes se fizer necessário, sem que haja problema de se perder ao longo do caminho. É necessário pensar a rota de forma sistemática e estabelecer claramente um início, pontos intermediários e o destino. As informações ao longo do caminho têm também o sentido de marcar estrategicamente determinados pontos, estabelecendo as referências que auxiliarão o individuo a se situar e compreender o caminho de ida e de volta. O reconhecimento do destino configura-se no êxito de todo o processo que envolve as três modalidades de comportamento, seja indo ou retornando. Nesse processo o conhecimento da tarefa e o conhecimento dos locais para onde se dirigir são mais duas variáveis imprescindíveis a esse êxito. Essas modalidades de comportamento são passíveis de observação quando delas se extraem ações comportamentais ou comportamentos observáveis passíveis de descrição e de categorização. 4. OBSERVAÇÃO E REGISTRO DO COMPORTAMENTO Além disso, deve se manter a uma distância razoável do observado em uma atitude neutra e discreta, e não interferir na situação, a não ser que a interferência seja objeto do estudo. Além das questões supracitadas para a eficácia da observação, Fagundes (2006) entende ser importante para o registro comportamental a marcação do tempo de duração, e o esclarecimento da técnica de registro adotada. Dos estudos apresentados por Fagundes (2006) e Danna e Matos (2006), destacamos duas modalidades de registro de comportamento: o registro cursivo e os registros categorizados. O registro cursivo trata de descrever os comportamentos sequencialmente, ou seja, na medida em que os fatos ocorrem (FAGUNDES, 2006). Para os registros categorizados, o observador utiliza categorias pré-definidas. As categorias emergem do conhecimento prévio do pesquisador e de seus autores (DANNA E MATOS, 2006). Isso implica, conforme apresentado acima, a definição do o quê observar. 4.1. O QUE OBSERVAR? No contexto desse estudo, o que observar, de maneira objetiva e sistematizada, abarca questões pertinentes ao comportamento espacial do usuário. O olhar volta-se para as ações comportamentais, frente à decisão da rota, o monitoramento da rota e o reconhecimento do destino. A postura, a exploração visual, as tomadas de informações, as comunicações e os deslocamentos (MORAES E MONT’ALVÃO, 2003, pp. 39 e 40) são comportamentos observáveis relacionados aos comportamentos de orientação, de localização e de exploração (ZINGALE, 2010). É possível observar nesses comportamentos a situação de orientação, de indecisão e de desorientação do usuário, conforme apresentado na tabela 1. Tabela 1 – Comportamentos espaciais observáveis Fonte: a autora Conforme apresentado na tabela 1, os comportamentos são categorizados de acordo com os estudos de Moraes e Mont’Alvão (2003), Ribeiro (2004) e Rangel (2011). São quatro (04) categorias, cujas ações são pertinentes a três situações elencadas à orientação espacial. Então, as categorias são (1) andar; (2) olhar; (3) parar e (4) expressões faciais e verbais durante as situações de orientação, indecisão e desorientação nos deslocamentos, A partir do levantamento e da categorização do que observar, e após o mapeamento do ambiente no qual são levantados os setores/destino, as rotas dos usuários referentes às tarefas pesquisadas, os nós, marcos referenciais e a rota da sinalização pertinente aos deslocamentos, é necessário apontar como a observação deve acontecer. A técnica da observação sistemática do comportamento do usuário é apresentada a seguir. Conforme apresentado na tabela 1, os comportamentos são categorizados de acordo com os estudos de Moraes e Mont’Alvão (2003), Ribeiro (2004) e Rangel (2011). São quatro (04) categorias, cujas ações são pertinentes a três situações elencadas à orientação espacial. Então, as categorias são (1) andar; (2) olhar; (3) parar e (4) expressões faciais e verbais durante as situações de orientação, indecisão e desorientação nos deslocamentos, A partir do levantamento e da categorização do que observar, e após o mapeamento do ambiente no qual são levantados os setores/destino, as rotas dos usuários referentes às tarefas pesquisadas, os nós, marcos referenciais e a rota da sinalização pertinente aos deslocamentos, é necessário apontar como a observação deve acontecer. A técnica da observação sistemática do comportamento do usuário é apresentada a seguir. Conforme apresentado na tabela 1, os comportamentos são categorizados de acordo com os estudos de Moraes e Mont’Alvão (2003), Ribeiro (2004) e Rangel (2011). 4.1. O QUE OBSERVAR? São quatro (04) categorias, cujas ações são pertinentes a três situações elencadas à orientação espacial. Então, as categorias são (1) andar; (2) olhar; (3) parar e (4) expressões faciais e verbais durante as situações de orientação, indecisão e desorientação nos deslocamentos, Conforme apresentado na tabela 1, os comportamentos são categorizados de acordo com os estudos de Moraes e Mont’Alvão (2003), Ribeiro (2004) e Rangel (2011). São quatro (04) categorias, cujas ações são pertinentes a três situações elencadas à orientação espacial. Então, as categorias são (1) andar; (2) olhar; (3) parar e (4) expressões faciais e verbais durante as situações de orientação, indecisão e desorientação nos deslocamentos, A partir do levantamento e da categorização do que observar, e após o mapeamento do ambiente no qual são levantados os setores/destino, as rotas dos usuários referentes às tarefas pesquisadas, os nós, marcos referenciais e a rota da sinalização pertinente aos deslocamentos, é necessário apontar como a observação deve acontecer. A técnica da observação sistemática do comportamento do usuário é apresentada a seguir. A partir do levantamento e da categorização do que observar, e após o mapeamento do ambiente no qual são levantados os setores/destino, as rotas dos usuários referentes às tarefas pesquisadas, os nós, marcos referenciais e a rota da sinalização pertinente aos deslocamentos, é necessário apontar como a observação deve acontecer. A técnica da observação sistemática do comportamento do usuário é apresentada a seguir. 4.2. COMO OBSERVAR A observação do comportamento do usuário utiliza técnica para o registro comportamental. Tal registro deve seguir a sequencia dos fatos, para registrar as ações categorizadas, portanto, há uma aproximação das duas modalidades propostas por Fagundes (2006) e Danna e Matos (2006). Em um levantamento prévio com as técnicas dos autores Bins Ely et al. (2002), Atkins (2008), Ribeiro (2004), Baptista (2011) relativas a tal registro, foi verificada também certa aproximação entre as mesmas. As variações mais significativas para esta pesquisa versam sobre: (1) maior ou menor controle do tempo de registro; (2) maior ou menor aproximação do pesquisador durante o preparo do registro; (3) maior ou menor aproximação do pesquisador ao usuário durante o registro. Para este estudo, a técnica proposta advém de adaptação a partir dos estudos dos autores pesquisados, sendo denominada de mapeamento do comportamento espacial. Nesta técnica a observação abarca as ações dos usuários em interação com a informação do ambiente, durante o seu deslocamento. O pesquisador ao preparar esta observação/ registros de comportamento, define: (1) a rota de observação; (2) delimita na planta baixa arquitetônica o trajeto, os nós, o suporte informacional durante o caminho, sobretudo os marcos referenciais; (3) categoriza os comportamentos observáveis. O usuário será definido conforme perfil previamente delineado pelo pesquisador e em conformidade com a tarefa. Nesta proposta, o pesquisador escolhe o usuário randomicamente no ambiente, de acordo com as características pré-estabelecidas. Durante a observação, o pesquisador busca se manter neutro o suficiente para não ser notado. Observa a certa distância o comportamento do usuário e registra as ações observáveis categorizadas. Ao final, o usuário é convidado a fazer um breve relato sobre as facilidades e dificuldades do seu deslocamento. Essa medida visa esclarecer possíveis dúvidas na observação. Os dados são registrados em planilha previamente preparada (Figuras 2 e 3), fotografias e/ou vídeos. 4.2. COMO OBSERVAR Figura 2 – Planilha de registro do comportamento do usuário (página 1) Fonte: a autora Figura 3 – Planilha de registro do comportamento do usuário (página 2) Fonte: a autora a constam dados acerca do setor da tarefa; da data hora de início e hora Figura 2 – Planilha de registro do comportamento do usuário (página 1) Fonte: a autora Figura 3 – Planilha de registro do comportamento do usuário (página 2) Fonte: a autora Figura 2 – Planilha de registro do comportamento do usuário (página 1) Fonte: a autora Figura 3 – Planilha de registro do comportamento do usuário (página 2) Fonte: a autora Na planilha constam dados acerca do setor, da tarefa; da data, hora de início e hora do fim da avaliação, e o nome do avaliador. Ainda possui campo para a planta baixa ou plantas baixas, abarcando todas as circulações possíveis para o desenvolvimento da tarefa. O avaliador fará marcações com linhas para apontar as rotas dos usuários, e marcações com numerais dos pontos pré-estabelecidos como importantes para registrar os comportamentos. Na planilha constam dados acerca do setor, da tarefa; da data, hora de início e hora do fim da avaliação, e o nome do avaliador. Ainda possui campo para a planta baixa ou plantas baixas, abarcando todas as circulações possíveis para o desenvolvimento da tarefa. O avaliador fará marcações com linhas para apontar as rotas dos usuários, e marcações com numerais dos pontos pré-estabelecidos como importantes para registrar os comportamentos. Os comportamentos previamente categorizados são registrados nas rotas e nos pontos pré- determinados na planta baixa. Como os comportamentos são complexos e possuem dados de imprevisibilidade, são considerados na planilha campos para pontos não previstos e que se apresentem relevantes na observação. 4.3. OS RESULTADOS Os resultados a partir do cruzamento das variáveis: comportamento (andar/ olhar/ parar/ expressões) x situação (orientação/ indecisão/ desorientação) x local ocorrido, poderão apresentar dados quantitativos e qualitativos acerca da informação do ambiente construído na orientação espacial do usuário. A tabulação irá evidenciar não só os locais no ambiente que apresentam falhas na informação – layout e/ou sinalização, como também aqueles em que a informação está melhor configurada. É possível verificar dentre os observados, o número de usuários que se encontram orientados, indecisos ou desorientados em cada ponto. Além disso, podem indicar, por meio das expressões verbais, a satisfação/insatisfação dos observados com o ambiente. Os dados levantados são relevantes para mensurar a visibilidade do ambiente nas rotas observadas, sendo incorporados ao corpus de dados relativos à observação do ambiente construído, para a aplicação da técnica do índice de visibilidade (VI). O índice de visibilidade (VI) é uma técnica que quantifica a visibilidade do ambiente. Foi desenvolvida por Braaksma e Cook (1980) para aferir o grau de visibilidade do ambiente de aeroportos. Lam et. al. (2002) e Ribeiro (2004 e 2009) utilizaram o VI para quantificar a visibilidade de aeroportos. Rangel (2011) a utilizou no estudo de um hospital. O VI avalia o layout e a sinalização do ambiente, por meio do cálculo matemático da propriedade de visibilidade desses elementos alocados na rota dos usuários. A visibilidade é calculada ponto a ponto, e a soma da visibilidade dos pontos indica o grau de visibilidade do ambiente. Os dados levantados com o registro do comportamento do usuário são a base para ratificar os nós pertinentes à rota pesquisada (FIGURA 4), e para desenvolver o gráfico linear com as linhas de visão (FIGURA 5). Tais linhas mostram se existe visão (direta ou indireta) de um ponto ao outro da rota. A visão direta é propiciada pelos elementos da arquitetura e pelo layout do espaço, e a indireta advém de informações adicionais tais como a sinalização. As linhas de visão estabelecem a rede de conexões com a visibilidade entre os nós. Para analisar com maior clareza tal rede de conexões, essa é desdobrada em uma matriz binária quadrática – matriz de visibilidade. Para o nó percebido a partir de outro ponto o valor registrado é 1. Caso contrário o valor é 0 (BRAAKSMA E COOK, 1980). O VI induz a análise mais criteriosa dos dados levantados nos registros de comportamento. Visa verificar o desempenho dos deslocamentos e da informação ponto a ponto. 5. O comportamento espacial decorre de mecanismos naturais, contudo, emerge da consciência dos seus movimentos e da atenção aos detalhes do ambiente. Durante a navegação, o usuário reage, consciente ou inconscientemente, frente às facilidades e dificuldades para iniciar, continuar e concluir sua rota. Estas reações expansivas ou contidas são ações observáveis, que podem ser indicadoras de problemas de wayfinding. Entendemos ser de grande importância ao projeto que envolve o wayfinding no ambiente construído, categorizar e mapear esse comportamento ponto a ponto no ambiente. Este estudo, portanto, visa contribuir com a pesquisa em ergonomia por apresentar os comportamentos passíveis de serem observados em ambientes diversificados e pertinentes a diversos perfis de usuários. Conforme apresentado, a observação é a base para a compreensão da situação que envolve a tarefa pesquisada, como também, para o planejamento e desenvolvimento de outros métodos e técnicas. Ocorrem, dessa forma, que os dados coletados por meio dos registros de comportamento aqui apresentado, devam ser utilizados em outras técnicas, com destaque para o VI. Os resultados cruzados ofertarão, certamente, maior efetividade para a pesquisa. 6. ARTHUR P, PASSINI, R. Wayfinding: people, signs, and architecture. 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Anais do 70 Congresso de Pesquisa & Desenvolvimento em Design, 2006. RIBEIRO, L.; MONT’ALVÃO, C. Habilidades espaciais e estratégias de navegação que influenciam a efetividade do wayfinding. Anais do 70 Congresso de Pesquisa & Desenvolvimento em Design, 2006. SEBEOK, Th. A. Signs. An introduction to Semiotics. Toronto Buffalo London: University of Toronto Press, 2001. ZINGALE, Salvatore. Wayfinding using colour: A semiotic research hypothesis. In CHEN, Lin-Lin; DJAJADINIGRAT, Tom; FEIJS, Loe; KYFFIN, Steven; STEFFEN, Dagmar; YOUNG, Bob. Design and semantics of form and movement – DeSForM 2010. Lucerne: 2010. Págs.22-32. ZINGALE, Salvatore. Wayfinding using colour: A semiotic research hypothesis. In CHEN, Lin-Lin; DJAJADINIGRAT, Tom; FEIJS, Loe; KYFFIN, Steven; STEFFEN, Dagmar; YOUNG, Bob. Design and semantics of form and movement – DeSForM 2010. Lucerne: 2010. Págs.22-32.
https://openalex.org/W2024859375	https://verbumetecclesia.org.za/index.php/ve/article/download/345/333	English	null	The beauty of theology	Verbum et Ecclesia	2,010	cc-by	1,842	The beauty of theology Book Title: anselm of canterbury: The beauty of theology Book Cover: David S. Hogg points out that although a lot of research work has already been done on Anselm’s views concerning the existence of God and the atonement, the surrounding and indeed related areas have been left largely untouched. This discovery led Hogg to examine the works of Anselm on a much broader scale and then come to new conclusions. Hogg argues that there is a disadvantage in a too narrow focus on particular themes in Anselm’s works; we run the risk of losing the real essence of Anselm’s thinking when not taking the wider context into account. For example, Anselm’s understanding of the atonement might not be rightly understood when we fail to look further and take an adequate account of his understanding of the Trinity. To this, Hogg adds that Anselm’s interpretation of the Trinity, as it relates to the incarnation and atonement, is further explained and enhanced when we read the Proslogion. In the Proslogion Anselm not only attends to the existence of God, but also to the nature of God in whom all humans and all other things live and find their being. In the same way Hogg argues that the Cur Deus Homo can be best explained when this work is studied along with the Monologion (especially chapter 39 and following). Book Title: anselm of canterbury: The beauty of theology Book Cover: The common way of interpreting the Proslogion is to do it on the basis of the logical argument in chapters two, three and four, but what if the emphasis does not rightly lie there? Hogg works on the possibility of the revelation, outlined in chapter one and developed from there in chapters five to twenty-six, as the real basis for the argument in chapters two to four. Hogg comes to the conclusion that our contemporary convictions colour our comprehension of medieval concepts. He says that it is indeed typical of our contemporary mindset to prefer a logical argument that develops sequentially from one proposition to the next. Though medieval theological and philosophical thinking do sometimes develop in this way, Hogg finds evidence that for the medieval mind this was not the most desirable means of expressing an argument. Hogg discovers, as one characteristically medieval way of communicating truth and conviction, the explaining and development of ideas on the basis of an aesthetic ideal. The beauty of theology For the medieval mind it is the presupposition of ‘beauty and fittingness’, as seen in the relationship between creator and creation, that provides the only foundation upon which our argument or series of propositions can be built. For Anselm and medieval thinking the basis of all our argumentation must be that there is something or indeed someone who holds everything together. And this is what Hogg wants to emphasise. Order and harmony and beauty, as guaranteed by the nature of God who created in his own image and sustains by his own power, is the needed precondition for medieval rational and logical thinking. Publisher: Ashgate Publishing Group, United Kingdom; 2004, p. 218, £16.99/£15.29 * Book price at time of Review Publisher: Ashgate Publishing Group, United Kingdom; 2004, p. 218, £16.99/£15.29 *Book price at time of Review Verbum et Ecclesia Article #345 Verbum et Ecclesia Article #345 Verbum et Ecclesia Article #345 Review Title: The beauty of theology Reviewer: Jacobus P. Labuschagne1 Affiliation: 1Department of Church History and Polity, University of Pretoria, South Africa emails: kobus.labuschagne@up.ac.za natasha.felix@up.ac.za Postal address: PO Box 38046, Garsfontein- East 0060, South Africa How to cite this book review: Labuschagne, J.P., 2010, The beauty of theology’, Verbum et Ecclesia 31(1), Art. #345, 2 pages. DOI: 10.4102/ ve.v31i1.345 This review is available at: http://www.ve.org.za © 2010. The Authors. Licensee: OpenJournals Publishing. This work is licensed under the Creative Commons Attribution License. Review Title: The beauty of theology Reviewer: Jacobus P. Labuschagne1 Affiliation: 1Department of Church History and Polity, University of Pretoria, South Africa emails: kobus.labuschagne@up.ac.za natasha.felix@up.ac.za Postal address: PO Box 38046, Garsfontein- East 0060, South Africa How to cite this book review: Labuschagne, J.P., 2010, The beauty of theology’, Verbum et Ecclesia 31(1), Art. #345, 2 pages. DOI: 10.4102/ ve.v31i1.345 This review is available at: http://www.ve.org.za © 2010. The Authors. Licensee: OpenJournals Publishing. This work is licensed under the Creative Commons Attribution License. In his Cur Deus Homo Hogg discovers in Anselm an overarching principle of God as by nature the God of order, harmony and beauty, and that God necessarily acts in accordance to these aspects of his nature. He recognises in Anselm’s thought as such an aesthetic dimension, which is a fully integrated part of creator and creation. The beauty of theology Hogg found that many scholars, while aware of Anselm’s aesthetic dimension, do not as yet fully realise that this aesthetic dimension is indeed inextricably interwoven in the whole of Anselm’s thinking. This means then that when Anselm deals, for instance, with the subject of the atonement in his Cur Deus Homo, he is not merely interested in explaining a rational and objective view of the atonement. His point of departure remains the aesthetic dimension. Therefore, all goodness, beauty and order around us are the results of God’s sustaining presence, God who in his uniqueness is the One, the Good and the Beautiful. Ugliness, wrong doings or disorder, are all deviations from the aesthetic dimension, and they are there because of sin. This, however, can be and indeed is rectified by God in his goodness. From this foundation follows Anselm’s explanation of the atonement and the new creation effected by God. Hogg found beauty and order as inherent in Anselm’s perception of reality. Understandably Hogg discovered that this then holds good not only for the Cur Deus Homo, but also for Anselm’s Monologion, his Proslogion, his Prayers and Meditations, and his De Incarnatione Verbi, also his De Grammatico, De Veritate, De Libertate Arbitrii, and De Casu Diaboli. For Anselm the person who seeks to understand something of God’s revelation is also the person who will somehow find himself rejoicing in the experience of the divine, experiencing the depth and the mystery of God, and becoming aware of the beatific vision. The church has the task to let the whole of humanity hear about the atoning sacrifice of Christ and about reconciliation through renewal so that every human being may regain the beatific vision. Hogg criticises Southern who refers to beauty in Anselm’s prayers as overdoing it. Hogg is convinced that Anselm’s prayers are not only intended to be beautiful, because they also bear witness to Anselm’s perception of reality. Anselm’s prayers are a manifestation of his existential awareness of the reality of the reconciliation achieved by Jesus Christ on the cross, and he hopes that also his readers will experience the same. The call of God, to be holy as he is holy, is so much alive in the mind of Anselm and he wishes to make his own all that God offers. Book Review Book Review The beauty of theology It is often said that Anselm has no central theme in his writings and that the subjects he wrote about were purely occasional. Hogg opposes this common view, by now not searching for a unity in the topics Anselm wrote about, but by discovering the weltbild (the hermeneutical framework, the particular understanding of reality) behind the thinking, as the real unifying factor. The weltbild, once discovered, without being repeatedly mentioned, nevertheless becomes conspicuous in the way Anselm said things and why he said them. The most pervasive element in his weltbild, which affects all 1 Vol. 31 No. 1 Page 1 of 2 Verbum et Ecclesia (page number not for citation purposes) http://www.ve.org.za Book Review Labuschagne of his thinking, bringing together particularity and diversity, is aesthetics. Hogg wishes to emphasise that Anselm indeed had an aesthetic model of reality in his mind. This understanding of reality was not uncommon in the literature of the world of which Anselm was part. This was an understanding of reality built upon the aesthetic view that all was wonderful, in harmony and unity, beauty and fittingness, attributes of God’s being and attributes imprinted on the whole of creation and therefore also imprinted on man’s way of reasoning, even the Christian’s prayers and meditation. of his thinking, bringing together particularity and diversity, is aesthetics. Hogg wishes to emphasise that Anselm indeed had an aesthetic model of reality in his mind. This understanding of reality was not uncommon in the literature of the world of which Anselm was part. This was an understanding of reality built upon the aesthetic view that all was wonderful, in harmony and unity, beauty and fittingness, attributes of God’s being and attributes imprinted on the whole of creation and therefore also imprinted on man’s way of reasoning, even the Christian’s prayers and meditation. medieval literature would clearly indicate that aesthetic ideals influenced all thinking in the medieval mind. It is generally accepted that medieval theologians depended heavily on classical authors and the early church fathers. Hogg points out that Anselm more than once indicated his preference for Augustine, and Augustine also revealed his appreciation for aesthetic thinking. medieval literature would clearly indicate that aesthetic ideals influenced all thinking in the medieval mind. It is generally accepted that medieval theologians depended heavily on classical authors and the early church fathers. The beauty of theology Hogg points out that Anselm more than once indicated his preference for Augustine, and Augustine also revealed his appreciation for aesthetic thinking. Hogg hopes to provide a renewed appreciation for beauty and fittingness as foundational in Anselm’s thinking, and to reveal that Anselm teaches us that the logic of human reason is not the only way of understanding God in his relation to man. Though we might today be hermeneutically far from the presuppositions of Anselm’s mind, there is still a lot to be learned from his understanding and a lot to appreciate in his devotion. Beauty is for Anselm indeed a multifaceted expression, and he can therefore even refer to the person and work of Christ as supreme beauty. ‘Aesthetics’ is for Hogg the all-embracing concept, including a wide range of issues connected to beauty. Hogg uses aesthetics as an explanatory concept, when describing Anselm’s beatific vision. Hogg does this in spite of the fact that aesthetics was a term that came 650 years later than Anselm’s time, originating from Baumgarten in the 18th century, and also in spite of a notably different direction taken by Baumgarten and Kant with the concept of aesthetics within the study of epistemology. He is convinced that even a cursory study of The book offers a new insight into Anselm’s thinking, and is a valuable and important contribution to the studies of medieval theologians. It is therefore recommended for theologians, students and ordinary Christians interested in medieval studies. Verbum et Ecclesia Verbum et Ecclesia Article #345 Article #345 Vol. 31 No. 2 Page 2 of 2 2 Verbum et Ecclesia 2 (page number not for citation purposes) http://www.ve.org.za
https://openalex.org/W2596479943	https://europepmc.org/articles/pmc5354280?pdf=render	English	null	Assessment of optimal strategies in a two-patch dengue transmission model with seasonality	PloS one	2,017	cc-by	10,503	Assessment of optimal strategies in a two- patch dengue transmission model with Jung Eun Kim1☯, Hyojung Lee1☯, Chang Hyeong Lee1, Sunmi Lee2,3 1 Department of Mathematical Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea, 2 Department of Applied Mathematics, Kyung Hee University, Yongin, Republic of Korea, 3 Institute of Natural Sciences, Kyung Hee University, Yongin, Republic of Korea Jung Eun Kim1☯, Hyojung Lee1☯, Chang Hyeong Lee1, Sunmi Lee2,3 1 Department of Mathematical Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea, 2 Department of Applied Mathematics, Kyung Hee University, Yongin, Republic of Korea, 3 Institute of Natural Sciences, Kyung Hee University, Yongin, Republic of Korea ☯These authors contributed equally to this work. * chlee@unist.ac.kr (CL); sunmilee@khu.ac.kr (SL) ☯These authors contributed equally to this work. * chlee@unist.ac.kr (CL); sunmilee@khu.ac.kr (SL) a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Editor: Jeffrey Shaman, Columbia University, UNITED STATES Received: July 22, 2016 Accepted: February 26, 2017 Published: March 16, 2017 Copyright: © 2017 Kim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. OPEN ACCESS Citation: Kim JE, Lee H, Lee CH, Lee S (2017) Assessment of optimal strategies in a two-patch dengue transmission model with seasonality. PLoS ONE 12(3): e0173673. https://doi.org/10.1371/ journal.pone.0173673 Citation: Kim JE, Lee H, Lee CH, Lee S (2017) Assessment of optimal strategies in a two-patch dengue transmission model with seasonality. PLoS ONE 12(3): e0173673. https://doi.org/10.1371/ journal.pone.0173673 Editor: Jeffrey Shaman, Columbia University, UNITED STATES RESEARCH ARTICLE Abstract Emerging and re-emerging dengue fever has posed serious problems to public health offi- cials in many tropical and subtropical countries. Continuous traveling in seasonally varying areas makes it more difficult to control the spread of dengue fever. In this work, we consider a two-patch dengue model that can capture the movement of host individuals between and within patches using a residence-time matrix. A previous two-patch dengue model without seasonality is extended by adding host demographics and seasonal forcing in the transmis- sion rates. We investigate the effects of human movement and seasonality on the two-patch dengue transmission dynamics. Motivated by the recent Peruvian dengue data in jungle/ rural areas and coast/urban areas, our model mimics the seasonal patterns of dengue out- breaks in two patches. The roles of seasonality and residence-time configurations are highlighted in terms of the seasonal reproduction number and cumulative incidence. More- over, optimal control theory is employed to identify and evaluate patch-specific control mea- sures aimed at reducing dengue prevalence in the presence of seasonality. Our findings demonstrate that optimal patch-specific control strategies are sensitive to seasonality and residence-time scenarios. Targeting only the jungle (or endemic) is as effective as control- ling both patches under weak coupling or symmetric mobility. However, focusing on inter- vention for the city (or high density areas) turns out to be optimal when two patches are strongly coupled with asymmetric mobility. Assessment of optimal strategies in a two-patch dengue transmission model with seasonality Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. syndrome [4, 5]. Despite intensive vector control programs, many countries have experienced dengue reemergence over the last few decades [6, 7]. In 2015, the first dengue vaccine was used in Mexico, however, the effectiveness of the dengue vaccine must be further investigated and assessed [8, 9]. Modeling vector-borne diseases is a challenge for many researchers due to complex factors, including the interplay between vector and host dynamics, spatial or multi-strain, immunity levels, or vaccination [10, 11]. Mathematical modeling has evolved from simpler models to more complex models that include climate changes, socio-economic changes, urbanization and transportation [12, 13]. Particularly, geographic heterogeneity and climate change are some of the key factors when modeling recurrent vector-borne diseases. The spatio-temporal dynamics of infectious disease has been studied using partial differential equations and meta- population models [14, 15]. Human movement plays a significant role on disease reemergence and persistence [16–18]. There are several approaches to model the effect of human movement [17] (references are therein). In that work, two different frameworks have been employed: the Lagrangian framework mimics human commuting behavior, and the Eulerian framework mimics human migration. A discrete-time multi-patch model was used to study the transmis- sion dynamics of dengue in multi-locations by incorporating the movement of people between villages and a city [19]. The impact of commuters between patches are well studied in star net- works of villages and a city [20], and quantifying the impact of human mobility on the spread of malaria has also been studied [21]. In these studies, the role of human movement was highlighted in the vector-born disease spread in multi-locations. Recurrent dengue outbreaks have been commonly observed in South America and other areas of the world [12, 22–24]. Many of these regions have shown seasonal patterns that directly influence the dynamics of dengue transmission [25]. A number of mathematical mod- els have been developed to understand the complexity of dengue transmission dynamics [11, 26]. It was shown that seasonality plays a major role in the size of the mosquito population, which influences the decision of effective strategies to control the disease [27, 28]. Introduction Funding: Chang Hyeong Lee was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2014R1A1A2054976). Sunmi Lee was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (No.20152194). Dengue is one of the most important vector-borne diseases, affecting more than 50 million people all around the world [1, 2]. Each year, there are 2.5 billion individuals at risk, including approximately 500,000 severe cases and 22,000 deaths, mostly involving children [3]. A. aegypti is the main vector that transmits dengue and it carries four different virus serotypes of the genus Flavivirus. Dengue fever is a mild disease; however, prior strain-specific infections may progress to increased susceptibility to severe dengue hemorrhagic fever and dengue shock PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 1 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 Two-patch dengue transmission model with seasonality A two-patch dengue transmission model has been developed for a short time scale (less than one year) in the absence of seasonality [31]. Since recurrent dengue outbreaks occur in many tropical and sub-tropical countries [33], it is more realistic to consider seasonality factors for the long-term dynamics of dengue transmission. For each patch, we add demographics to the host population and seasonality to the vector population and the transmission rates, as pro- posed in our baseline model [30]. The variables of the vectors are Sv for the susceptible class, Ev for the exposed class and Iv for the infected class, with the total vector population Nv Sv + Ev + Iv. The variables for the human classes are Sh for the susceptible class, Eh for the exposed class, Ih for the infected class, and Rh for the recovered class, with Nh Sh + Eh + Ih + Rh. Since the dengue fatality rate is below 1% under proper medical cares [34], deaths due to dengue are assumed to be negligible, that is, the total human population is assumed to be constant. How- ever, seasonality evidently affects the total population size of the vectors [32, 35–37]. Hence, the seasonality of the vector population is modeled by a periodic vector birth rate. The two patches are coupled via a residence-time matrix P = (pij)2×2 for i, j = 1, 2. It is assumed that the human residence-time matrix is not affected by seasonality, so pij is constant in [0, 1] satisfying the condition P2 j¼1 pij ¼ 1 for i = 1, 2. The residence-time matrix can model the virtual movement of humans between/within patches. More precisely, the coupling param- eter pij represents the proportion of time that a person residing in patch i visits patch j. Hence, our model is based on the Lagrangian framework that mimics human commuting behavior [17]. It is assumed that vectors do not move, that is, only humans can move across patches. More details of the residence-time matrix are found in the previous work [31]. Several ento- mological factors are included in the temperature-dependent entomological parameters such as mortality rates and oviposition rates in a mosquito life-cycle [10]. Recently, ten Bosch et al carried out extensive studies for the dengue transmission dynamics with multiple strains and seasonality [29]. They proposed six models by employing a pattern oriented modeling approach and identified the parameter space so that all proposed models reproduced the observed patterns of dengue outbreaks. Furthermore, they demonstrated that seasonal forcing played a key role in their model selection. These studies highlight the importance of seasonality in the dynamics of dengue transmission. Therefore, it is critical to incorporate the environmental and seasonal effects into dengue transmission modeling. As reported in previous research [30], the 2000 dengue outbreaks in Peru were examined by using a two-patch model where the jungle areas were always endemic, observing how human movement caused epidemics in the coastal cities. Based on the work in [30], the transmission dynamics of dengue was investigated in a two-patch model [31]. Two patches represented two interconnected locations (a jungle/rural area and a city/urban area) and they were coupled by a residence-time matrix assuming constant transmission rates in both the host and vector populations. The focus was on the overall transmission dynamics between patches under different residence-time configurations for a short-time scale (less than a year). In the present work, we formulate a non-autonomous system to investigate a relatively longer-term dengue dynamics by incorporating seasonality into the two-patch system. The effect of seasonality on patch-specific dynamics including the seasonal basic reproduction number and cumulative incidence is highlighted under various scenarios [32]. We formulate an optimal control problem in a host-vector population in two-patches. The goal is to identify PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 2 / 21 Assessment of optimal strategies in a two-patch dengue transmission model with seasonality patch-specific strategies that minimize the total number of infected humans and vectors in the presence of seasonality. The effects of seasonality and residence-time configurations are explored on patch-specific preventive controls for a longer time scale. PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 3 / 21 Two-patch dengue transmission model with seasonality The two-patch dengue transmission dynamics is captured by the following patch-specific system of nonlinear ordinary differential equations: _Sv1 ¼ mv1ðtÞNv1 bv1ðtÞðp11Ih1=Nh1 þ p21Ih2=Nh2ÞSv1 mvSv1 _Ev1 ¼ bv1ðtÞðp11Ih1=Nh1 þ p21Ih2=Nh2ÞSv1 mvEv1 kEv1 _I v1 ¼ kEv1 mvIv1 _Sh1 ¼ mhNh1 Sh1ðbh1ðtÞp11Iv1=Nv1 þ bh2p12Iv2=Nv2Þ mhSh1 _Eh1 ¼ Sh1ðbh1ðtÞp11Iv1=Nv1 þ bh2p12Iv2=Nv2Þ gEh1 mhEh1 _I h1 ¼ gEh1 dIh1 mhIh1 _Rh1 ¼ dIh1 mhRh1 ð1Þ _Sh2 ¼ mhNh2 Sh2ðbh1ðtÞp21Iv1=Nv1 þ bh2ðtÞp22Iv2=Nv2Þ mhSh2 _Eh2 ¼ Sh2ðbh1ðtÞp21Iv1=Nv1 þ bh2ðtÞp22Iv2=Nv2Þ gEh2 mhEh2 _I h2 ¼ gEh2 dIh2 mhIh2 _ d _Rh2 ¼ dIh2 mhRh2; _Rh2 ¼ dIh2 mhRh2; 3 / 21 Assessment of optimal strategies in a two-patch dengue transmission model with seasonality where where mv1ðtÞ ¼ mv 1 ε1 sin 2pt 365 ; mv2ðtÞ ¼ mv 1 ε2 sin 2pt 365 : ð2Þ ð2Þ The vector birth rate, μvi is modeled as a sinusoidal function with a distinct amplitude, which results in the varying vector population size over time (high in the summer and low in the winter) [32, 37, 38]. Dengue is transmitted through two types of interactions: from hosts to vectors and from vectors to hosts. Susceptible vectors acquire infections through contact with infected human individuals at the per-infective and per-capita rate βvi(t). It is also transmitted when susceptible individuals are infected via contacts with infected vectors at the per-infective and per-capita rate βhi(t). The dengue data in Peru from 2001 to 2008 shows recurrent seasonal patterns in Fig 1 (den- gue cases in the jungle and the coastal city are shown [25]). Dengue is endemic in the jungle, which is presumed as the driving force of dengue in Peru. In coastal cities, the number of den- gue cases are very small in the winter due to the low vector population Hence we consider the The dengue data in Peru from 2001 to 2008 shows recurrent seasonal patterns in Fig 1 (den- gue cases in the jungle and the coastal city are shown [25]). Dengue is endemic in the jungle, The dengue data in Peru from 2001 to 2008 shows recurrent seasonal patterns in Fig 1 (den- gue cases in the jungle and the coastal city are shown [25]). Two-patch dengue transmission model with seasonality Seasonality scenario II In both patches, βv1(t) and βv2(t) are sinusoidal type functions with two distinct ampli- tudes (higher in Patch 1 than Patch 2). The transmission rate in Patch 1 oscillates throughout the year but never goes to zero, in contrast to the one in Patch 2, which is almost zero in the winter [32, 40–42]. atch-specific transmission rate functions are given by (S2). Seasonality scenario II In both patches, βv1(t) and βv2(t) are sinusoidal type functions with two distinct ampli- tudes (higher in Patch 1 than Patch 2). The transmission rate in Patch 1 oscillates throughout the year but never goes to zero, in contrast to the one in Patch 2, which is almost zero in the winter [32, 40–42]. atch-specific transmission rate functions are given by bv1ðtÞ ¼ 0:07 cos 2pt 365 þ 1 þ 0:1; bv2ðtÞ ¼ 0:21 cos 2pt 365 þ 0:04; if t 2 365 n 1 4 ; 365 n þ 1 4 for n ¼ 0; 1; 2; ; 0:01; otherwise: ð4Þ 8 < : bv1ðtÞ ¼ 0:07 cos 2pt 365 þ 1 þ 0:1; bv1ðtÞ ¼ 0:07 cos 2pt 365 þ 1 þ 0:1; bv2ðtÞ ¼ 0:21 cos 2pt 365 þ 0:04; if t 2 365 n 1 4 ; 365 n þ 1 4 for n ¼ 0; 1; 2; ; 0:01; otherwise: ð4Þ 8 < : bv2ðtÞ ¼ 0:21 cos 2pt 365 þ 0:04; if t 2 365 n 1 4 ; 365 n þ 1 4 for n ¼ 0; 1; 2; ; 0:01; otherwise: ð4Þ 8 < : ð4Þ Also, the patch-specific transmission rates from vectors to hosts are defined as βhi(t) = mi(t)βvi(t), where mi(t) = Nvi(t)/Nhi is the ratio of vector to host [43, 44]. It is assumed that the average vector population size is approximately twice of the human population size (Nvi Nhi 2) [45]. Note that these parameters (μvi(t), βvi(t), βhi(t)) are seasonally varying time-dependent (see Fig A in S1 Appendix). The annual oscillations in dengue incidence are caused by complex fac- tors including the seasonality of the vector population and the transmission rates. It is well known that temperature and precipitation play a key role in the seasonal patterns of dengue incidence [32, 46–48]. Two-patch dengue transmission model with seasonality In general, these parameters increase as temperature or precipitation increases to a certain extent. In our work, we assumed that temperature and precipitation in a jungle/rural area (one patch) are greater than a city/urban area (the other patch). Also, this holds for any two locations with one higher than the other. It has been studied in the previous work when the climate in two locations is similar [31]. The system of nonlinear ordinary differential equations is non-autonomous with periodic forcing terms μvi(t), βvi(t) and βhi(t) for a 1-year period. Numerical simulations show that the system has an asymptotically stable limit cycle, which is a forced-period solution of period 1-year (See Fig B in S1 Appendix). Since recurrent epidemics are considered here, the focus is on the dynamics of periodic solutions, which is independent of the initial values. Descriptions of the parameters are given in Tables 1 and 2. Two-patch dengue transmission model with seasonality Dengue is endemic in the jungle, which is presumed as the driving force of dengue in Peru. In coastal cities, the number of den- gue cases are very small in the winter due to the low vector population. Hence, we consider the following two different types of transmission rates, βvi(t) (i = 1, 2) motivated by the work [30]. which is presumed as the driving force of dengue in Peru. In coastal cities, the number of den- gue cases are very small in the winter due to the low vector population. Hence, we consider the following two different types of transmission rates, βvi(t) (i = 1, 2) motivated by the work [30]. (S1). Seasonality scenario I For Patch 1, βv1(t) is assumed to be a constant function to model an endemic situation in Patch 1. For Patch 2, the transmission rate has a positive value in the summer, describing that vectors are active and contact hosts, and it is almost zero in the winter, resulting in no incidence rates between hosts and vectors. Thus, βv2(t) is modeled as a square wave function, which is frequently used for seasonally varying epidemic models [39, 40]. Patch-specific transmission rate functions are given by (S1). Seasonality scenario I For Patch 1, βv1(t) is assumed to be a constant function to model an endemic situation in Patch 1. For Patch 2, the transmission rate has a positive value in the summer, describing that vectors are active and contact hosts, and it is almost zero in the winter, resulting in no incidence rates between hosts and vectors. Thus, βv2(t) is modeled as a square wave function, which is frequently used for seasonally varying epidemic models [39, 40]. Patch-specific transmission rate functions are given by bv1ðtÞ ¼ 0:21; bv2ðtÞ ¼ 0:21; if t 2 ð365 ðn 1 4Þ; 365 ðn þ 1 4ÞÞ for n ¼ 0; 1; 2; ; 0:01; otherwise: ð3Þ 8 < : ð3Þ ð3Þ Fig 1. Weekly number of dengue cases in the jungle and the coast in Peru. https://doi.org/10.1371/journal.pone.0173673.g001 Fig 1. Weekly number of dengue cases in the jungle and the coast in Peru. https://doi.org/10.1371/journal.pone.0173673.g001 Fig 1. Weekly number of dengue cases in the jungle and the coast in Peru. https://doi.org/10.1371/journal.pone.0173673.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 4 / 21 Assessment of optimal strategies in a two-patch dengue transmission model with seasonality (S2). Assessment of optimal strategies in a two-patch dengue transmission model with seasonality Table 2. Definitions and baseline values of parameters used in numerical simulations. Parameters Description Value Ref pij Proportion of time for hosts visiting patch j from patch i 0–1 [31] γ Progression rate from latent to infectious for host (days−1) 1/5.5 [45] κ Progression rate from latent to infectious for vector (days−1) 1/12 [45] δ Recovery rate (days−1) for infectious class for host (days−1) 1/5.0 [45] ε1 Amplitude of oscillations in vector birth rate in Patch 1 0, 0.1 [49–51] ε2 Amplitude of oscillations in vector birth rate in Patch 2 0.2 [49–51] μh Host birth/death rate (days−1) 1/(65 * 365) [45] μv Average vector birth/death rate (days−1) 1/14 [45] Nhi Total number of hosts in patch i 100000 Assumption Nvi Total number of vectors in patch i [45] μvi Vector birth rate in patch i (days−1) [49–51] mi Number of vectors per host in patch i [43, 44] βvi Transmission rate from host to vector in patch i (days−1) [43] βhi Transmission rate from vector to host in patch i (days−1) ** [43, 44] b The upper bound of control (efﬁcient reduction rates, days−1) 0.35 Wi Weight constants on controls i = 3, 4 5000, 10000, 50000 ble 2. Definitions and baseline values of parameters used in numerical simulations. pesticide (sprays), mosquito repellents, reduction of the impact of vector breeding grounds, or the results of education campaigns, which increase personal protection. It is assumed that these preventive interventions do not reduce the total vector population significantly, and the effect of these interventions implicitly translates in reductions of transmission between vec- tors and hosts per unit time. In particular, incidence rates including controls are modified as bvið1 uiðtÞÞSvi P2 j¼1 pjiIhj and Shi P2 j¼1 bhjð1 ujðtÞÞpijIvj for i = 1, 2. The controlled two- patch system is given as pesticide (sprays), mosquito repellents, reduction of the impact of vector breeding grounds, or the results of education campaigns, which increase personal protection. It is assumed that these preventive interventions do not reduce the total vector population significantly, and the effect of these interventions implicitly translates in reductions of transmission between vec- tors and hosts per unit time. Optimal controls in two-patch dengue transmission model More recently, optimal control theory has been successfully employed in many biological and epidemiological models [52–54]. Optimal control problems have been formulated to identify optimal strategies and study the impact of control measures for vector-borne diseases [55, 56]. In this section, we formulate an optimal control problem in order to implement effective patch-specific control measures that take into account different coupling and sea- sonality cases. The two-patch dengue model is modified by incorporating the patch-specific control functions (1 −ui(t)) into the incidence rates at which humans and vectors get infected for patch i (i = 1, 2) in Eq (1). Preventive control efforts may involve the application of Table 1. Parameter values in a residence-time matrix. Coupling intensity Weak coupling Strong coupling Symmetric coupling p11 = 0.99 p12 = 0.01 p21 = 0.01 p22 = 0.99 p11 = 0.7 p12 = 0.3 p21 = 0.3 p22 = 0.7 Asymmetric coupling p11 = 0.9 p12 = 0.1 p21 = 0.001 p22 = 0.999 p11 = 0.7 p12 = 0.3 p21 = 0.001 p22 = 0.999 https://doi.org/10.1371/journal.pone.0173673.t001 Table 1. Parameter values in a residence-time matrix. 5 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 https://doi.org/10.1371/journal.pone.0173673.t002 Simulation results In this section, we present the two-patch dengue transmission dynamics in the absence of con- trols and in the presence of controls. The roles of a residence-time matrix and seasonality are investigated. More specifically, the residence-time matrix configurations include the coupling intensity and the mobility patterns. For instance, weak coupling implies that most humans stay in their own patch while strong coupling implies that certain proportions of humans visit the other patch. Mobility patterns represent the symmetry of human movement between the two patches. For example, if the proportion of humans visiting from patch i to patch j is the same as for patch j to patch i, then it is symmetric. Asymmetric mobility implies that the time spent for each proportion becomes more asymmetric. For the asymmetric mobility pattern, we assume that more humans from the jungle/rural area (Patch 1) visit the city/urban area (Patch 2) than the other way around. We first focus on the case when the two patches have the similar population size (Nh1 = Nh2). Then, we will discuss the impact of different subpopulation sizes later. The population size of the coastal area is about twice as the population size of the jungle area in Peru [59], hence we consider the second case of Nh1 < Nh2 with Nh2 = 2Nh1. The objective functional to be minimized is defined as Jðu1ðtÞ; u2ðtÞÞ ¼ Z tf 0 W1ðIh1ðtÞ þ Iv1ðtÞÞ þ W2ðIh2ðtÞ þ Iv2ðtÞÞ þ 1 2 W3u2 1ðtÞ þ 1 2 W4u2 2ðtÞdt; ð6Þ where W1 and W2 are weight constants on the infected hosts and vectors for Patch 1 and Patch 2, respectively. Weight constants, W3 and W4 are the relative costs of the implementation of the preventive controls for Patch 1 and Patch 2, respectively. We model the control efforts as a linear combination of quadratic terms, u2 i ðtÞ (i = 1, 2) due to the convexity of the controls in the objective functional. Then, we seek an optimal pair (U, X) such that JðUÞ ¼ minfJðUÞjU 2 Og; ð7Þ ð7Þ JðUÞ ¼ minfJðUÞjU 2 Og; where O = {(u1(t), u2(t)) 2 (L1(0, tf))2 k a ui(t) b, t 2 [0, tf], i = 1, 2} subject to the state sys- tem Eq (5) with X = (Sv1, Ev1, Iv1, Sh1, Eh1, Ih1, Rh1, Sv2, Ev2, Iv2, Sh2, Eh2, Ih2, Rh2) and U = (u1, u2). The existence of optimal controls is guaranteed from standard results on optimal control theory [57]. Pontryagin’s Maximum Principle is used to establish necessary conditions that must be satisfied by an optimal solution [58]. Derivations of the necessary conditions are shown in Section C in S1 Appendix. In particular, incidence rates including controls are modified as bvið1 uiðtÞÞSvi P2 j¼1 pjiIhj and Shi P2 j¼1 bhjð1 ujðtÞÞpijIvj for i = 1, 2. The controlled two- patch system is given as _Sv1 ¼ mv1ðtÞNv1 bv1ðtÞð1 u1ðtÞÞðp11Ih1=Nh1 þ p21Ih2=Nh2ÞSv1 mvSv1 _Ev1 ¼ bv1ðtÞð1 u1ðtÞÞðp11Ih1=Nh1 þ p21Ih2=Nh2ÞSv1 mvEv1 kEv1 _I v1 ¼ kEv1 mvIv1 _Sv1 ¼ mv1ðtÞNv1 bv1ðtÞð1 u1ðtÞÞðp11Ih1=Nh1 þ p21Ih2=Nh2ÞSv1 mvSv1 _Ev1 ¼ bv1ðtÞð1 u1ðtÞÞðp11Ih1=Nh1 þ p21Ih2=Nh2ÞSv1 mvEv1 kEv1 _I v1 ¼ kEv1 mvIv1 _Sv1 ¼ mv1ðtÞNv1 bv1ðtÞð1 u1ðtÞÞðp11Ih1=Nh1 þ p21Ih2=Nh2ÞSv1 mvSv1 _Ev1 ¼ bv1ðtÞð1 u1ðtÞÞðp11Ih1=Nh1 þ p21Ih2=Nh2ÞSv1 mvEv1 kEv1 _I v1 ¼ kEv1 mvIv1 _Sh1 ¼ mhNh1 Sh1ðbh1ðtÞð1 u1ðtÞÞp11Iv1=Nv1 þ bh2ðtÞð1 u2ðtÞÞp12Iv2=Nv2Þ mhSh1 _Eh1 ¼ Sh1ðbh1ðtÞð1 u1ðtÞÞp11Iv1=Nv1 þ bh2ðtÞð1 u2ðtÞÞp12Iv2=Nv2Þ gEh1 mhEh1 _I h1 ¼ gEh1 dIh1 mhIh1 _Rh1 ¼ dIh1 mhRh1 _Sv2 ¼ mv2ðtÞNv2 bv2ðtÞð1 u2ðtÞÞðp12Ih1=Nh1 þ p22Ih2=Nh2ÞSv2 mvSv2 _Ev2 ¼ bv2ðtÞð1 u2ðtÞÞðp12Ih1==Nh1 þ p22Ih2=Nh2ÞSv2 mvEv2 kEv2 _I v2 ¼ kEv2 mvIv2 _Sh2 ¼ mhNh2 Sh2ðbh1ðtÞð1 u1ðtÞÞp21Iv1=Nv1 þ bh2ðtÞð1 u2ðtÞÞp22Iv2=Nv2Þ mhSh2 _Eh2 ¼ Sh2ðbh1ðtÞð1 u1ðtÞÞp21Iv1=Nv1 þ bh2ðtÞð1 u2ðtÞÞp22Iv2=Nv2Þ gEh2 mhEh2 _I h2 ¼ gEh2 dIh2 mhIh2 _Rh2 ¼ dIh2 mhRh2: ð v1 v1 mv v1 _Sh1 ¼ mhNh1 Sh1ðbh1ðtÞð1 u1ðtÞÞp11Iv1=Nv1 þ bh2ðtÞð1 u2ðtÞÞp12Iv2=Nv2Þ mhSh1 _Eh1 ¼ Sh1ðbh1ðtÞð1 u1ðtÞÞp11Iv1=Nv1 þ bh2ðtÞð1 u2ðtÞÞp12Iv2=Nv2Þ gEh1 mhEh1 _I h1 ¼ gEh1 dIh1 mhIh1 _R ¼ dI m R _Rh1 ¼ dIh1 mhRh1 _Sv2 ¼ mv2ðtÞNv2 bv2ðtÞð1 u2ðtÞÞðp12Ih1=Nh1 þ p22Ih2=Nh2ÞSv2 mvSv2 _Ev2 ¼ bv2ðtÞð1 u2ðtÞÞðp12Ih1==Nh1 þ p22Ih2=Nh2ÞSv2 mvEv2 kEv2 _I v2 ¼ kEv2 mvIv2 ð5Þ ð5Þ PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 6 / 21 Assessment of optimal strategies in a two-patch dengue transmission model with seasonality Patch-specific optimal controls are obtained by minimizing the number of both infected hosts and vectors and the cost of implementation strategies over a finite time interval. PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 Two-patch dengue transmission dynamics in the absence of controls The seasonal reproduction number. The basic reproduction number R0 is the average number of secondary infectious cases when one infectious individual is introduced into a whole susceptible population. It can be calculated as the dominant eigenvalue of the next gen- eration matrix for an autonomous system [60]. However, due to several time-dependent parameters in our system, the basic reproduction number varies in time. Therefore, the sea- sonal reproduction number Rs is computed by the same procedure as for time-dependent parameters [32, 61]. Rs ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ kg 2C ð1 þ ﬃﬃﬃﬃﬃ 2 p Þ r ð8Þ ð8Þ 7 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 Assessment of optimal strategies in a two-patch dengue transmission model with seasonality where 1 ¼ B1 þ B2 2 ¼ B3ðA1 þ A2 þ A3Þ: 2 ¼ B3ðA1 þ A2 þ A3Þ: C ¼ mv k þ mv ð Þ g þ mh ð Þ d þ mh ð Þ B1 ¼ ðbh1bv1p2 11Sh1Sv1Þ=ðNh1Nv1Þ þ ðbh1bv1p2 21Sh2Sv1Þ=ðNh2Nv1Þ B2 ¼ ðbh2bv2p2 12Sh1Sv2Þ=ðNh1Nv2Þ þ ðbh2bv2p2 22Sh2Sv2Þ=ðNh2Nv2Þ B3 ¼ 1=ðN2 h1N2 h2N2 v1N2 v2Þ A1 ¼ b2 h1b2 v1N2 v2ðNh2p2 11Sh1 þ Nh1p2 21Sh2Þ 2S2 v1 A2 ¼ 2bh1bh2bv1bv2Nv1Nv2ðN2 h2p2 11p2 12S2 h1 þ Nh1Nh2ð4p11p12p21p22 p2 12p2 21 p2 11p2 22ÞSh1Sh2 þ N2 h1p2 21p2 22S2 h2ÞSv1Sv2 A3 ¼ b2 h2b2 v2N2 v1ðNh2p2 12Sh1 þ Nh1p2 22Sh2Þ 2S2 v2 C ¼ mv k þ mv ð Þ g þ mh ð Þ d þ mh ð Þ B1 ¼ ðbh1bv1p2 11Sh1Sv1Þ=ðNh1Nv1Þ þ ðbh1bv1p2 21Sh2Sv1Þ=ðNh2Nv1Þ B1 ¼ ðbh1bv1p2 11Sh1Sv1Þ=ðNh1Nv1Þ þ ðbh1bv1p2 21Sh2Sv1Þ=ðNh2Nv1Þ B2 ¼ ðbh2bv2p2 12Sh1Sv2Þ=ðNh1Nv2Þ þ ðbh2bv2p2 22Sh2Sv2Þ=ðNh2Nv2Þ A1 ¼ b2 h1b2 v1N2 v2ðNh2p2 11Sh1 þ Nh1p2 21Sh2Þ 2S2 v1 A2 ¼ 2bh1bh2bv1bv2Nv1Nv2ðN2 h2p2 11p2 12S2 h1 þ Nh1Nh2ð4p11p12p21p22 p2 12p2 21 p2 11p2 22ÞSh1Sh2 þ N2 h1p2 21p2 22S2 h2ÞSv1Sv2 A3 ¼ b2 h2b2 v2N2 v1ðNh2p2 12Sh1 þ Nh1p2 22Sh2Þ 2S2 v2 More details of the calculation of Rs are given in Section B in S1 Appendix. In Fig C in S1 Appendix, the time series of the global seasonal reproduction number Rs and human inci- dence are displayed for symmetric coupling. The incidence of Patch 2 increases when Rs > 1 and decreases when Rs < 1. The time series of Rs follows βv2(t) rather than βv1(t). This implies that the global seasonal reproduction number is more sensitive to βv2(t). The effects of residence-time matrix configurations and seasonality scenarios. PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 Two-patch dengue transmission dynamics in the absence of controls First, the impacts of a residence-time matrix and seasonality scenarios are presented on patch-spe- cific incidences. Fig 2 shows the results under different coupling intensities using symmetric mobility: weakly coupled (left panel) and strongly coupled (right panel). As coupling becomes stronger, the peak sizes of both patches become more similar. This can be interpreted as the population from different patches being well mixed, and so the dynamics of each patch become similar. Fig 3 presents the effects of mobility patterns: symmetric coupling (p12 = p21 on the left) and asymmetric coupling (p12 > p21 on the right). The peak sizes in both patches become higher as the coupling becomes more asymmetric, that is, the proportion of humans visiting the city from the jungle becomes larger. The effect of seasonality scenarios is presented in Fig D in S1 Appendix using symmetric mobility. This figure shows slightly stronger syn- chronization between two patches under the sinusoidal type scenario (S2) than for under the square wave type scenario (S1). The qualitative behaviors in both patches are not significantly sensitive to the seasonality scenario. Cumulative incidence. The final epidemic size is one of the most important epidemiolog- ical quantities for the standard SIR model with a constant transmission in the absence of the demographic effect [62]. For seasonally varying epidemic models, cumulative incidence (CI) for a finite time interval can be computed instead of the final epidemic size [41, 42]. Through- out our manuscript, the cumulative incidence is computed as R 365 0 kEhiðtÞdt for a one-year period. We present the effects of various coupling and seasonality scenarios on CI in Fig 4. Cumulative incidence in Patch 1 is always larger than that in Patch 2, even though peak sizes in Patch 2 are higher than for Patch 1 (see Figs 2 and 3). This is due to the fact that the vector population remains constantly high in the endemic region. It is also observed that the dynam- ics in Patch 2 is more sensitive to residence-time configurations since people tend to move from the jungle to the city (Patch 2 has a higher density). The impact of different residence-time configurations on cumulative incidence is shown in Fig E of S1 Appendix. Clearly, CI on Patch 1 decreases as p12 increases in a linear fashion, that PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 8 / 21 https://doi.org/10.1371/journal.pone.0173673.g004 i l f P t h 1 i it P t h 2 H CI P t h 2 b l Fig 4. The effect of seasonality and coupling scenarios on patch-specific cumulative incidence for one year. #1–#4: Square-wave type seasonality (S1), #5–#8: Sinusoidal type seasonality (S2); #1, #5: p12 = 0.01, p21 = 0.01 (weak / symmetric); #2, #6: p12 = 0.1, p21 = 0.001 (weak / asymmetric); #3, #7: p12 = 0.3, p21 = 0.3 (strong / symmetric); #4, #8: p12 = 0.3, p21 = 0.001 (strong / asymmetric). https://doi.org/10.1371/journal.pone.0173673.g004 Fig 4. The effect of seasonality and coupling scenarios on patch-specific cumulative incidence for one year. #1–#4: Square-wave type seasonality (S1), #5–#8: Sinusoidal type seasonality (S2); #1, #5: p12 = 0.01, p21 = 0.01 (weak / symmetric); #2, #6: p12 = 0.1, p21 = 0.001 (weak / asymmetric); #3, #7: p12 = 0.3, p21 = 0.3 (strong / symmetric); #4, #8: p12 = 0.3, p21 = 0.001 (strong / asymmetric). https://doi.org/10.1371/journal.pone.0173673.g004 is, as more people from Patch 1 visit Patch 2. However, CI on Patch 2 becomes more complex as p12 increases. For the case when p21 is small (p21 0.2), CI on Patch 2 increases as p12 gets larger. On the other hand, when there is more visiting from Patch 2 to Patch 1, then CI on Patch 2 decreases even for a lager value of p12. Overall CI (Patch 1 + Patch 2) shows a similar tendency as that for CI on Patch 1, that is, an increase in visiting from Patch 1 to Patch 2 makes CI smaller, while the opposite is true for more people visiting from Patch 2 to Patch 1. Moreover, we have carried out sensitivity analysis for seasonality parameters, εi, μv, βv and βh, and the effects of these parameters on CI are shown in Fig I of S1 Appendix. Assessment of optimal strategies in a two-patch dengue transmission model with seasonality Fig 2. The effect of coupling strength on patch-specific incidence. (A) Strong coupling (p12 = p21 = 0.3) and (B) weak coupling (p12 = p21 = 0.01) for the case of symmetric coupling and the sinusoidal type seasonality (S2). https://doi org/10 1371/journal pone 0173673 g002 Fig 2. The effect of coupling strength on patch-specific incidence. (A) Strong coupling (p12 = p21 = 0.3) and (B) weak coupling (p12 = p21 = 0.01) for the case of symmetric coupling and the sinusoidal type seasonality (S2). https://doi.org/10.1371/journal.pone.0173673.g002 Fig 3. The effect of symmetry of movement on patch-specific incidence. (A) Symmetric coupling (p12 = p21 = 0.3) and (B) asymmetric coupling (p12 = 0.3 and p21 = 0.001) for the case of strong coupling and the square-wave type seasonality (S1). https://doi.org/10.1371/journal.pone.0173673.g003 Fig 2. The effect of coupling strength on patch-specific incidence. (A) Strong coupling (p12 = p21 = 0.3) and (B) weak coupling (p12 = p21 = 0.01) for the case of symmetric coupling and the sinusoidal type seasonality (S2). https://doi.org/10.1371/journal.pone.0173673.g002 Fig 2. The effect of coupling strength on patch-specific incidence. (A) Strong coupling (p12 = p21 = 0.3) and (B) weak coupling (p12 = p21 = 0.01) for the case of symmetric coupling and the sinusoidal type seasonality (S2). Fig 3. The effect of symmetry of movement on patch-specific incidence. (A) Symmetric coupling (p12 = p21 = 0.3) and (B) asymmetric coupling (p12 = 0.3 and p21 = 0.001) for the case of strong coupling and the square-wave type seasonality (S1). https://doi.org/10.1371/journal.pone.0173673.g003 Fig 3. The effect of symmetry of movement on patch-specific incidence. (A) Symmetric coupling (p12 = p21 = 0.3) and (B) asymmetric coupling (p12 = 0.3 and p21 = 0.001) for the case of strong coupling and the square-wave type seasonality (S1). https://doi.org/10.1371/journal.pone.0173673.g003 https://doi.org/10.1371/journal.pone.0173673.g003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 9 / 21 Assessment of optimal strategies in a two-patch dengue transmission model with seasonality Fig 4. The effect of seasonality and coupling scenarios on patch-specific cumulative incidence for one year. #1–#4: Square-wave type seasonality (S1), #5–#8: Sinusoidal type seasonality (S2); #1, #5: p12 = 0.01, p21 = 0.01 (weak / symmetric); #2, #6: p12 = 0.1, p21 = 0.001 (weak / asymmetric); #3, #7: p12 = 0.3, p21 = 0.3 (strong / symmetric); #4, #8: p12 = 0.3, p21 = 0.001 (strong / asymmetric). Controlled two-patch dengue transmission dynamics We present the two-patch dengue transmission dynamics in the presence of optimal controls. Numerical solutions to Eq (7) are obtained by the standard scheme (a two point boundary 10 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 Assessment of optimal strategies in a two-patch dengue transmission model with seasonality method [52]), which is employed as follows. First, the state system Eq (5) is solved forward in time with initial conditions and an initial guess for the control. Second, the adjoint system with transversality conditions is solved backward in time. Third, the optimality condition is updated. These three steps are iterated until convergence is achieved. There are some critical control parameter values that affect optimal solutions greatly such as weight constants and a simulation time duration. For the weight constants, W1 = W2 = 1 and W3 = W4 = 10000 are used throughout the simulations and two different simulation time durations (three and six years) are used. Parameter values are given in Tables 1 and 2. The effect of coupling and seasonality scenarios. We consider three distinct control strategies: (1) both patches, (2) only Patch 1 and (3) only Patch 2. First, the impact of coupling intensity is explored under the same coupling symmetry and seasonality. Fig 5 shows human incidence and optimal controls for p12 = 0.1, p21 = 0.001 (weak) and p12 = 0.3, p21 = 0.001 (strong) using S1 (asymmetric mobility). Obviously, applying control strategies to both patches gives the greatest incidence reduction, but it is worth noting that controlling only Patch 1 is as effective as controlling both patches under weak coupling. This is in contrast to strong cou- pling, where controlling only Patch 2 is more effective than controlling Patch 1. This can be interpreted such that when mobility between the two patches is relatively small, it is effective to control the endemic region (Patch 1). While the residents in two patches are well mixed due to higher mobility, targeting the region where the population density is higher (Patch 2) is optimal. For symmetric mobility, the results are not significantly sensitive to coupling inten- sity; for weak or strong coupling, focusing on Patch 1 is as effective as controlling both patches. The effectiveness of the controls becomes higher as coupling is weaker (i.e., most people stay in their own patch). PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 Controlled two-patch dengue transmission dynamics The impact of the mobility pattern on the controlled dynamics is investigated in Fig 6. Results are compared for p12 = 0.3, p21 = 0.3 (symmetric) and p12 = 0.3, p21 = 0.001 (asymmet- ric) using S2. Again, controlling both patches shows the greatest incidence reduction in both patches. For symmetric coupling, applying control strategies for only Patch 1 (endemic area) is more effective than controlling for only on Patch 2 due to the fact that there is a higher rate of movement between the patches and almost identical human densities on both patches. For asymmetric coupling, there are more people from Patch 1 visiting Patch 2 than from Patch 2 to Patch 1, hence, controlling only Patch 2 (higher density) is more effective. Next, we com- pared the results with two distinct seasonality scenarios, S1 (a square-wave type) and S2 (a sinu- soidal type), under the same coupling strength and symmetry mobility. Fig F in S1 Appendix shows human incidence and controls for (S1) and (S2) using p12 = 0.1 and p21 = 0.001. Patch 1 is more sensitive to the change of seasonality scenario than Patch 2 due to a bigger change of βv1 than βv2. Our results show that the seasonality scenario does not significantly affect the qualitative behavior of the dynamics. The controlled dynamics is more sensitive to coupling scenarios than the seasonality scenario. Regardless of all residence-time configurations or seasonality scenarios, in general, (the overall profiles of controls) intensive controls should be given at the first year in both patches. Moreover, due to the endemicity of Patch 1, it turns out that continuous control in a decreas- ing manner during the entire time duration is necessary in Patch 1. However, for Patch 2, intermittent control is more effective, that is, control should be concentrated during the sum- mer (right before the outbreak) due to seasonal patterns in Patch 2. If there are sufficient resources available, control measures can be applied to both patches, and if resources are not sufficient, it is better to directly target the location of interest. Therefore, under limited resources available, it becomes more critical to take into consideration the residence-time con- figurations and seasonality scenarios when public health officials make a decision on which area should be targeted. PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 Controlled two-patch dengue transmission dynamics 11 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 Assessment of optimal strategies in a two-patch dengue transmission model with seasonality Fig 5. The effect of coupling intensity on patch-specific incidence and optimal control function (D) Weak coupling (p12 = 0.1, p21 = 0.001) and (E)-(H) strong coupling (p12 = 0.3, p21 = 0.001) for the ca asymmetric coupling and the sinusoidal type seasonality (S2). https://doi.org/10.1371/journal.pone.0173673.g005 Fig 5. The effect of coupling intensity on patch-specific incidence and optimal control functions. (A)- (D) Weak coupling (p12 = 0.1, p21 = 0.001) and (E)-(H) strong coupling (p12 = 0.3, p21 = 0.001) for the case of asymmetric coupling and the sinusoidal type seasonality (S2). https://doi.org/10.1371/journal.pone.0173673.g005 https://doi.org/10.1371/journal.pone.0173673.g005 PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 12 / 21 Assessment of optimal strategies in a two-patch dengue transmission model with seasonality Fig 6. The effect of symmetry of movement on patch-specific incidence and optimal control functions. (A)-(D) Symmetric movement (p12 = p21 = 0.3) and (E)-(H) asymmetric movement (p12 = 0.3, p21 = 0.001) for the case of strong coupling and the sinusoidal type seasonality (S2). Fig 6. The effect of symmetry of movement on patch-specific incidence and optimal control functions. (A)-(D) Symmetric movement (p12 = p21 = 0.3) and (E)-(H) asymmetric movement (p12 = 0.3 0.001) for the case of strong coupling and the sinusoidal type seasonality (S2). https://doi.org/10.1371/journal.pone.0173673.g006 Fig 6. The effect of symmetry of movement on patch-specific incidence and optimal control functions. (A)-(D) Symmetric movement (p12 = p21 = 0.3) and (E)-(H) asymmetric movement (p12 = 0.3, p21 = 0.001) for the case of strong coupling and the sinusoidal type seasonality (S2). https://doi.org/10.1371/journal.pone.0173673.g006 https://doi.org/10.1371/journal.pone.0173673.g006 PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 13 / 21 Assessment of optimal strategies in a two-patch dengue transmission model with seasonality The effects of duration and weight constants. In the previous section, all results are com- puted under a simulation duration of three years. The simulation duration is doubled to six years to investigate the effect of time duration on the two-patch controlled dynamics. Fig 7 dis- plays the human incidence and optimal controls under three years and six years using p12 = 0.3, p21 = 0.001. Putting controls in both patches is expected to best reduce the incidences. Controlled two-patch dengue transmission dynamics For the case of limited resources available, controlling only Patch 2 is sufficiently effective to reduce dengue incidence on both patches as we observed in the three year duration case. Next, Fig 8 shows cumulative incidence for each patch, when both patches are controlled for three years and six years, which are compared with CI without control. It is observed that under the duration of six years, cumulative incidence has been reduced significantly. The weight con- stant can be considered as the relative cost of control implementation, and a larger value repre- sents a relatively higher cost. The impact of control weight constants is illustrated under several values of weight constants. Fig G in S1 Appendix shows human incidence and optimal controls using W3 = W4 = 5000, 10000, 50000 (W3 = W4 = 10000 is taken as the baseline value, which is used throughout this paper). Obviously, the impact is straightforward; for higher costs, the control decreases, which leads to larger incidences. The impact of different subpopulation sizes. In the previous sections, we have focused on the effect of coupling and seasonality scenarios when each patch has the same host popula- tion size. Here we have investigated the impact of different patch-sizes of human individuals by comparing the results of two scenarios (Case 1: Nh1 = Nh2 and Case 2: Nh1 < Nh2 with Nh2 = 2Nh1). Patch-specific incidences are illustrated in Fig H in S1 Appendix. As Nh2 is doubled, Patch 1 incidence barely changes, but Patch 2 incidence increases almost twice. Patch-specific incidences and controls are presented in Fig 9. As the population size of Patch 2 becomes larger, Patch 2 incidence increases, hence, control efforts increase in both patches. Note that more intensive efforts should be implemented in Patch 2. The effect of coupling scenarios on the results of Case 2 remains qualitatively similar as the ones of Case 1. PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 Discussions We have investigated the dynamics of dengue transmission in a seasonally varying two-patch dengue system. It is assumed that the two patches represent two locations that have a constant and well-defined visiting relationship modeled by a residence-time matrix. Motivated by the recurrent dengue outbreaks in Peru, host demographics and seasonality have been included in the previous model [31]. We assumed that one patch is endemic (jungle/rural areas), and we modeled how human visiting between the two patches caused epidemics in the other patch (coast/urban areas) [30]. The effects of two distinct seasonality scenarios have been investi- gated under different residence-time configurations. Stronger synchronization occurs for the sinusoidal type transmission rate function than for the square-wave type. Regardless of resi- dence-time configurations or seasonality, the dengue dynamics in both patches become similar as coupling strength becomes stronger. Also, human incidences in both patches have higher peaks as the residence-time matrix becomes more asymmetric regardless of seasonality. The seasonal reproduction number and cumulative incidence are investigated under various sce- narios. Overall cumulative incidence increases as coupling intensity becomes stronger for both the symmetric and asymmetric cases. However, overall cumulative incidence decreases as p12 increases, that is, more people visit the city from the jungle. We developed an optimal control framework to identify optimal patch-specific control strategies under various scenarios. First, we identify optimal strategies and compare the con- trolled dynamics with the results in the absence of controls. Our results indicate that, as expected, controlling the two patches simultaneously gives the best reduction in dengue PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 14 / 21 Assessment of optimal strategies in a two-patch dengue transmission model with seasonality Fig 7. The effect of control duration on patch-specific incidence and optimal control functions. ( Duration of three years and (E)-(H) duration of six years for the case of strong and asymmetric coupling 0.3, p21 = 0.001) with the sinusoidal type seasonality (S2). https://doi.org/10.1371/journal.pone.0173673.g007 Fig 7. The effect of control duration on patch-specific incidence and optimal control functions. (A)-(D) Duration of three years and (E)-(H) duration of six years for the case of strong and asymmetric coupling (p12 = 0.3, p21 = 0.001) with the sinusoidal type seasonality (S2). https://doi.org/10.1371/journal.pone.0173673.g007 https://doi.org/10.1371/journal.pone.0173673.g007 PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 15 / 21 Assessment of optimal strategies in a two-patch dengue transmission model with seasonality Fig 8. Cumulative incidence for one year under different control duration. Discussions When both patches are controlled for (A) three years and (B) six years, cumulative incidence for one year is compared with cumulative incidence for one year without control (displayed on time 0) using p12 = 0.1, p21 = 0.001 and the sinusoidal type seasonality (S2). https://doi.org/10.1371/journal.pone.0173673.g008 Fig 8. Cumulative incidence for one year under different control duration. When both patches are controlled for (A) three years and (B) six years, cumulative incidence for one year is compared with cumulative incidence for one year without control (displayed on time 0) using p12 = 0.1, p21 = 0.001 and the sinusoidal type seasonality (S2). https://doi.org/10.1371/journal.pone.0173673.g008 prevalence. However, for the case when only one patch can be controlled due to limited resources, the resulting control strategies become more sensitive to residence-time configura- tions. For instance, focusing on Patch 1 (the endemic area) is turned out to be optimal under weak coupling or symmetric mobility patterns. However, focusing only on Patch 2 (the city with higher human density) is more effective under strong coupling with asymmetric mobility patterns. Moreover, the results of optimal problems are sensitive to the subpopulation sizes. As the population size of Patch 2 is increased, more intensive control efforts should be imple- mented in both patches. Even though our work is motivated by dengue incidence in Peru, we aim to build a general model that can provide an experimental tool for any two interconnected locations with well defined commuting or visiting relationships. Human mobility patterns are one of critical fac- tors for dengue transmission dynamics, however, we rather use a residence-time matrix to cap- ture the effect of virtual human movements due to the lack of real commuting data. Dengue fever is a challenge for vector control and education program, even with the joint efforts of government and community, and the potential use of partially effective vaccines at the population level. More advanced mathematical modeling should involve vector and host interactions, dynamics of circulating dengue serotypes, and geographical and demographical/ behavioral factors for both vector and host. Hence, understanding the mechanism behind the complex spatial-temporal dynamics of dengue disease requires multidisciplinary and transdis- ciplinary efforts. Identification and evaluation of optimal strategies that minimize the spread of dengue have been explored through the use of mathematical models. The work presented here can model the dengue transmission dynamics in two seasonally varying locations that are geographically close and similar in population size. PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 Discussions Our results show the challenges that public health officials face in how resources should be allocated in heterogeneous environments. Our findings sug- gest that public health officials should focus on combating dengue in the area with a higher PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 16 / 21 Assessment of optimal strategies in a two-patch dengue transmission model with seasonality Fig 9. The effect of different patch sizes on patch-specific incidence and optimal controls. (A)-(D) Nh1 = Nh2 = 105 and (E)-(H) Nh1 = 105, Nh2 = 2Nh1 for the case of asymmetric and weak coupling (p12 = 0.1, p21 = 0.001) with the sinusoidal type seasonality (S2). Control efforts increase in both patches due to the increment of Nh2 in (E)-(H). More intensive efforts should be implemented in Patch 2 (H). https://doi.org/10.1371/journal.pone.0173673.g009 Fig 9. The effect of different patch sizes on patch-specific incidence and optimal controls. (A)-(D) Nh1 = Nh2 = 105 and (E)-(H) Nh1 = 105, Nh2 = 2Nh1 for the case of asymmetric and weak coupling (p12 = 0.1, p21 = 0.001) with the sinusoidal type seasonality (S2). Control efforts increase in both patches due to the increment of Nh2 in (E)-(H). More intensive efforts should be implemented in Patch 2 (H). https://doi.org/10.1371/journal.pone.0173673.g009 PLOS ONE \| https://doi.org/10.1371/journal.pone.0173673 March 16, 2017 17 / 21 Assessment of optimal strategies in a two-patch dengue transmission model with seasonality population density (cities) or in the region with a higher transmission rate where dengue is endemic, depending on the residence-time configurations and the amount of available resources. Validation: SL CL. Validation: SL CL. Validation: SL CL. Writing – original draft: JK HL SL. Writing – review & editing: JK HL SL CL. Writing – review & editing: JK HL SL CL. 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https://openalex.org/W2081064539	https://advancesindifferenceequations.springeropen.com/counter/pdf/10.1155/2011/784161	English	null	Nonlocal Impulsive Cauchy Problems for Evolution Equations	Advances in difference equations	2,011	cc-by	8,697	Hindawi Publishing Corporation Advances in Diﬀerence Equations Volume 2011, Article ID 784161, 17 pages doi:10.1155/2011/784161 Hindawi Publishing Corporation Advances in Diﬀerence Equations Volume 2011, Article ID 784161, 17 pages doi:10.1155/2011/784161 Correspondence should be addressed to Jin Liang, jinliang@sjtu.edu.cn Correspondence should be addressed to Jin Liang, jinliang@sjtu.edu.cn Received 17 October 2010; Accepted 19 November 2010 Academic Editor: Toka Diagana Copyright q 2011 J. Liang and Z. Fan. 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. Of concern is the existence of solutions to nonlocal impulsive Cauchy problems for evolution equations. Combining the techniques of operator semigroups, approximate solutions, noncompact measures and the ﬁxed point theory, new existence theorems are obtained, which generalize and improve some previous results since neither the Lipschitz continuity nor compactness assumption on the impulsive functions is required. An application to partial diﬀerential equations is also presented. Jin Liang1 and Zhenbin Fan1, 2 1 Department of Mathematics, Shanghai Jiao Tong University, Shanghai 200240, China 2 Department of Mathematics, Changshu Institute of Technology, Suzhou, Jiangsu 215500, China F, f, g, Ii are given continuous functions to be speciﬁed later. By going a new way, that is, by combining operator semigroups, the techniques of approximate solutions, noncompact measures, and the ﬁxed point theory, we obtain new existence results for problem 1.1, which generalize and improve some previous theorems since neither the Lipschitz continuity nor compactness assumption on the impulsive functions is required in the present paper. The organization of this work is as follows. In Section 2, we recall some deﬁnitions, and facts about fractional powers of operators, mild solutions and Hausdorﬀmeasure of noncompactness. In Section 3, we give the existence results for problem 1.1 when the nonlocal item and impulsive functions are only assumed to be continuous. In Section 4, we give an example to illustrate our abstract results. 1. Introduction , p, 0 < t1 < t2 < · · · < tp < K, where −A : DA ⊆X →X is the inﬁnitesimal generator of an analytic semigroup {Tt; t ≥ 0} and X is a real Banach space endowed with the norm ∥· ∥, where −A : DA ⊆X →X is the inﬁnitesimal generator of an analytic semigroup {Tt; t ≥ 0} and X is a real Banach space endowed with the norm ∥· ∥, Δuti ut i −ut− i , ut i lim t →t i ut, ut− i lim t →t− i ut , 1.2 1.2 F, f, g, Ii are given continuous functions to be speciﬁed later. 1. Introduction Impulsive equations arise from many diﬀerent real processes and phenomena which appeared in physics, chemical technology, population dynamics, biotechnology, medicine, and economics. They have in recent years been an object of investigations with increasing interest. For more information on this subject, see for instance, the papers cf., e.g., 1–6 and references therein. On the other hand, Cauchy problems with nonlocal conditions are appropriate models for describing a lot of natural phenomena, which cannot be described using classical Cauchy problems. That is why in recent years they have been studied by many researchers cf., e.g., 4, 7–12 and references therein. In 4, the authors combined the two directions and studied ﬁrstly a class of nonlocal impulsive Cauchy problems for evolution equations by investigating the existence for mild in generalized sense solutions to the problems. In this paper, we study further the existence of solutions to the following nonlocal impulsive Cauchy problem for evolution equations: Advances in Diﬀerence Equations Advances in Diﬀerence Equations 2 Advances in Diﬀerence Equations Advances in Diﬀerence Equations d dtut Ft, ut Aut ft, ut, 0 ≤t ≤K, t / ti, d dtut Ft, ut Aut ft, ut, 0 ≤t ≤K, t / ti, d dtut Ft, ut Aut ft, ut, 0 ≤t ≤K, t / ti, u0 gu u0, Δuti Iiuti, i 1, 2, . . . , p, 0 < t1 < t2 < · · · < tp < K, 1.1 u0 gu u0, Δuti Iiuti i 1 2 p 0 < t1 < t2 < · · · < t < K 1.1 1.1 u0 gu u0, Δuti Iiuti, i 1, 2, . . . 2. Preliminaries Let X, ∥· ∥ be a real Banach space. We denote by C0, K, X the space of X-valued continuous functions on 0, K with the norm ∥u∥ max{∥ut∥; t ∈0, K}, 2.1 2.1 and by L10, K, X the space of X-valued Bochner integrable functions on 0, K with the norm ∥f∥L1 K 0 ∥ft∥dt. Let PC0, K, X : {u : 0, K →X; ut is continuous at t / ti, left continuous at t ti, and the right limit ut i exists for i 1, 2, . . . , p. X : {u : 0, K →X; ut is continuous at t / ti, left continuous at t ti, and the right limit ut i exists for i 1, 2, . . . , p. 2 2 2.2 g i p 2.2 2.2 It is easy to check that PC0, K, X is a Banach space with the norm ∥u∥PC sup t∈0,K ∥ut∥. 2.3 2.3 Advances in Diﬀerence Equations 3 In this paper, for r > 0, let Br : {x ∈X; ∥x∥≤r} and Wr : {u ∈PC0, K, X; ut ∈Br, ∀t ∈0, K}. 2.4 In this paper, for r > 0, let Br : {x ∈X; ∥x∥≤r} and Wr : {u ∈PC0, K, X; ut ∈Br, ∀t ∈0, K}. 2.4 In this paper, for r > 0, let Br : {x ∈X; ∥x∥≤r} and Wr : {u ∈PC0, K, X; ut ∈Br, ∀t ∈0, K}. 2.4 2.4 hroughout this paper, we assume the following. Throughout this paper, we assume the following. H1 The operator −A : DA ⊆X →X is the inﬁnitesimal generator of a compact analytic semigroup {Tt : t ≥0} on Banach space X and 0 ∈ρA the resolvent set of A. H1 The operator −A : DA ⊆X →X is the inﬁnitesimal generator of a compact analytic semigroup {Tt : t ≥0} on Banach space X and 0 ∈ρA the resolvent set of A. In the remainder of this work, M : sup0≤t≤K∥Tt∥< ∞. ≤≤ Under the above conditions, it is possible to deﬁne the fractional power Aα : DAα ⊂ X →X, 0 < α < 1, of A as closed linear operators. And it is known that the following properties hold. Theorem 2.1 see 13, Pages 69–75. 2. Preliminaries Let 0 < α < 1 and assume that (H1) holds. Then, 1 DAα is a Banach space with the norm ∥x∥α ∥Aαx∥for x ∈DAα, 2 Tt : X →DAα for t > 0, 3 AαTtx TtAαx for x ∈DAα and t ≥0, DAα is a Banach space with the norm ∥x∥α ∥Aαx∥for x ∈DAα, AαTtx TtAαx for x ∈DAα and t ≥0, 4 for every t > 0, AαTt is bounded on X and there exists Cα > 0 such that 4 for every t > 0, AαTt is bounded on X and there exists Cα > 0 such that ∥AαTt∥≤Cα tα , 0 < t ≤K, 2.5 2.5 5 A−α is a bounded linear operator in X with DAα ImA−α, 6 if 0 < α < β ≤1, then DAβ →DAα. 5 A−α is a bounded linear operator in X with DAα ImA−α, 6 if 0 < α < β ≤1, then DAβ →DAα. We denote by Xα that the Banach space DAα endowed the graph norm from now on. Deﬁnition 2.2. A function u ∈PC0, K, X is said to be a mild solution of 1.1 on 0, K if the function s →ATt −sFs, us is integrable on 0, t for all t ∈0, K and the following integral equation is satisﬁed: ut Ttu0 F0, u0 −gu −Ft, ut t 0 ATt −sFs, usds t 0 Tt −sfs, usds 0<ti<t Tt −tiIiuti, 0 ≤t ≤K. 2.6 2.6 To discuss the compactness of subsets of PC0, K, X, we let t0 0, tp1 K, J0 t0, t1, J1 t1, t2, . . . , Jp tp, tp1 . 2.7 J0 t0, t1, J1 t1, t2, . . . , Jp tp, tp1 . 2.7 For D ⊆PC0, K, X, we denote by D\|Ji the set For D ⊆PC0, K, X, we denote by D\|Ji the set D\|Ji u ∈Cti, ti1, X; uti vt i , ut vt, t ∈Ji, v ∈D, 2.8 D\|Ji u ∈Cti, ti1, X; uti vt i , ut vt, t ∈Ji, v ∈D, 2.8 2.8 i 0, 1, 2, . . . , p. Then it is easy to see that the following result holds. i 0, 1, 2, . . . , p. Some basic properties of α· are given in the following Lemma. Lemma 2.4 see 14. Let Y be a real Banach space and let B, C ⊆Y be bounded. Then, 1 B is precompact if and only if αB 0; 1 B is precompact if and only if αB 0; 2 αB αB αconvB, where B and convB mean the closure and convex hull of B, respectively; 3 αB ≤αC when B ⊆C; 4 αB C ≤αB αC, where B C {x y; x ∈B, y ∈C}; 5 αB ∪C ≤max{αB, αC}; 6 αλB \|λ\|αB for any λ ∈R; 7 let Z be a Banach space and Q : DQ ⊆Y →Z Lipschitz continuous with constant k. 4 αB C ≤αB αC, where B C {x y; x ∈B, y ∈C}; 6 αλB \|λ\|αB for any λ ∈R; 7 let Z be a Banach space and Q : DQ ⊆Y →Z Lipschitz continuous with constant k. Then αQB ≤kαB for all B ⊆DQ being bounded. We note that a continuous map Q : W ⊆Y →Y is an α-contraction if there exists a positive constant k < 1 such that αQC ≤kαC for all bounded closed C ⊆W. Lemma 2.5 see Darbo-Sadovskii’s ﬁxed point theorem in 14. If W ⊆Y is bounded closed and convex, and Q : W →W is an α-contraction, then the map Q has at least one ﬁxed point in W. 2. Preliminaries Then it is easy to see that the following result holds. i 0, 1, 2, . . . , p. Then it is easy to see that the following result holds. 4 q Lemma 2.3. A set D ⊆PC0, K, X is precompact in PC0, K, X if and only if the set D\|Ji is precompact in Cti, ti1, X for every i 0, 1, 2, . . . , p. Lemma 2.3. A set D ⊆PC0, K, X is precompact in PC0, K, X if and only if the set D\|Ji is precompact in Cti, ti1, X for every i 0, 1, 2, . . . , p. Next, we recall that the Hausdorﬀmeasure of noncompactness α· on each bounded subset Ω of Banach space Y is deﬁned by αΩ inf{ε > 0; Ω has a ﬁnite ε-net in Y}. 2.9 2.9 αΩ inf{ε > 0; Ω has a ﬁnite ε-net in Y}. 2.9 3. Main Results Moreover, there exist L2, L3 > 0 such that AβFt, x1 −AβFt, x2 ≤L2∥x1 −x2∥ 3.2 3.2 AβFt, x ≤L3∥x∥ 1 AβFt, x ≤L3∥x∥ 1 3.3 3.3 3. Main Results In this section, by using the techniques of approximate solutions and ﬁxed points, we establish a result on the existence of mild solutions for the nonlocal impulsive problem 1.1 when the nonlocal item g and the impulsive functions Ii are only assumed to be continuous in PC0, K, X and X, respectively. In practical applications, the values of ut for t near zero often do not aﬀect gu. For example, it is the case when gu q j1 cjusj , 0 < s1 < s2 < · · · < sq < K. 3.1 3.1 So, to prove our main results, we introduce the following assumptions. So, to prove our main results, we introduce the following assumptions. So, to prove our main results, we introduce the following assumptions. H2 g : PC0, K, X →X is a continuous function, and there is a δ ∈0, t1 such that gu gv for any u, v ∈PC0, K, X with us vs, s ∈δ, K. Moreover, there exist L1, L′ 1 > 0 such that ∥gu∥≤L1∥u∥PC L′ 1 for any u ∈PC0, K, X. H2 g : PC0, K, X →X is a continuous function, and there is a δ ∈0, t1 such that gu gv for any u, v ∈PC0, K, X with us vs, s ∈δ, K. Moreover, there exist L1, L′ 1 > 0 such that ∥gu∥≤L1∥u∥PC L′ 1 for any u ∈PC0, K, X. dvances in Diﬀerence Equations 5 5 Advances in Diﬀerence Equations Advances in Diﬀerence Equations H3 There exists a β ∈0, 1 such that F : 0, K × X →Xβ is a continuous function, and F·, u· F·, v· for any u, v ∈PC0, K, X with us vs, s ∈δ, K. Moreover, there exist L2, L3 > 0 such that H3 There exists a β ∈0, 1 such that F : 0, K × X →Xβ is a continuous function, and F·, u· F·, v· for any u, v ∈PC0, K, X with us vs, s ∈δ, K. Moreover, there exist L2, L3 > 0 such that H3 There exists a β ∈0, 1 such that F : 0, K × X →Xβ is a continuous function, and F·, u· F·, v· for any u, v ∈PC0, K, X with us vs, s ∈δ, K. for any 0 ≤t ≤K, x ∈X. 3.7 3.7 6 Advances in Diﬀerence Eq Advances in Diﬀerence Equations Advances in Diﬀerence Equations 6 In addition, we introduce the decomposition Qn Qn1 Qn2 Qn3 Qn4, where Qn1ut Tt u0 −T 1 n gu , Qn2ut 0<ti<t Tt −tiT 1 n Iiuti, 3 8 n Qn2ut 0<ti<t Tt −tiT 1 n Iiuti, Qn3ut TtF0, u0 −Ft, ut t 0 ATt −sFs, usds, Qn4ut t 0 Tt −sfs, usds 3.8 3.8 0<ti<t Qn3ut TtF0, u0 −Ft, ut t 0 ATt −sFs, usds, Qn4ut t 0 Tt −sfs, usds 3.8 0<ti<t Qn3ut TtF0, u0 −Ft, ut t 0 ATt −sFs, usds, t 3.8 Qn3ut TtF0, u0 −Ft, ut t 0 ATt −sFs, usds, 3.8 Qn4ut t 0 Tt −sfs, usds Qn4ut t 0 Tt −sfs, usds for u ∈PC0, K, X and t ∈0, K. for any 0 ≤t ≤K, x ∈X. for any 0 ≤t ≤K, x ∈X. for any 0 ≤t ≤K, x ∈X. H4 The function ft, · : X →X is continuous a.e. t ∈0, K; the function f·, x : 0, K →X is strongly measurable for all x ∈X. Moreover, for each l ∈N, there exists a function ρl ∈L10, K, R such that ∥ft, x∥≤ρlt for a.e. t ∈0, K and all x ∈Bl, and γ : lim inf l →∞ 1 l K 0 ρlsds < ∞. 3.4 3.4 H5 Ii : X →X is continuous for every i 1, 2, . . . , p, and there exist positive numbers L4, L′ 4 such that ∥Iix∥≤L4∥x∥ L′ 4 for any x ∈X and i 1, 2, . . . , p. H5 Ii : X →X is continuous for every i 1, 2, . . . , p, and there exist positive numbers L4, L′ 4 such that ∥Iix∥≤L4∥x∥ L′ 4 for any x ∈X and i 1, 2, . . . , p. We note that, by Theorem 2.1, there exist M0 > 0 and C1−β > 0 such that M0 ∥A−β∥and A1−βTt ≤C1−β t1−β , 0 < t ≤K. 3.5 3.5 For simplicity, in the following we set L max{L1, L2, L3, L4} and will substitute L1, L2, L3, L4 by L below. Theorem 3.1. Let (H1)–(H5) hold. Then the nonlocal impulsive Cauchy problem 1.1 has at least one mild solution on 0, K, provided L0 ML M0L γ pL M0L LC1−βKβ β < 1. 3.6 3.6 To prove the theorem, we need some lemmas. Next, for n ∈N, we denote by Qn the maps Qn : PC0, K, X →PC0, K, X deﬁned by Qnut Tt u0 F0, u0 −T 1 n gu −Ft, ut t 0 ATt −sFs, usds t 0 Tt −sfs, usds 0<ti<t Tt −tiT 1 n Iiuti, 0 ≤t ≤K. for u ∈PC0, K, X and t ∈0, K. for u ∈PC0, K, X and t ∈0, K. Lemma 3.2. Assume that all the conditions in Theorem 3.1 are satisﬁed. Then for any n ≥1, the map Qn deﬁned by 3.7 has at least one ﬁxed point un ∈PC0, K, X. Proof. To prove the existence of a ﬁxed point for Qn, we will use Darbu-Sadovskii’s ﬁxed point theorem. Firstly, we prove that the map Qn3 is a contraction on PC0, K, X. For this purpose, let u1, u2 ∈PC0, K, X. Then for each t ∈0, K and by condition H3, we have ∥Qn3u1t −Qn3u2t∥ ≤M∥F0, u10 −F0, u20∥ ∥Ft, u1t −Ft, u2t∥ ∥Qn3u1t −Qn3u2t∥ ≤M∥F0, u10 −F0, u20∥ ∥Ft, u1t −Ft, u2t∥ t 0 ∥ATt −sFs, u1s −Fs, u2s∥ds ≤M A−βAβF0, u10 −A−βAβF0, u20 A−βAβFt, u1t −A−βAβFt, u2t t 0 A1−βTt −s AβFs, u1s −AβFs, u2s ds ≤M A−βAβF0, u10 −A−βAβF0, u20 A−βAβFt, u1t −A−βAβFt, u2t t 0 A1−βTt −s AβFs, u1s −AβFs, u2s ds ≤MM0L∥u1 −u2∥ M0L∥u1t −u2t∥ t 0 C1−β t −s1−β L∥u1s −u2s∥ds. 3.9 Thus, Thus, ∥Qn3u1 −Qn3u2∥PC ≤ M 1M0L LC1−βKβ β ∥u1 −u2∥, 3.10 3.10 which implies that Qn3 is a contraction by condition 3.6. which implies that Qn3 is a contraction by condition 3.6. which implies that Qn3 is a contraction by condition 3.6. 7 Advances in Diﬀerence Equations Secondly, we prove that Qn4, Qn1, Qn2 are completely continuous operators. Let {um}∞ m1 be a sequence in PC0, K, X with lim m →∞um u 3.11 3.11 in PC0, K, X. By the continuity of f with respect to the second argument, we deduce that for each s ∈0, K, fs, ums converges to fs, us in X, and we have ∥Qn4um −Qn4u∥PC ≤M K 0 fs, ums −fs, us ds, ∥Qn1um −Qn1u∥PC ≤M gum −gu , ∥Qn2um −Qn2u∥PC ≤M p i1 ∥Iiumti −Iiuti∥. 3.12 3.12 Then by the continuity of f, g, Ii, and using the dominated convergence theorem, we get Then by the continuity of f, g, Ii, and using the dominated convergence theorem, we get Then by the continuity of f, g, Ii, and using the dominated convergence theorem, we get lim m →∞Qn4um Qn4u, lim m →∞Qn1um Qn1u, lim m →∞Qn2um Qn2u 3.13 lim m →∞Qn1um Qn1u, lim m →∞Qn2um Qn2u 3.13 lim m →∞Qn4um Qn4u, 3.13 in PC0, K, X, which implies that Qn4, Qn1, Qn2 are continuous on PC0, K, X. Next, for the compactness of Qn4 we refer to the proof of 4, Theorem 3.1. For Qn1 and any bounded subset W of PC0, K, X, we have Qn1ut Ttu0 −T 1 n Ttgu, t ∈0, K, u ∈W, 3.14 3.14 which implies that Qn1Wt is relatively compact in X for every t ∈0, K by the compactness of T1/n. On the other hand, for 0 ≤s ≤t ≤K, we have which implies that Qn1Wt is relatively compact in X for every t ∈0, K by the compactness of T1/n. On the other hand, for 0 ≤s ≤t ≤K, we have ∥Qn1ut −Qn1us∥≤ Tt −Ts u0 −T 1 n gu . 3.15 3.15 Since {T1/ngu; u ∈W} is relatively compact in X, we conclude that g y p ∥Qn1ut −Qn1us∥−→0 uniformly as t −→s and u ∈W, 3.16 ∥Qn1ut −Qn1us∥−→0 uniformly as t −→s and u ∈W, 3.16 3.16 which implies that Qn1W is equicontinuous on 0, K. Therefore, Qn1 is a compact operator. Now, we prove the compactness of Qn2. For this purpose, let hich implies that Qn1W is equicontinuous on 0, K. Therefore, Qn1 is a compact operator. Now, we prove the compactness of Qn2. For this purpose, let J0 0, t1, J1 t1, t2, . . . , Jp tp, K . 3.17 3.17 8 Advances in Diﬀerenc 8 Advances in Diﬀerence Equations Note that Advances in Diﬀerence Equations 8 8 Note that Note that Note that Qn2ut ⎧ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎩ 0, t ∈J0, Tt −t1T 1 n I1ut1, t ∈J1, · · · p i1 Tt −tiT 1 n Iiuti, t ∈Jp. Then by the continuity of f, g, Ii, and using the dominated convergence theorem, we get 3.18 3.18 Thus according to Lemma 2.3, we only need to prove that Thus according to Lemma 2.3, we only need to prove that Thus according to Lemma 2.3, we only need to prove that {Qn2u; u ∈W}\|J1 T· −t1T 1 n I1ut1; · ∈J1, u ∈W 3.19 3.19 is precompact in Ct1, t2, X, as the remaining cases for t ∈Ji, i 2, 3, . . . , p, can be dealt with in the same way; here W is any bounded subset in PC0, K, X. And, we recall that v Qn2u\|J1, u ∈W, which means that is precompact in Ct1, t2, X, as the remaining cases for t ∈Ji, i 2, 3, . . . , p, can be dealt with in the same way; here W is any bounded subset in PC0, K, X. And, we recall that v Qn2u\|J1, u ∈W, which means that vt1 Qn2ut 1 T 1 n I1ut1, vt Qn2ut Tt −t1T 1 n I1ut1, t ∈J1. 3.20 3.20 Thus, by the compactness of T1/n, we know that {Qn2u; u ∈W}\|J1t is relatively compact in X for every t ∈J1. Next, for t1 ≤s ≤t ≤t2, we have Thus, by the compactness of T1/n, we know that {Qn2u; u ∈W}\|J1t is relatively compact in X for every t ∈J1. p y Next, for t1 ≤s ≤t ≤t2, we have y Next, for t1 ≤s ≤t ≤t2, we have Tt −t1T 1 n I1ut1 −Ts −t1T 1 n I1ut1 Ts −t1Tt −s −T0T 1 n I1ut1 ≤M Tt −s −T0T 1 n I1ut1 . 3.21 3.21 Thus, the set {Qn2u; u ∈W}\|J1 ⊆Ct1, t2, X is equicontinuous due to the compactness of {T1/nI1ut1; u ∈W} and the strong continuity of operator T·. By the Arzela-Ascoli theorem, we conclude that {Qn2u; u ∈W}\|J1 is precompact in Ct1, t2, X. The same idea can be used to prove that {Qn2u; u ∈W}\|Ji is precompact for each i 2, 3, . . . , p. Therefore, {Qn2u; u ∈W} is precompact in PC0, K, X, that is, the operator Qn2 : PC0, K, X → PC0, K, X is compact. Then by the continuity of f, g, Ii, and using the dominated convergence theorem, we get Thus, the set {Qn2u; u ∈W}\|J1 ⊆Ct1, t2, X is equicontinuous due to the compactness of {T1/nI1ut1; u ∈W} and the strong continuity of operator T·. By the Arzela-Ascoli theorem, we conclude that {Qn2u; u ∈W}\|J1 is precompact in Ct1, t2, X. The same idea can be used to prove that {Qn2u; u ∈W}\|Ji is precompact for each i 2, 3, . . . , p. Therefore, {Qn2u; u ∈W} is precompact in PC0, K, X, that is, the operator Qn2 : PC0, K, X → PC0, K, X is compact. Thus, the set {Qn2u; u ∈W}\|J1 ⊆Ct1, t2, X is equicontinuous due to the compactness of {T1/nI1ut1; u ∈W} and the strong continuity of operator T·. By the Arzela-Ascoli theorem, we conclude that {Qn2u; u ∈W}\|J1 is precompact in Ct1, t2, X. The same idea can be used to prove that {Qn2u; u ∈W}\|Ji is precompact for each i 2, 3, . . . , p. Therefore, {Qn2u; u ∈W} is precompact in PC0, K, X, that is, the operator Qn2 : PC0, K, X → PC0, K, X is compact. Thus, the set {Qn2u; u ∈W}\|J1 ⊆Ct1, t2, X is equicontinuous due to the compactness of {T1/nI1ut1; u ∈W} and the strong continuity of operator T·. By the Arzela-Ascoli theorem, we conclude that {Qn2u; u ∈W}\|J1 is precompact in Ct1, t2, X. The same idea can be used to prove that {Qn2u; u ∈W}\|Ji is precompact for each i 2, 3, . . . , p. Therefore, {Qn2u; u ∈W} is precompact in PC0, K, X, that is, the operator Qn2 : PC0, K, X → PC0, K, X is compact. Advances in Diﬀerence Equations 9 Thus, for any bounded subset W ⊆PC0, K, X, we have by Lemma 2.4, , for any bounded subset W ⊆PC0, K, X, we have by Lemma 2.4, αQnW ≤αQn1W αQn3W αQn4W αQn2W ≤L0αW. 3.22 3.22 Hence, the map Qn is an α-contraction in PC0, K, X. Now, in order to apply Lemma 2.5, it remains to prove that there exists a constant r > 0 such that QnWr ⊆Wr. Suppose this is not true; then for each positive integer r, there are ur ∈Wr and tr ∈0, K such that ∥Qnurtr∥> r. Then by the continuity of f, g, Ii, and using the dominated convergence theorem, we get Then r < ∥Qnurtr∥ Ttr u0 −T 1 n gur F0, ur0 −Ftr, urtr tr 0 ATtr −sFs, ursds Ttr u0 −T 1 n gur F0, ur0 −Ftr, urtr tr 0 ATtr −sFs, ursds tr 0 Ttr −sfs, ursds 0<ti<tr Ttr −tiT 1 n Iiurti n 0 tr 0 Ttr −sfs, ursds 0<ti<tr Ttr −tiT 1 n Iiurti ≤M∥u0∥ Lr L′ 1 M0Lr 1 M0Lr 1 t 0 C1−β t −s1−β Lr 1ds M t 0 ρrsds MpLr L′ 4 M t 0 ρrsds MpLr L′ 4 ≤M∥u0∥ Lr L′ 1 1 MM0Lr 1 LC1−βKβ β r 1 M K 0 ρrsds MpLr L′ 4 . M K 0 ρrsds MpLr L′ 4 . 3.23 3.23 Dividing on both sides by r and taking the lower limit as r →∞, we obtain that Dividing on both sides by r and taking the lower limit as r →∞, we obtain that L0 ML M0L γ pL M0L LC1−βKβ β ≥1. 3.24 3.24 This is a contradiction with inequality 3.6. Therefore, there exists r > 0 such that the mapping Qn maps Wr into itself. By Darbu-Sadovskii’s ﬁxed point theorem, the operator Qn has at least one ﬁxed point in Wr. This completes the proof. Lemma 3.3. Assume that all the conditions in Theorem 3.1 are satisﬁed. Then the set D\|h,K is precompact in PCh, K, X for all h ∈0, δ, where D : {un; un ∈PC0, K, X coming from Lemma 3.2, n ≥1}, 3.25 3.25 and δ is the constant in (H2). and δ is the constant in (H2). Proof. The proof will be given in several steps. In the following h is a number in 0, δ. 10 Advances in Diﬀerence Equations Advances in Diﬀerence Equations Step 1. D\|h,t1 is precompact in Ch, t1, X. For u ∈PC0, K, X, deﬁne QF1 : PC0, K, X →PC0, K, X by QF1ut TtF0, u0, t ∈0, K. Then by the continuity of f, g, Ii, and using the dominated convergence theorem, we get 3.26 3.26 For u ∈Ch, t1, X, let ut ut, t ∈h, t1, ut uh, t ∈0, h, and we deﬁne QF2 : Ch, t1, X →Ch, t1, X by For u ∈Ch, t1, X, let ut ut, t ∈h, t1, ut uh, t ∈0, h, and we deﬁne QF2 : Ch, t1, X →Ch, t1, X by For u ∈Ch, t1, X, let ut ut, t ∈h, t1, ut uh, t ∈0, h, and we deﬁne QF2 : Ch, t1, X →Ch, t1, X by QF2ut −Ft, ut t 0 ATt −sFs, usds, t ∈h, t1. 3.27 3.27 By condition H3, QF2 is well deﬁned and for u ∈D, we have Qn3ut QF1ut QF2u\|h,t1 t, t ∈h, t1. 3.28 3.28 On the other hand, for un ∈D, n ≥1, we have Qn2unt 0, t ∈h, t1. So, unt Qn1unt QF1unt QF2un\|h,t1 t Qn4unt, t ∈h, t1. 3.29 n the other hand, for un ∈D, n ≥1, we have Qn2unt 0, t ∈h, t1. So, On the other hand, for un ∈D, n ≥1, we have Qn2unt 0, t ∈h, t1. So, unt Qn1unt QF1unt QF2un\|h,t1 t Qn4unt, t ∈h, t1. 3.29 3.29 Now, for {Qn1un; n ≥1}, we have Now, for {Qn1un; n ≥1}, we have Qn1unt Ttu0 −TtT 1 n gun, t ∈h, t1. 3.30 3.30 By the compactness of Tt, t > 0, we get that {Qn1unt; n ≥1} is relatively compact in X for every t ∈h, t1 and {Qn1un; n ≥1}\|h,t1 is equicontinuous on h, t1, which implies that {Qn1un; n ≥1}\|h t1 is precompact in Ch, t1, X. {Qn1 n; ≥}\|h,t1 p p , 1, By the same reasoning, {QF1un; n ≥1}\|h,t1 is precompact in Ch, t1, X. For QF2, we claim that QF2 : Ch, t1, X →Ch, t1, X is Lipschitz continuous with constant M0L LC1−βKβ/β. Then by the continuity of f, g, Ii, and using the dominated convergence theorem, we get In fact, H3 implies that for every u, v ∈Ch, t1, X and t ∈h, t1, ∥QF2ut −QF2vt∥ ≤∥Ft, ut −Ft, vt∥ t 0 ∥ATt −sFs, us −Fs, vs∥ds ≤M0L∥ut −vt∥ t 0 C1−β t −s1−β L ds max 0≤t≤t1∥ut −vt∥ ≤M0L∥ut −vt∥ LC1−βKβ β max h≤t≤t1∥ut −vt∥, 3.31 3.31 ≤M0L∥ut −vt∥ t 0 C1−β t −s1−β L ds max 0≤t≤t1∥ut −vt∥ 3.31 0 t −s1 β 0≤t≤t1 ≤M0L∥ut −vt∥ LC1−βKβ β max h≤t≤t1∥ut −vt∥, ≤M0L∥ut −vt∥ LC1−βKβ β max h≤t≤t1∥ut −vt∥, Advances in Diﬀerence Equations 11 that is, iﬀerence Equations 11 nces in Diﬀerence Equations 11 Advances in Diﬀerence Equations 11 nces in Diﬀerence Equations 11 dvances in Diﬀerence Equations 11 that is, at is, ∥QF2u −QF2v∥Ch,t1,X ≤ M0L LC1−βKβ β ∥u −v∥Ch,t1,X. 3.32 3.32 Therefore, QF2 : Ch, t1, X →Ch, t1, X is Lipschitz continuous with constant M0L LC1−βKβ/β. Clearly, {Qn4un; n ≥1} is precompact in PC0, K, X, and so is {Qn4un; n ≥1}\|h,t1 in Ch, t1, X. Thus, by 3.29 and Lemma 2.4, we obtain Therefore, QF2 : Ch, t1, X →Ch, t1, X is Lipschitz continuous with constant M0L LC1−βKβ/β. Clearly, {Qn4un; n ≥1} is precompact in PC0, K, X, and so is {Qn4un; n ≥1}\|h,t1 in Ch, t1, X. Thus, by 3.29 and Lemma 2.4, we obtain Therefore, QF2 : Ch, t1, X →Ch, t1, X is Lipschitz continuous with constant M0L LC1−βKβ/β. Clearly, {Qn4un; n ≥1} is precompact in PC0, K, X, and so is {Qn4un; n ≥1}\|h,t1 in Ch, t1, X. Thus, by 3.29 and Lemma 2.4, we obtain α D\|h,t1 ≤ M0L LC1−βKβ β α D\|h,t1 . 3.33 3.33 By 3.6, M0L LC1−βKβ/β < 1, which implies αD\|h,t1 0. Consequently, D\|h,t1 is precompact in Ch, t1, X. By 3.6, M0L LC1−βKβ/β < 1, which implies αD\|h,t1 0. Consequently, D\|h,t1 is precompact in Ch, t1, X. Step 2. D\|h,t2 is precompact in PCh, t2, X. For u ∈PCh, t2, X, let ut ut, t ∈h, t2, ut uh, t ∈0, h, 3.34 3.34 and deﬁne Q′ F2 : PCh, t2, X →PCh, t2, X by and deﬁne Q′ F2 : PCh, t2, X →PCh, t2, X by Q′ F2ut −Ft, ut t 0 ATt −sFs, usds, t ∈h, t2. 3.35 Q′ F2ut −Ft, ut t 0 ATt −sFs, usds, t ∈h, t2. Then by the continuity of f, g, Ii, and using the dominated convergence theorem, we get 3.35 3.35 By H3, Q′ F2 is well deﬁned and for u ∈D, we have By H3, Q′ F2 is well deﬁned and for u ∈D, we have By H3, Q′ F2 is well deﬁned and for u ∈D, we have By H3, Q′ F2 is well deﬁned and for u ∈D, we have Qn3ut QF1ut Q′ F2u\|h,t2 t, t ∈h, t2. 3.36 unt Qn1unt QF1unt Q′ F2un\|h,t2 t Qn4unt Qn2unt, t ∈h, t2, 3.37 where Qn2unt ⎧ ⎪ ⎨ ⎪ ⎩ 0, t ∈h, t1, Tt −t1T 1 n I1unt1, t ∈J1. 3.38 t ∈h, t1, Qn2unt ⎧ ⎪ ⎨ ⎪ ⎩ 0, t ∈h, t1, Tt −t1T 1 n I1unt1, t ∈J1. 3.38 3.38 12 Advances in Diﬀerence Equations According to the proof of Step 1, we know that According to the proof of Step 1, we know that {Qn1un; n ≥1}\|h,t2, {QF1un; n ≥1}\|h,t2, {Qn4un; n ≥1}\|h,t2 3.39 3.39 are all precompact in PCh, t2, X and Q′ F2 : PCh, t2, X →PCh, t2, X is Lipschitz continuous with constant M0L LC1−βKβ/β. are all precompact in PCh, t2, X and Q′ F2 : PCh, t2, X →PCh, t2, X is Lipschitz continuous with constant M0L LC1−βKβ/β. β β Next, we will show that {Qn2un; n ≥1}\|h,t2 is precompact in PCh, t2, X. Firstly, it is easy to see that {Qn2un; n ≥1}\|h,t1 is precompact in Ch, t1, X. Thus according to Lemma 2.3, it remains to prove that {Qn2un; n ≥1}\|J1 T· −t1T 1 n I1unt1; · ∈J1, n ≥1 3.40 3.40 is precompact in Ct1, t2, X. And, we recall that vn Qn2un\|J1, n ≥1, which means that is precompact in Ct1, t2, X. And, we recall that vn Qn2un\|J1, n ≥1, which means that vnt1 Qn2unt 1 T 1 n I1unt1, vnt Qn2unt Tt −t1T 1 n I1unt1, t ∈J1. 3.41 3.41 By Step 1, D\|h,t1 is precompact in Ch, t1, X. Without loss of generality, we may suppose that un\|h,t1 −→w, as n −→∞in Ch, t1, X. 3.42 3.42 Therefore, unt1 →wt1, as n →∞in X. Thus, by the continuity of I1 and Tt, we get Therefore, unt1 →wt1, as n →∞in X. Thus, by the continuity of I1 and Tt, we get T 1 n I1unt1 −I1wt1 ≤ T 1 n I1unt1 −T 1 n I1wt1 T 1 n I1wt1 −I1wt1 ≤M∥I1unt1 −I1wt1∥ T 1 n I1wt1 −I1wt1 3.43 ≤ T 1 n I1unt1 −T 1 n I1wt1 T 1 n I1wt1 −I1wt1 ≤M∥I1unt1 −I1wt1∥ T 1 n I1wt1 −I1wt1 3.43 3.43 ≤M∥I1unt1 −I1wt1∥ T 1 n I1wt1 −I1wt1 −→0, −→0, as n →∞, which implies that {vnt1; n ≥1} is relatively compact in X. And, for t ∈J1, by the compactness of Tt, t > 0, {vnt; n ≥1} is also relatively compact in X. Therefore, {Qn2un; n ≥1}\|J1t is relatively compact in X for every t ∈J1. According to the proof of Step 1, we know that nces in Diﬀerence Equations 13 13 Advances in Diﬀerence Equations Next, for t1 ≤s ≤t ≤t2, we have t1 ≤s ≤t ≤t2, we have Next, for t1 ≤s ≤t ≤t2, we have Tt −t1T 1 n I1unt1 −Ts −t1T 1 n I1unt1 Ts −t1Tt −s −T0T 1 n I1unt1 ≤M Tt −s −T0T 1 n I1unt1 . 3.44 3.44 Thus, the set {Qn2un; n ≥1}\|J1 ⊆Ct1, t2, X is equicontinuous on J1 due to the compactness of {T1/nI1unt1; n ≥1} and the strong continuity of operator Tt, t ≥0. By the Arzela- Ascoli theorem, we conclude that {Qn2un; n ≥1}\|J1 is precompact in Ct1, t2, X. Therefore, {Qn2un; n ≥1}\|h,t2 is precompact in PCh, t2, X. Th b L 2 4 b i Thus, the set {Qn2un; n ≥1}\|J1 ⊆Ct1, t2, X is equicontinuous on J1 due to the compactness of {T1/nI1unt1; n ≥1} and the strong continuity of operator Tt, t ≥0. By the Arzela- Ascoli theorem, we conclude that {Qn2un; n ≥1}\|J1 is precompact in Ct1, t2, X. Therefore, {Qn2un; n ≥1}\|h,t2 is precompact in PCh, t2, X. Th b L 2 4 bt i , 2 Thus, by Lemma 2.4, we obtain , 2 Thus, by Lemma 2.4, we obtain α D\|h,t2 ≤ M0L LC1−βKβ β α D\|h,t2 . 3.45 3.45 By 3.6, M0L LC1−βKβ/β < 1, which implies αD\|h,t2 0. Consequently, D\|h,t2 is precompact in PCh, t2, X. By 3.6, M0L LC1−βKβ/β < 1, which implies αD\|h,t2 0. Consequently, D\|h,t2 is precompact in PCh, t2, X. Step 3. The same idea can be used to prove the compactness of D\|h,ti in PCh, ti, X for i 3, . . . , p, p 1, where tp1 K. This completes the proof. Proof of Theorem 3.1. For un ∈D, n ≥1, let unt ⎧ ⎨ ⎩ unt, t ∈δ, K, unδ, t ∈0, δ, 3.46 3.46 where δ comes from the condition H2. Then, by condition H2, gun gun. By Lemma 3.3, without loss of generality, we may suppose that un → u ∈ PC0, K, X, as n →∞. Thus, by the continuity of Tt and g, we get where δ comes from the condition H2. Then, by condition H2, gun gun. According to the proof of Step 1, we know that By Lemma 3.3, without loss of generality, we may suppose that un → u ∈ PC0, K, X, as n →∞. Thus, by the continuity of Tt and g, we get T 1 n gun −gu ≤ T 1 n gun −T 1 n gu T 1 n gu −gu ≤M gun −gu T 1 n gu −gu 3.47 3.47 −→0, 14 Advances in Diﬀerence Equations Advances in Diﬀerence Equations as n →∞. Thus, as n →∞. Thus, {Qn1un; n ≥1} T· u0 −T 1 n gun ; n ≥1 3.48 3.48 is precompact in PC0, K, X. Moreover, {Qn4un; n ≥1} and {Qn2un; n ≥1} are both precompact in PC0, K, X. And Qn3 : PC0, K, X → PC0, K, X is Lipschitz continuous with constant M 1M0L LC1−βKβ/β. Note that is precompact in PC0, K, X. Moreover, {Qn4un; n ≥1} and {Qn2un; n ≥1} are both precompact in PC0, K, X. And Qn3 : PC0, K, X → PC0, K, X is Lipschitz continuous with constant M 1M0L LC1−βKβ/β. Note that unt Qnunt Qn1unt Qn3unt Qn4unt Qn2unt, t ∈0, K. 3.49 Therefore, by Lemma 2.4, we know that the set D is precompact in PC0, K, X. Without loss of generality, we may suppose that un →u∗in PC0, K, X. On the other hand, we also have Therefore, by Lemma 2.4, we know that the set D is precompact in PC0, K, X. Without loss of generality, we may suppose that un →u∗in PC0, K, X. On the other hand, we also have unt Tt u0 F0, un0 −T 1 n gun −Ft, unt t 0 ATt −sFs, unsds t 0 Tt −sfs, unsds 0<ti<t Tt −tiT 1 n Iiunti, 0 ≤t ≤K. 3.50 3.50 Letting n →∞in both sides, we obtain Letting n →∞in both sides, we obtain Letting n →∞in both sides, we obtain u∗t Ttu0 F0, u∗0 −gu∗ −Ft, u∗t t 0 ATt −sFs, u∗sds t 0 Tt −sfs, u∗sds 0<ti<t Tt −tiIiu∗ti, 0 ≤t ≤K, 3.51 3.51 which implies that u∗is a mild solution of the nonlocal impulsive problem 1.1. This completes the proof. 4. Application In this section, to illustrate our abstract result, we consider the following diﬀerential system: ∂ ∂t wt, x π 0 λt, x, ywt, ydy ∂2 ∂x2 wt, x vt, wt, x, 0 ≤t ≤1, 0 ≤x ≤π, t / ti, wt, 0 wt, π 0, 0 ≤t ≤1, wt i −wt− i Iiwti, i 1, . . . , p, 0 < t1 < · · · < tp < 1, w0, x q j1 cjwsj, x w0x, 0 < s1 < · · · < sq < 1, 0 ≤x ≤π, 4.1 4.1 where w0 ∈L20, π, ti, sj, cj are given real numbers for i 1, . . . , p, j 1, . . . , q, and λ : 0, 1 × 0, π × 0, π →R and v : 0, 1 × R →R are functions to be speciﬁed below. where w0 ∈L20, π, ti, sj, cj are given real numbers for i 1, . . . , p, j 1, . . . , q, and λ : 0, 1 × 0, π × 0, π →R and v : 0, 1 × R →R are functions to be speciﬁed below. 2 p To treat the above system, we take X L20, π with the norm ∥· ∥and we consider the operator A : DA ⊆X →X deﬁned by Az −z′′ 4.2 4.2 with domain DA z ∈X; z, z′ area absolutely continuous, z′′ ∈X, z0 zπ 0. 4.3 DA z ∈X; z, z′ area absolutely continuous, z′′ ∈X, z0 zπ 0. 4.3 4.3 The operator −A is the inﬁnitesimal generator of an analytic compact semigroup Ttt≥0 on X. Moreover, A has a discrete spectrum, the eigenvalues are n2, n ∈N, with the corresponding normalized eigenvectors enx 2/π sinnx, and the following properties are satisﬁed. The operator −A is the inﬁnitesimal generator of an analytic compact semigroup Ttt≥0 on X. Moreover, A has a discrete spectrum, the eigenvalues are n2, n ∈N, with the corresponding normalized eigenvectors enx 2/π sinnx, and the following properties are satisﬁed. a If z ∈DA, then Az ∞ n1 n2⟨z, en⟩en. b For each z ∈X, Ttz ∞ n1 exp−n2t⟨z, en⟩en. According to the proof of Step 1, we know that which implies that u∗is a mild solution of the nonlocal impulsive problem 1.1. This completes the proof. Remark 3.4. From Lemma 3.3 and the above proof, it is easy to see that we can also prove Theorem 3.1 by showing that D\|0,h is precompact in PC0, h, X. The following results are immediate consequences of Theorem 3.5. Theorem 3.5. Assume (H1), (H3)–(H5) hold. If g ≡0, then the impulsive Cauchy problem 1.1 has at least one mild solution on 0, K, provided MM0L γ pL M0L LC1−βKβ β < 1. 3.52 3.52 Advances in Diﬀerence Equations 15 Theorem 3.6. Assume (H1), (H2), (H4), and (H5) hold. If F ≡0, then the nonlocal impulsive problem 1.1 has at least one mild solution on 0, K, provided ML γ pL < 1. Theorem 3.6. Assume (H1), (H2), (H4), and (H5) hold. If F ≡0, then the nonlocal impulsive problem 1.1 has at least one mild solution on 0, K, provided ML γ pL < 1. Theorem 3.7. Assume (H1), (H4), and (H5) hold. If g ≡0, F ≡0, then the impulsive problem 1.1 has at least one mild solution on 0, K, provided Mγ pL < 1. Remark 3.8. Theorems 3.5-3.6 are new even for many special cases discussed before, since neither the Lipschitz continuity nor compactness assumption on the impulsive functions is required. 4. Application Moreover, ∥Tt∥≤1 for all t ≥0. c For each z ∈X, A−1/2z ∞ n1 1/n⟨z, en⟩en. In particular, ∥A−1/2∥ 1. d A1/2 is given by A1/2z ∞ n1 n⟨z, en⟩en with the domain DA1/2 {z ∈ X; ∞ n1 n⟨z, en⟩en ∈X}. d A1/2 is given by A1/2z ∞ n1 n⟨z, en⟩en with the domain DA1/2 {z ∈ X; ∞ n1 n⟨z, en⟩en ∈X}. 16 Advances in Diﬀerence Equations Advances in Diﬀerence Equations Advances in Diﬀerence Equations Assume the following. Assume the following. Assume the following. 1 The function λ : 0, 1 × 0, π × 0, π →R is continuously diﬀerential with λt, 0, y λt, π, y 0 for t ∈0, 1, y ∈0, π, and there exists a real number δ ∈0, s1 such that λt, x, y 0 for t ∈0, δ, x, y ∈0, π. Moreover, Λ : sup t∈0,1 π 0 ∂ ∂x λt, x, y2 dx dy 1/2 < ∞. 4.4 4.4 2 For each t ∈0, 1, vt, · is continuous, and for each x ∈R, v·, x is measurable and, there exists a function a· ∈L10, 1, R such that \|vt, x\| ≤at\|x\| for a.e. t ∈0, 1 and all x ∈R. 2 For each t ∈0, 1, vt, · is continuous, and for each x ∈R, v·, x is measurable and, there exists a function a· ∈L10, 1, R such that \|vt, x\| ≤at\|x\| for a.e. t ∈0, 1 and all x ∈R. 3 Ii : X →X is a continuous function for each i 1, . . . , p, and there exist positive numbers L4, L′ 4 such that ∥Iiz∥≤L4∥z∥ L′ 4 for any z ∈X and i 1, 2, . . . , p. 3 Ii : X →X is a continuous function for each i 1, . . . , p, and there exist positive numbers L4, L′ 4 such that ∥Iiz∥≤L4∥z∥ L′ 4 for any z ∈X and i 1, 2, . . . , p. ne F, f : 0, 1×X →X and g : PC0, 1, X →X, respectively, as follows. For x ∈0, π, Ft, zx π 0 λt, x, yzydy, ft, zx vt, zx, gux q j1 cjusj x. 4. Application 4.5 4.5 From the deﬁnition of F and assumption 1, it follows that From the deﬁnition of F and assumption 1, it follows that From the deﬁnition of F and assumption 1, it follows that F·, u1· F·, u2· with u1t u2t, t ∈δ, 1, for u1, u2 ∈PC0, 1, X, F·, u1· F·, u2· with u1t u2t, t ∈δ, 1, for u1, u2 ∈PC0, 1, X, F·, u1· F·, u2· with u1t u2t, t ∈δ, 1, for u1, u2 ∈PC0, 1, X, F·, u1· F·, u2· with u1t u2t, t ∈δ, 1, for u1, u2 ∈PC0, 1, X, ⟨Ft, z, en⟩ π 0 enx π 0 λt, x, yzydy dx 1 n π 0 ∂ ∂xλt, x, yzydy, ! 2 π cosnx " , A1/2Ft, z1 −A1/2Ft, z2 ∞ n1 n⟨Ft, z1 −Ft, z2, en⟩en ≤ π 0 ∂ ∂x λt, x, y2 dy dx 1/2 × π 0 z1 y−z2 y2dy 1/2 ≤Λ∥z1 −z2∥. 4.6 4.6 Thus, system 4.1 can be transformed into the abstract problem 1.1, and conditions H2, H3, H4, and H5 are satisﬁed with Thus, system 4.1 can be transformed into the abstract problem 1.1, and conditions H2, H3, H4, and H5 are satisﬁed with Thus, system 4.1 can be transformed into the abstract problem 1.1, and conditions H2, H3, H4, and H5 are satisﬁed with L1 q j1 ##cj ##, L2 L3 Λ, ρlt lat, γ 1 0 atdt. 4.7 4.7 17 Advances in Diﬀerence Equations If 3.6 holds it holds when the related constants are small, then according to Theorem 3.1, the problem 4.1 has at least one mild solution in PC0, 1, X. Acknowledgments The authors would like to thank the referees for helpful comments and suggestions. J. Liang acknowledges support from the NSF of China 10771202 and the Specialized Research Fund for the Doctoral Program of Higher Education of China 2007035805. Z. Fan acknowledges support from the NSF of China 11001034 and the Research Fund for Shanghai Postdoctoral Scientiﬁc Program 10R21413700. References 1 N. U. Ahmed, “Optimal feedback control for impulsive systems on the space of ﬁnitely additive measures,” Publicationes Mathematicae Debrecen, vol. 70, no. 3-4, pp. 371–393, 2007. 2 T. Cardinali and P. Rubbioni, “Impulsive semilinear diﬀerential inclusions: topological structure of the solution set and solutions on non-compact domains,” Nonlinear Analysis: Theory, Methods & Applications, vol. 69, no. 1, pp. 73–84, 2008. 3 M. Eduardo Hern´andez and S. M. 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https://openalex.org/W3161113732	https://digital.csic.es/bitstream/10261/261522/5/Arrizabalaga%20et%20al.%20AJGWR.pdf	English	null	Growth and physiology of four<i>Vitis vinifera</i>L. cv. Tempranillo clones under future warming and water deficit regimes	Australian journal of grape and wine research	2,021	cc-by	18,756	Growth and physiology of four Vitis vinifera L. cv. 1 water deficit 2 3 M. ARRIZABALAGA-ARRIAZU1,2,3, F. MORALES4, J 4 1Universidad de Navarra. Faculty of Sciences, Plant Stress Physiology Grou 5 (CSIC) [Estación Experimental de Aula Dei (EEAD), Zaragoza, and Ins 6 Pamplona, Spain; 2Université de Bordeaux, Institut des Sciences de la V 7 Génomique Fonctionelle de la Vigne, 33883, Villenave d’Ornon, France 8 Fonctionnelle de la Vigne (EGFV), Bordeaux Sciences Agro, Institut Natio 9 Institut des Sciences de la Vigne et du Vin, 33883, Villenave d’Ornon, Fr 10 Navarra, 31192, M 11 12 13 14 Corresponding author: Associate Professor Inmacu 15 16 17 Running title: Response of Tempra 18 19 20 Growth and physiology of four Vitis vinifera L. cv. 1 water deficit 2 3 M. ARRIZABALAGA-ARRIAZU1,2,3, F. MORALES4, J 4 1Universidad de Navarra. Faculty of Sciences, Plant Stress Physiology Grou 5 (CSIC) [Estación Experimental de Aula Dei (EEAD), Zaragoza, and Ins 6 Pamplona, Spain; 2Université de Bordeaux, Institut des Sciences de la V 7 Génomique Fonctionelle de la Vigne, 33883, Villenave d’Ornon, France 8 Fonctionnelle de la Vigne (EGFV), Bordeaux Sciences Agro, Institut Natio 9 Institut des Sciences de la Vigne et du Vin, 33883, Villenave d’Ornon, Fr 10 Navarra, 31192, M 11 12 13 14 Corresponding author: Associate Professor Inmacu 15 16 17 Running title: Response of Tempra 18 19 20 Growth and physiology of four Vitis vinifera L. cv. Tempranillo clones und 1 water deficit regimes 2 3 M. ARRIZABALAGA-ARRIAZU1,2,3, F. MORALES4, J. J. IRIGOYEN1, G. HILBERT3 a 4 1Universidad de Navarra. Faculty of Sciences, Plant Stress Physiology Group, Associated Unit to Consejo Sup 5 (CSIC) [Estación Experimental de Aula Dei (EEAD), Zaragoza, and Instituto de Ciencias de la Vid y del V 6 Pamplona, Spain; 2Université de Bordeaux, Institut des Sciences de la Vigne et du Vin, Unité Mixte de Re 7 Génomique Fonctionelle de la Vigne, 33883, Villenave d’Ornon, France; 3Unité Mixte de Recherche, 128 8 Fonctionnelle de la Vigne (EGFV), Bordeaux Sciences Agro, Institut National de la Recherche Agronomique 9 Institut des Sciences de la Vigne et du Vin, 33883, Villenave d’Ornon, France; 4Instituto de Agrobiotecnol 10 Navarra, 31192, Mutilva, Spain 11 12 13 14 Corresponding author: Associate Professor Inmaculada Pascual Elizalde, email ipasc 15 16 17 Running title: Response of Tempranillo clones to climate change 18 19 M. ARRIZABALAGA-ARRIAZU1,2,3, F. MORALES4, J. J. IRIGOYEN1, G. HILBERT3 and I. PASCUAL1 GA-ARRIAZU1,2,3, F. MORALES4, J. J. IRIGOYEN1, G. HILBERT3 and I. PASCUAL1 RRIZABALAGA-ARRIAZU1,2,3, F. MORALES4, J. J. IRIGOYEN1, G. HILBERT3 and 1Universidad de Navarra. Faculty of Sciences, Plant Stress Physiology Group, Associated Unit to Consejo Superior de Investigaciones Científicas 5 (CSIC) [Estación Experimental de Aula Dei (EEAD), Zaragoza, and Instituto de Ciencias de la Vid y del Vino (ICVV), Logroño], 31008, 6 Pamplona, Spain; 2Université de Bordeaux, Institut des Sciences de la Vigne et du Vin, Unité Mixte de Recherche, 1287 Ecophysiologie et 7 Génomique Fonctionelle de la Vigne, 33883, Villenave d’Ornon, France; 3Unité Mixte de Recherche, 1287 Ecophysiologie et Génomique 8 Fonctionnelle de la Vigne (EGFV), Bordeaux Sciences Agro, Institut National de la Recherche Agronomique (INRA), Université de Bordeaux, 9 Institut des Sciences de la Vigne et du Vin, 33883, Villenave d’Ornon, France; 4Instituto de Agrobiotecnología (IdAB), CSIC – Gobierno de 10 Navarra, 31192, Mutilva, Spain 11 Corresponding author: Associate Professor Inmaculada Pascual Elizalde, email ipascual@unav.es Running title: Response of Tempranillo clones to climate change 20 1 1 Abstract 1 Abstract 1 Background and Aim: The interactive effects of simulated 2100 environmental conditions (air temperature and CO2 level) and w 2 four clones of Vitis vinifera cv. Tempranillo were investigated. 3 Methods and Results: Fruit-bearing cuttings were subjected to: (i) two temperature/CO2/RH regimes: climate change (CC) (28°C 4 µmol/mol CO2 and 33%/53% RH, day/night) vs current climatic conditions (CS) (24°C/14°C, 400 µmol/mol CO2 and 45%/65% R 5 with (ii) two water availabilities: well-watered (WW) vs water deficit (WD). Climate change increased net photosynthesis (An), tra 6 ameliorating the low carbon fixation rates under drought, but not the reduction in vegetative and reproductive growth. Current clim 7 intrinsic water use efficiency (An/gs), especially when combined with WD, but not the instantaneous water use efficiency (An/T). Th 8 exhibited differences in the ripening time, plant vigour and reproductive growth. Variability in the response of An, phenology and g 9 simulated conditions was observed among clones. 10 Conclusions: Differences in the length of the reproductive cycle conditioned, in part, the physiological response of the clones to th 11 environmental factors. 12 Significance of the Study: The study improves our understanding of the interactive effects of climate change factors and provides 13 the response of different clones, as the basis for the adaptation of cultivars in their traditional growing regions. M. ARRIZABALAGA-ARRIAZU1,2,3, F. MORALES4, J. J. IRIGOYEN1, G. HILBERT3 and I. PASCUAL1 14 15 Keywords: climate change, gas exchange, growth, phenology, Vitis vinifera L., water deficit 16 17 18 Background and Aim: The interactive effects of simulated 2100 environmental conditions (air temperature and CO2 level) and water deficit on 2 four clones of Vitis vinifera cv. Tempranillo were investigated. 3 Methods and Results: Fruit-bearing cuttings were subjected to: (i) two temperature/CO2/RH regimes: climate change (CC) (28°C/18°C, 700 4 µmol/mol CO2 and 33%/53% RH, day/night) vs current climatic conditions (CS) (24°C/14°C, 400 µmol/mol CO2 and 45%/65% RH), combined 5 with (ii) two water availabilities: well-watered (WW) vs water deficit (WD). Climate change increased net photosynthesis (An), transiently 6 ameliorating the low carbon fixation rates under drought, but not the reduction in vegetative and reproductive growth. Current climate increased 7 intrinsic water use efficiency (An/gs), especially when combined with WD, but not the instantaneous water use efficiency (An/T). The clones 8 exhibited differences in the ripening time, plant vigour and reproductive growth. Variability in the response of An, phenology and growth to the 9 simulated conditions was observed among clones. 10 Conclusions: Differences in the length of the reproductive cycle conditioned, in part, the physiological response of the clones to the 11 environmental factors. 12 Significance of the Study: The study improves our understanding of the interactive effects of climate change factors and provides insights into 13 the response of different clones, as the basis for the adaptation of cultivars in their traditional growing regions. 14 Keywords: climate change, gas exchange, growth, phenology, Vitis vinifera L., water deficit 16 2 2 Introduction 1 According to the 5th assessment report of the Intergovernmental Panel on Climate Change (IPCC), precipitation patterns are expected to be 2 altered, with likely more frequent drought events predicted for regions that are already arid (Intergovernmental Panel on Climate Change 2013). 3 These changes will be a consequence of the likely increase in the atmospheric CO2 concentration, among other greenhouse gases (GHG). The 4 IPCC has determined the magnitude of these changes according to different emission scenarios called Representative Concentration Pathways 5 (RCPs). According to these time-dependent projections of atmospheric GHG, the atmospheric CO2 concentration may increase up to between 670 6 and 936 µmol/mol, according to the RCP 6.0 and RCP 8.5, respectively, by the end of this century (Moss et al. 2008, 2010), thus contributing to 7 an expected increase in global mean temperature of 2.2 ± 0.5°C and 3.7 ± 0.7°C, respectively, according to the concentration-driven CMIP5 8 model simulations (Taylor et al. 2012, Intergovernmental Panel on Climate Change 2013). Recent models also report a decrease in near-surface 9 land RH with climate change (Byrne and O’Gorman 2016). Therefore, even if rainfall does not decrease locally, water deficit experienced by 10 crops will likely increase because of the impact of temperature and RH on evapotranspiration, especially in summer (van Leeuwen and Darriet 11 2016). 12 According to the 5th assessment report of the Intergovernmental Panel on Climate Change (IPCC), precipitation patterns are expected to be 2 altered, with likely more frequent drought events predicted for regions that are already arid (Intergovernmental Panel on Climate Change 2013). 3 These changes will be a consequence of the likely increase in the atmospheric CO2 concentration, among other greenhouse gases (GHG). The 4 IPCC has determined the magnitude of these changes according to different emission scenarios called Representative Concentration Pathways 5 (RCPs). According to these time-dependent projections of atmospheric GHG, the atmospheric CO2 concentration may increase up to between 670 6 and 936 µmol/mol, according to the RCP 6.0 and RCP 8.5, respectively, by the end of this century (Moss et al. 2008, 2010), thus contributing to 7 an expected increase in global mean temperature of 2.2 ± 0.5°C and 3.7 ± 0.7°C, respectively, according to the concentration-driven CMIP5 8 model simulations (Taylor et al. 2012, Intergovernmental Panel on Climate Change 2013). Recent models also report a decrease in near-surface 9 land RH with climate change (Byrne and O’Gorman 2016). Introduction 1 Am 23 Mediterranean area, Tempranillo is one of the most internationally recognised, with the highest number of certifi 24 Variability among Tempranillo clones for phenological development has previously been described (Arrizabalag 25 however, have assessed the performance of different clones of the same cultivar to climate conditions expected b 26 Tortosa et al. 2019). 27 2017), however, extreme heat events (temperature higher than 40°C), reduce stomatal conductance and photosynthesis rates, slowing down grape 1 ripening (Greer and Weedon 2013). As a consequence, vegetative growth and yield are also expected to decrease (Medrano et al. 2003, Webb et 2 al. 2009). 3 Plants will not experience climate change factors individually, but simultaneously. The combined impact of these factors is frequently 4 non-additive (Gray and Brady 2016), and can be either greater or lower than expected on single-factor studies (synergistic and antagonistic, 5 respectively). The number of studies, however, on the interactive effects of environmental factors on grapevine physiology is still relatively few. 6 Combined high temperature and elevated CO2 concentration have been reported to increase the photosynthetic capacity of grapevine, hastening 7 grape development (Martínez-Lüscher et al. 2015b, 2016). The interactive effects among water deficit and heat temperature on grapevine 8 physiology have been also investigated in short-term experiments, showing a more severe effect of high-temperature events on grapevines 9 experiencing water stress (Edwards et al. 2011, Galat Giorgi et al. 2019). The studies on the physiological and growth response of grapevine to 10 multiple stress factors associated with climate change (CO2, temperature and water deficit) remain limited due to their complexity. Authors report 11 that the beneficial effect of increased air CO2 and elevated temperature on the photosynthetic performance and growth of Tempranillo was 12 eliminated by water deficit (Leibar et al. 2015, Kizildeniz et al. 2018). 13 In order to reduce adverse climatic effects, some adaptation of future viticulture is needed. Within this context, the choice of adequate 14 plant material is one of the most powerful tools. Grapevine plants are propagated vegetatively, and new features can appear spontaneously in a 15 bud after accidental modifications in the DNA. These modifications include point mutations, large deletions, illegitimate recombinations or 16 variable number of repeats in microsatellite sequences (Pelsy 2010). This emergence of genetic variability leads to clonal variation within a 17 cultivar. Introduction 1 Therefore, even if rainfall does not decrease locally, water deficit experienced by 10 crops will likely increase because of the impact of temperature and RH on evapotranspiration, especially in summer (van Leeuwen and Darriet 11 2016). 12 Grapevine (Vitis vinifera L.) is one of the most widespread crops worldwide (7.5 Mha in 2016) (Organisation Internationale de la Vigne et 13 du Vin 2017). Europe represents the largest vineyard area in the world (39% of the world area), the main part being located in the Mediterranean 14 region (Organisation Internationale de la Vigne et du Vin 2017). The region is considered to be vulnerable to the impacts of climate change, 15 especially concerning water availability (Fraga et al. 2019). Grapevine development and grape ripening are sensitive to environmental factors. 16 Under conditions of water deficit, stomata closure is one of the early grapevine responses in order to prevent hydraulic failure (Charrier et al. 17 2018), thus restricting water loss, but also C assimilation (Flexas et al. 1998, Chaves et al. 2010). Consequently, drought has been reported to 18 decrease grapevine vigour and final fruit production (Chaves et al. 2007, Salazar-Parra et al. 2012, Kizildeniz et al. 2015), with variable effects 19 on phenology, depending on its intensity (van Leeuwen et al. 2009). Increases in temperature and atmospheric CO2 concentration have been 20 reported to impact the physiology and development of many plant species (Wang et al. 2012, Gray and Brady 2016, Kacienè et al. 2017). In the 21 case of grapevine, leaf photosynthetic activity (An) is stimulated under elevated CO2 conditions during the first days of exposure, then 22 experiencing in some cases an acclimation process resulting in down-regulated photosynthesis rates (Salazar-Parra et al. 2012, 2015). Elevated 23 CO2 also increased grape yield of cvs Sangiovese, Cabernet Sauvignon and Riesling (Bindi et al. 2001, Wohlfahrt et al. 2018), but this effect was 24 not so marked in other experiments with Tempranillo and Touriga Franca (Moutinho-Pereira et al. 2009, Kizildeniz et al. 2015, 2018). Studies of 25 elevated temperature have mainly focused on grape ripening and composition (Kuhn et al. 2014, Gouot et al. 2019). Warm temperature has been 26 reported to accelerate grapevine phenology, and to increase photosynthesis performance (Greer and Weedon 2012, De Cortázar-Atauri et al. 27 3 2017), however, extreme heat events (temperature higher than 40°C), reduce stomatal conductance and photosyn 1 ripening (Greer and Weedon 2013). Introduction 1 As a consequence, vegetative growth and yield are also expected to decrease 2 al. 2009). 3 Plants will not experience climate change factors individually, but simultaneously. The combined impact 4 non-additive (Gray and Brady 2016), and can be either greater or lower than expected on single-factor studies (sy 5 respectively). The number of studies, however, on the interactive effects of environmental factors on grapevine p 6 Combined high temperature and elevated CO2 concentration have been reported to increase the photosynthetic ca 7 grape development (Martínez-Lüscher et al. 2015b, 2016). The interactive effects among water deficit and heat te 8 physiology have been also investigated in short-term experiments, showing a more severe effect of high-tempera 9 experiencing water stress (Edwards et al. 2011, Galat Giorgi et al. 2019). The studies on the physiological and gr 10 multiple stress factors associated with climate change (CO2, temperature and water deficit) remain limited due to 11 that the beneficial effect of increased air CO2 and elevated temperature on the photosynthetic performance and gr 12 eliminated by water deficit (Leibar et al. 2015, Kizildeniz et al. 2018). 13 In order to reduce adverse climatic effects, some adaptation of future viticulture is needed. Within this co 14 plant material is one of the most powerful tools. Grapevine plants are propagated vegetatively, and new features 15 bud after accidental modifications in the DNA. These modifications include point mutations, large deletions, ille 16 variable number of repeats in microsatellite sequences (Pelsy 2010). This emergence of genetic variability leads t 17 cultivar. The existing collections of clones can be explored to detect any phenotypic variation that could be usefu 18 climate change in their traditional growing region (Duchêne 2016), without changing wine typicity, and giving ri 19 accepted by the corresponding protection figure (Ibáñez et al. 2015). 20 Phenology is considered the first biological indicator of climate change, this trait being considered one of 21 for cultivar adaptation (Duchêne et al. 2010). In this sense, the selection of late-ripening clones has been propose 22 alterations of grape quality caused by high temperature during fruit ripening (van Leeuwen and Darriet 2016). Introduction 1 The existing collections of clones can be explored to detect any phenotypic variation that could be useful in the adaptation of cultivars to 18 climate change in their traditional growing region (Duchêne 2016), without changing wine typicity, and giving rise to plant material immediately 19 accepted by the corresponding protection figure (Ibáñez et al. 2015). 20 Phenology is considered the first biological indicator of climate change, this trait being considered one of the main factors to be explored 21 for cultivar adaptation (Duchêne et al. 2010). In this sense, the selection of late-ripening clones has been proposed as a strategy to mitigate the 22 alterations of grape quality caused by high temperature during fruit ripening (van Leeuwen and Darriet 2016). Among the cultivars grown in the 23 Mediterranean area, Tempranillo is one of the most internationally recognised, with the highest number of certified clones (Ibáñez et al. 2015). 24 Variability among Tempranillo clones for phenological development has previously been described (Arrizabalaga et al. 2018). Few studies, 25 however, have assessed the performance of different clones of the same cultivar to climate conditions expected by 2100 (Torres et al. 2015, 2016, 26 Tortosa et al. 2019). 27 4 The purpose of our study was to evaluate the response of four Tempranillo clones, differing in their maturation times, to the combined 1 effects of elevated CO2, elevated temperature and reduced RH in plants exposed to two irrigation regimes. The hypothesis behind this study is 2 that differences among clones in the phenological development may condition their physiological and growth response to future climate 3 conditions. One of the strengths of this study lies in the assessment of three-way interactions among clones, T/CO2/RH regimes, and water 4 availability. Considering that it is not possible to extrapolate plant responses to combined environmental conditions from the response derived 5 from a single condition (Rampino et al. 2012), the information obtained from multi-stress approaches is crucial to predict the impact of projected 6 climate change on grapevines and to develop appropriate adaptation strategies. 7 The purpose of our study was to evaluate the response of four Tempranillo clones, differing in their maturation times, to the combined 1 effects of elevated CO2, elevated temperature and reduced RH in plants exposed to two irrigation regimes. Introduction 1 The hypothesis behind this study is 2 that differences among clones in the phenological development may condition their physiological and growth response to future climate 3 conditions. One of the strengths of this study lies in the assessment of three-way interactions among clones, T/CO2/RH regimes, and water 4 availability. Considering that it is not possible to extrapolate plant responses to combined environmental conditions from the response derived 5 from a single condition (Rampino et al. 2012), the information obtained from multi-stress approaches is crucial to predict the impact of projected 6 climate change on grapevines and to develop appropriate adaptation strategies. 7 Material and methods 10 They grew up to fruitset at 25°C/15°C (day/night), air 4 RH of 50% and natural light supplemented with high-pressure metal halide lamps (POWERSTAR HQI-TS 400W/D PRO, OSRAM, Augsburg, 5 Germany) with a photosynthetic photon flux density of 500 µmol/(m2 · s) at plant level for 15 h a day. Plants were irrigated with the nutritive 6 solution described in Ollat et al. (1998). Vegetative growth was controlled by manual pruning, and a single flowering stem was allowed to 7 develop on each plant in order to obtain a single berry bunch per plant. Plants were maintained with four leaves up to fruitset. At this moment, 8 and for standardisation purposes, plants of each clone showing similar phenological development were transferred to 7 L pots with a mixture of 9 2:1 peat:sand (v/v). 10 At fruitset, plants of each of the four clones were transferred to growth chamber greenhouses (GCGs), and divided into four homogeneous 12 groups. Plants were selected to have a similar number of berries. From fruitset to maturity (considered at 23°Brix), plants were subjected to two 13 temperature, CO2 concentration and RH (T/CO2/RH) regimes: climate change conditions (CC: 28°C/18°C, day/night, 700 µmol/mol CO2 and 14 33%/53% RH, day/night) vs current climatic conditions (CS: 24°C/14°C, 400 µmol/mol CO2 and 45%/65% RH). Moreover, plants within each 15 greenhouse were subjected to two water regimes: well-watered (WW) vs water deficit (WD, i.e. 60% of the irrigation received by the WW 16 plants). The combination of clones, T/CO2/RH regimes and water availability made a total of 16 treatments with eight replicates each. 17 The CO2 and temperature conditions in the CC treatment (temperature 4ºC warmer and atmospheric CO2 concentration 300 µmol/mol higher 18 than in the CS treatment) approached the projections for the year 2100, as per the RCP 8.5 greenhouse emission scenario, derived from the 19 concentration-driven CMIP5 model simulations (Taylor et al. 2012, Intergovernmental Panel for Climate Change 2013). Regarding RH, 20 ENSEMBLES models (based on IPCC data), according to the Max Planck Institute model [MPI-ECHAM5 (Roeckner et al. 2003)], state that the 21 RH for the summer period will likely be 12% lower at the end of the present century in the area of study (Navarra and La Rioja regions) (Leibar 22 et al. 2015). For details about the motorisation and control of the environmental parameters within the GCGs, readers are referred to Morales et 23 al. (2014). Material and methods 10 The water deficit level was chosen to match conditions predicted for the end of the present century 25 and La Rioja by the model of the Max Planck Institute, that is likely 40% lower precipitation in the summer (Leibar et 26 An adapted protocol from Mullins and Rajasekaran (1981) was used to produce fruit-bearing cuttings, as described in Morales et al. (2016). 1 Cuttings were treated with a solution of indole butyric acid (300 mg/L), kept for several weeks in a hot-bed at 27°C, and placed in a cold room 2 at 5°C, until they developed roots. Then, cuttings were set in pots of 0.8 L with a mixture of 2:1 peat:sand (v/v) and transferred to growth 3 chamber greenhouses (GCGs) (Morales et al. 2014) located at the University of Navarra. They grew up to fruitset at 25°C/15°C (day/night), air 4 RH of 50% and natural light supplemented with high-pressure metal halide lamps (POWERSTAR HQI-TS 400W/D PRO, OSRAM, Augsburg, 5 Germany) with a photosynthetic photon flux density of 500 µmol/(m2 · s) at plant level for 15 h a day. Plants were irrigated with the nutritive 6 solution described in Ollat et al. (1998). Vegetative growth was controlled by manual pruning, and a single flowering stem was allowed to 7 develop on each plant in order to obtain a single berry bunch per plant. Plants were maintained with four leaves up to fruitset. At this moment, 8 and for standardisation purposes, plants of each clone showing similar phenological development were transferred to 7 L pots with a mixture of 9 2:1 peat:sand (v/v). 10 An adapted protocol from Mullins and Rajasekaran (1981) was used to produce fruit-bearing cuttings, as described in Morales et al. (2016). 1 Cuttings were treated with a solution of indole butyric acid (300 mg/L), kept for several weeks in a hot-bed at 27°C, and placed in a cold room 2 at 5°C, until they developed roots. Then, cuttings were set in pots of 0.8 L with a mixture of 2:1 peat:sand (v/v) and transferred to growth 3 chamber greenhouses (GCGs) (Morales et al. 2014) located at the University of Navarra. Material and methods 10 Dormant cuttings of four clones of Vitis vinifera L. cv. Tempranillo were used in this experiment: three certified clones (CL306, RJ43 and VN31) 12 and one non-commercialised clone (1084). Clones CL306 and RJ43 are two Spanish clones widely distributed during the previous decade (Ibáñez 13 et al. 2015) and considered as short and intermediate cycle variants (Yuste et al. 2001, Estación de Viticultura y Enología de Navarra 2009, 14 Vicente Castro 2012). According to the agronomic characterisation, 5-year study, done by the Instituto Tecnológico Agrario de Castilla y León 15 (Junta de Castilla y León, Valladolid, Spain), the CL306 clone has a budburst to maturity period 5 days shorter than the average of the 16 Tempranillo (Tinta Toro) cultivar, as well as higher potential alcohol concentration (Yuste et al. 2001). In a recent study, RJ43 and CL306 17 showed a complete cycle (budburst–maturity) of 166 and 155 days, respectively, and a flowering–maturity period of 107 and 102 days [1-year 18 study, Instituto de Ciencias de la Vid y del Vino (ICVV), Logroño, Spain]. The plant material of these two clones was provided by Estación de 19 Viticultura y Enología de Navarra (EVENA, Olite, Spain). Vitis Navarra nursery (Larraga, Spain) supplied the VN31, a certified clone 20 characterised (5-year field study, unpublished data, 2016). It is described as a long-phenological development clone, based on its lower potential 21 alcohol concentration at harvest, compared with that of the average of Tempranillo genotypes assessed (García García 2014). The non- 22 commercialised accession 1084 was provided by the Instituto de Ciencias de la Vid y del Vino (ICVV, Logroño, Spain) and it is classified as a 23 long-phenological development clone. This clone showed a complete cycle of 172 days (14 days longer than the average for the cultivar) and a 24 flowering-maturity period of 117 days (11 days longer than the average of the cultivar) (data of a 3-year field study by ICVV). 25 5 6 An adapted protocol from Mullins and Rajasekaran (1981) was used to produce fruit-bearing cuttings, as described 1 Cuttings were treated with a solution of indole butyric acid (300 mg/L), kept for several weeks in a hot-bed at 27°C 2 at 5°C, until they developed roots. Then, cuttings were set in pots of 0.8 L with a mixture of 2:1 peat:sand (v/v) and tra 3 chamber greenhouses (GCGs) (Morales et al. 2014) located at the University of Navarra. Material and methods 10 They grew up to fruitset at 25 4 RH of 50% and natural light supplemented with high-pressure metal halide lamps (POWERSTAR HQI-TS 400W/D P 5 Germany) with a photosynthetic photon flux density of 500 µmol/(m2 · s) at plant level for 15 h a day. Plants were irri 6 solution described in Ollat et al. (1998). Vegetative growth was controlled by manual pruning, and a single flowering s 7 develop on each plant in order to obtain a single berry bunch per plant. Plants were maintained with four leaves up to f 8 and for standardisation purposes, plants of each clone showing similar phenological development were transferred to 7 9 2:1 peat:sand (v/v). 10 Experimental design 11 At fruitset, plants of each of the four clones were transferred to growth chamber greenhouses (GCGs), and divided into 12 groups. Plants were selected to have a similar number of berries. From fruitset to maturity (considered at 23°Brix), pla 13 temperature, CO2 concentration and RH (T/CO2/RH) regimes: climate change conditions (CC: 28°C/18°C, day/night, 7 14 33%/53% RH, day/night) vs current climatic conditions (CS: 24°C/14°C, 400 µmol/mol CO2 and 45%/65% RH). Mor 15 greenhouse were subjected to two water regimes: well-watered (WW) vs water deficit (WD, i.e. 60% of the irrigation r 16 plants). The combination of clones, T/CO2/RH regimes and water availability made a total of 16 treatments with eight 17 The CO2 and temperature conditions in the CC treatment (temperature 4ºC warmer and atmospheric CO2 concentra 18 than in the CS treatment) approached the projections for the year 2100, as per the RCP 8.5 greenhouse emission scenar 19 concentration-driven CMIP5 model simulations (Taylor et al. 2012, Intergovernmental Panel for Climate Change 2013 20 ENSEMBLES models (based on IPCC data), according to the Max Planck Institute model [MPI-ECHAM5 (Roeckner 21 RH for the summer period will likely be 12% lower at the end of the present century in the area of study (Navarra and 22 et al. 2015). For details about the motorisation and control of the environmental parameters within the GCGs, readers a 23 al. (2014). Irrigation regimes were designed following our previous experience with grapevine fruit-bearing cuttings (K 24 Leibar et al. 2015). Material and methods 10 18 Leaf gas exchange and chlorophyll content 19 Net photosynthesis (An), transpiration (T) and stomatal conductance (gs) were measured at mid-veraison and 2 weeks after mi 20 fully expanded leaves (four–eight plants per treatment), using a portable photosynthesis system (LCi-SD with the PLUS5 com 21 ADC BioScientific, Hoddesdon, England). Measurements started 3 h after sunrise and extended over about 3 h. Temperature, 22 conditions used in the measurement chamber corresponded to the respective growth conditions (400 µmol/mol CO2, 24°C and 23 sensors (EC-5 Soil Moisture Sensors, Decagon Devices, Pullman, WA, USA) were placed in the substrate to monitor soil water content. Well- 1 watered plants were maintained at about 90% of the substrate field capacity (sensor value between 40–50%, m3 H2O/100 m3 substrate), 2 equivalent to 400–500 g H2O/L substrate. In the water deficit treatment, plants were subjected to a drought that consisted of withholding 3 irrigation until the soil moisture sensors reached a value of ca. 10% (m3 H2O/100 m3), equivalent to 100 g H2O/L substrate. Then, plants were 4 irrigated with 60% of the volume received by the WW plants during the corresponding drought period. These periods of drought and re-watering 5 were repeated successively throughout the experiment. In order to provide the same amount of nutrients to all the treatments, the WW plants wer 6 irrigated with a nutrient solution (Ollat et al. 1998) alternated with water, whereas WD plants were irrigated only with nutrient solution. The 7 plants were drip irrigated, either with nutrient solution or water with, two drippers of 60 mL/min per pot. Pots had free drainage at the bottom. 8 Pre-dawn leaf water potential 9 Pre-dawn leaf water potential (Ψleaf) was measured at mid-veraison (half of the berries in the bunch had started to change colour) and 2 weeks 10 after mid-veraison (56.0 ± 1.5 and 70.0 ± 1.5 days on average after fruitset, respectively), in young fully expanded leaves (three–five plants per 11 clone and treatment), using a pressure chamber (SKYE SKPM 1400, Skye Instruments, Llandrindod, Wales) and according to the methodology 12 described by Scholander et al. (1965). 13 The number of days between fruitset (E-L 27) and maturity (TSS of ca. 23°Brix, E-L 38) (Coombe 1995) was determined. Fruitset was assessed 15 visually (young berries between 1–2 mm diameter). Material and methods 10 Irrigation regimes were designed following our previous experience with grapevine fruit-bearing cuttings (Kizildeniz et al. 2015, 24 Leibar et al. 2015). The water deficit level was chosen to match conditions predicted for the end of the present century in the regions of Navarra 25 and La Rioja by the model of the Max Planck Institute, that is likely 40% lower precipitation in the summer (Leibar et al. 2015). Soil water 26 6 sensors (EC-5 Soil Moisture Sensors, Decagon Devices, Pullman, WA, USA) were placed in the substrate to monitor soil wat 1 watered plants were maintained at about 90% of the substrate field capacity (sensor value between 40–50%, m3 H2O/100 m3 s 2 equivalent to 400–500 g H2O/L substrate. In the water deficit treatment, plants were subjected to a drought that consisted of w 3 irrigation until the soil moisture sensors reached a value of ca. 10% (m3 H2O/100 m3), equivalent to 100 g H2O/L substrate. T 4 irrigated with 60% of the volume received by the WW plants during the corresponding drought period. These periods of drou 5 were repeated successively throughout the experiment. In order to provide the same amount of nutrients to all the treatments, 6 irrigated with a nutrient solution (Ollat et al. 1998) alternated with water, whereas WD plants were irrigated only with nutrien 7 plants were drip irrigated, either with nutrient solution or water with, two drippers of 60 mL/min per pot. Pots had free draina 8 Pre-dawn leaf water potential 9 Pre-dawn leaf water potential (Ψleaf) was measured at mid-veraison (half of the berries in the bunch had started to change colo 10 after mid-veraison (56.0 ± 1.5 and 70.0 ± 1.5 days on average after fruitset, respectively), in young fully expanded leaves (thr 11 clone and treatment), using a pressure chamber (SKYE SKPM 1400, Skye Instruments, Llandrindod, Wales) and according to 12 described by Scholander et al. (1965). 13 Phenological development 14 The number of days between fruitset (E-L 27) and maturity (TSS of ca. 23°Brix, E-L 38) (Coombe 1995) was determined. Fru 15 visually (young berries between 1–2 mm diameter). Maturity was established by sampling periodically two berries (from the 16 portion of the bunch, which allocate the highest number of berries) and measuring the TSS in the must using a refractometer ( 17 315RS, Zuzi, Beriain, Spain). Every plant was assessed individually. Material and methods 10 Maturity was established by sampling periodically two berries (from the top and middle 16 portion of the bunch, which allocate the highest number of berries) and measuring the TSS in the must using a refractometer (Abbe Digital 17 315RS, Zuzi, Beriain, Spain). Every plant was assessed individually. 18 Net photosynthesis (An), transpiration (T) and stomatal conductance (gs) were measured at mid-veraison and 2 weeks after mid-veraison in young 20 fully expanded leaves (four–eight plants per treatment), using a portable photosynthesis system (LCi-SD with the PLUS5 compact light unit, 21 ADC BioScientific, Hoddesdon, England). Measurements started 3 h after sunrise and extended over about 3 h. Temperature, CO2 and RH 22 conditions used in the measurement chamber corresponded to the respective growth conditions (400 µmol/mol CO2, 24°C and 45% RH for CS 23 7 condition, and 700 µmol/mol CO2, 28°C and 35% RH for CC condition). The photosynthetic photon flux d 1 With the values of gas exchange parameters measured, intrinsic and instantaneous water use efficiencies we 2 An/T (WUEinst), respectively. 3 The concentration of chlorophyll was assessed in young, fully expanded leaves by non-destructive fluor 4 multiparametric portable optical sensor (Multiplex_Research, FORCE-A, Orsay, France). Chlorophyll conc 5 resulting from the ratio of far-red to red fluorescence (SFR_R index) (Gitelson et al. 1999). 6 Plant growth and fruit production 7 Leaf area was determined at mid-veraison, 2 weeks after mid-veraison and maturity, by measuring the shoo 8 adapted for Tempranillo by Arrizabalaga et al. (2018). The model relates leaf area measured using a leaf ar 9 Biosciences, Lincoln, NE, USA) (y) and total shoot length (x): leaf area (dm2) = 13.859x + 200.33; R2 = 0. 10 vegetative production, expressed as dry matter mass, was measured at maturity by weighing the oven-dried 11 was done by placing the plant material in an oven at 80°C until constant mass. The fresh mass of individual 12 ripening process (mid-veraison, 1 week after mid-veraison, 2 weeks after mid-veraison and maturity). At m 13 number of berries per bunch were determined. 14 Statistical analysis 15 The software used for the statistical analysis was R (version 3.5.1, Lucent Technologies, Murray Hill, NJ, U 16 three-way ANOVA (three factors: clone, T/CO2/RH regime and water availability) in order to determine th 17 possible interactions. The Fisher’s least significant difference (LSD) test was used as a post-hoc. Results w 18 at P<0.05. Material and methods 10 Results were also analysed using a principal component analysis (PCA). 19 Results 20 condition, and 700 µmol/mol CO2, 28°C and 35% RH for CC condition). The photosynthetic photon flux density was set at 1200 µmol/(m2 · s). 1 With the values of gas exchange parameters measured, intrinsic and instantaneous water use efficiencies were calculated as An/gs (WUEi) and 2 An/T (WUEinst), respectively. 3 With the values of gas exchange parameters measured, intrinsic and instantaneous water use efficiencies were calculated as An/gs (WU 2 An/T (WUEinst), respectively. 3 The concentration of chlorophyll was assessed in young, fully expanded leaves by non-destructive fluorescence measurements, using a 4 multiparametric portable optical sensor (Multiplex_Research, FORCE-A, Orsay, France). Chlorophyll concentration is correlated to a parameter 5 resulting from the ratio of far-red to red fluorescence (SFR_R index) (Gitelson et al. 1999). 6 Leaf area was determined at mid-veraison, 2 weeks after mid-veraison and maturity, by measuring the shoot length, and using a regression model 8 adapted for Tempranillo by Arrizabalaga et al. (2018). The model relates leaf area measured using a leaf area meter (LI-300 model; Li-COR 9 Biosciences, Lincoln, NE, USA) (y) and total shoot length (x): leaf area (dm2) = 13.859x + 200.33; R2 = 0.9239 (x = shoot length). The 10 vegetative production, expressed as dry matter mass, was measured at maturity by weighing the oven-dried leaves, stem and roots. The drying 11 was done by placing the plant material in an oven at 80°C until constant mass. The fresh mass of individual berries was measured throughout the 12 ripening process (mid-veraison, 1 week after mid-veraison, 2 weeks after mid-veraison and maturity). At maturity, the fresh bunch mass and the 13 number of berries per bunch were determined. 14 The software used for the statistical analysis was R (version 3.5.1, Lucent Technologies, Murray Hill, NJ, USA). Data were first analysed using a 6 three-way ANOVA (three factors: clone, T/CO2/RH regime and water availability) in order to determine the effects of the treatments and their 7 possible interactions. The Fisher’s least significant difference (LSD) test was used as a post-hoc. Results were considered statistically significant 8 at P<0.05. Results were also analysed using a principal component analysis (PCA). 9 The software used for the statistical analysis was R (version 3.5.1, Lucent Technologies, Murray Hill, NJ, USA). Leaf gas exchange parameters and photosynthetic pigments 5 The clones showed similar An, T and gs values at mid-veraison, considering all the T/CO2/RH and water regime situations altogether (PCL values 6 of 0.295, 0.222 and 0.160 for An, T, and gs, respectively). Nevertheless, under WW conditions, 1084 tended to have lower An, T and, especially, gs 7 values than the other clones (Figure 2a). Two weeks after mid-veraison, a significant interaction between clone and irrigation level was observed 8 for An, T and gs. At this time, comparing the four clones under WW conditions, the 1084 accession showed significantly lower An and T compared 9 with RJ43 and VN31 (CS conditions) and with VN31 (CC conditions), as well as the lowest gs values among the four clones (CS conditions). 10 These differences, however, disappeared under WD conditions, which drastically reduced the values of gas exchange parameters. Considering the 11 clones altogether, CC conditions enhanced significantly An at mid-veraison, regardless of the water regime applied (PT/CO2/RH=0.001, Figure 2b). 12 Two weeks after mid-veraison, differences in the An between CS and CC plants were maintained in WW plants, but these disappeared under WD 13 (significant interaction between T/CO2/RH and water availability). This effect was especially evident in VN31 and 1084 (Figure 2a). Stomatal 14 conductance was reduced by CC conditions in WW plants, both at mid-veraison and 2 weeks later, but these differences disappeared under WD, 15 with a significant interaction between T/CO2/RH and water availability. The clones studied showed some variability in their gas exchange 16 response to climate change both at mid-veraison and 2 weeks later, 1084 showing the greatest increase in An and minor changes in gs between CC 17 and CS compared with the other clones (Figure 2a). 18 Clones did not differ systematically either in WUEi (An/gs) or WUEinst (An/T), but 2 weeks after mid-veraison, plants under WD of CL306 19 and VN31 accessions showed higher WUEinst values (CS and CC, respectively) (Table 1). Water deficit significantly increased both WUEi and 20 WUEinst at mid-veraison and 2 weeks after mid-veraison, regardless of the clone and the T/CO2/RH regime. Climate change conditions 21 significantly increased WUEi, especially under WD (significant interaction observed 2 weeks after mid-veraison), but they did not affect WUEinst. 22 The SFR_R index, parameter correlated with the leaf chlorophyll concentration, did not show significant differences among clones either at 23 mid-veraison (PCL=0.334) or 2 weeks after mid-veraison (PCL=0.253) (data not shown). Material and methods 10 Data were first analysed using a 6 three-way ANOVA (three factors: clone, T/CO2/RH regime and water availability) in order to determine the effects of the treatments and their 7 possible interactions. The Fisher’s least significant difference (LSD) test was used as a post-hoc. Results were considered statistically significant 8 at P<0.05. Results were also analysed using a principal component analysis (PCA). 9 8 Pre-dawn leaf water potential 1 Clones had similar Ψleaf values (PCL=0.243 at mid-veraison and PCL=0.765 2 weeks after mid-veraison). Water deficit (WD) significantly reduced 2 Ψleaf compared with vines grown under WW conditions (Figure 1). A significant interaction between the T/CO2/RH and irrigation regimes was 3 observed at mid-veraison, when, under WD conditions, CS plants had lower Ψleaf than the CC vines. 4 Clones had similar Ψleaf values (PCL=0.243 at mid-veraison and PCL=0.765 2 weeks after mid-veraison). Water deficit (WD) significantly reduced 2 Ψleaf compared with vines grown under WW conditions (Figure 1). A significant interaction between the T/CO2/RH and irrigation regimes was 3 observed at mid-veraison, when, under WD conditions, CS plants had lower Ψleaf than the CC vines. 4 Leaf gas exchange parameters and photosynthetic pigments 5 2 Considering the clones altogether, CC conditions significantly shortened the elapsed time between fruitset and maturity (9 days on average), 4 especially under WD (up to 13 days), with slight differences among clones (VN31 being less impacted under WW) (Figure 3a). A significant 5 interaction was observed between clones and water availability, where WD significantly delayed maturity in RJ43, CL306 and VN31 clones, but 6 not in the 1084 accession (Figure 3b). The elapsed time between fruitset and maturity was significantly different among clones, 1084 showing a 7 grape developmental period 25.8 days longer on average than that of RJ43, CL306 and VN31 clones (Figure 3b). 8 A significant interaction between clone and irrigation level was observed for total leaf area measured at mid-veraison, 2 weeks after mid-veraison 10 and at maturity, as leaf area was significantly reduced by WD throughout the experiment, with this reduction being more noticeable in the 1084 11 accession (Figure 4a). Clones showed significant differences in total leaf area at maturity in WW plants (higher in 1084), but these differences 12 disappeared under WD conditions. The T/CO2/RH regime also interacted with the irrigation level, and CC conditions significantly reduced leaf 13 area at mid-veraison and 2 weeks after mid-veraison, compared with CS, only in WW plants (Figure 4b). No differences between T/CO2/RH 14 regimes were observed at maturity (Figure 4b). 15 Climate change conditions increased total leaf dry mass at maturity, when considering all the clones as a whole, but especially in VN31, but it 16 did not affect either stem or root growth (Figure 5a,b). Clones showed significant differences in the final dry matter production of the different 17 organs analysed, 1084 having the highest dry mass under WW conditions (Figure 5b). Water deficit reduced the final dry mass of all the organs, 18 regardless of the T/CO2/RH regime applied. There was a significant interaction between water deficit and clone for leaves, stem and total dry 19 mass, with clone 1084 being the most negatively affected by drought (Figure 5b). 20 No significant interactions among factors were observed for yield and grape characteristics (Table 2). The RJ43 accession had the highest bunch 22 mass and number of berries per bunch, whereas 1084 had higher individual berry mass during the ripening period (significant differences with 23 VN31, which had the lowest values). Leaf gas exchange parameters and photosynthetic pigments 5 The RJ43 accessi 22 mass and number of berries per bunch, whereas 1084 had higher individual berry mass during the ripening period (signi 23 VN31, which had the lowest values). Climate change conditions did not affect either grape yield or yield components at 24 SFR_R index compared with CS (2.09 ± 0.05 and 2.21 ± 0.03 relative units, CS a 1 and the irrigation regime. 2 Phenological development 3 Considering the clones altogether, CC conditions significantly shortened the elaps 4 especially under WD (up to 13 days), with slight differences among clones (VN3 5 interaction was observed between clones and water availability, where WD signif 6 not in the 1084 accession (Figure 3b). The elapsed time between fruitset and matu 7 grape developmental period 25.8 days longer on average than that of RJ43, CL30 8 Total leaf area and vegetative growth 9 A significant interaction between clone and irrigation level was observed for total 10 and at maturity, as leaf area was significantly reduced by WD throughout the expe 11 accession (Figure 4a). Clones showed significant differences in total leaf area at m 12 disappeared under WD conditions. The T/CO2/RH regime also interacted with the 13 area at mid-veraison and 2 weeks after mid-veraison, compared with CS, only in W 14 regimes were observed at maturity (Figure 4b). 15 Climate change conditions increased total leaf dry mass at maturity, when con 16 did not affect either stem or root growth (Figure 5a,b). Clones showed significant 17 organs analysed, 1084 having the highest dry mass under WW conditions (Figure 18 regardless of the T/CO2/RH regime applied. There was a significant interaction be 19 mass, with clone 1084 being the most negatively affected by drought (Figure 5b). 20 Bunch characteristics 21 No significant interactions among factors were observed for yield and grape chara 22 mass and number of berries per bunch, whereas 1084 had higher individual berry 23 VN31, which had the lowest values). Climate change conditions did not affect eit 24 SFR_R index compared with CS (2.09 ± 0.05 and 2.21 ± 0.03 relative units, CS and CC, respectively, PT/CO2/RH=0.042), regardless of the clone 1 and the irrigation regime. 2 SFR_R index compared with CS (2.09 ± 0.05 and 2.21 ± 0.03 relative units, CS and CC, respectively, PT/CO2/RH=0.042), regardless of the clone 1 and the irrigation regime. Leaf gas exchange parameters and photosynthetic pigments 5 The RJ43 access 22 mass and number of berries per bunch, whereas 1084 had higher individual berry mass during the ripening period (sign 23 VN31, which had the lowest values). Climate change conditions did not affect either grape yield or yield components a 24 SFR_R index compared with CS (2.09 ± 0.05 and 2.21 ± 0.03 relative units, CS and CC, respectively, PT/CO2/RH=0.042) 1 and the irrigation regime. 2 Phenological development 3 Considering the clones altogether, CC conditions significantly shortened the elapsed time between fruitset and maturity 4 especially under WD (up to 13 days), with slight differences among clones (VN31 being less impacted under WW) (Fig 5 interaction was observed between clones and water availability, where WD significantly delayed maturity in RJ43, CL3 6 not in the 1084 accession (Figure 3b). The elapsed time between fruitset and maturity was significantly different among 7 grape developmental period 25.8 days longer on average than that of RJ43, CL306 and VN31 clones (Figure 3b). 8 Total leaf area and vegetative growth 9 A significant interaction between clone and irrigation level was observed for total leaf area measured at mid-veraison, 2 10 and at maturity, as leaf area was significantly reduced by WD throughout the experiment, with this reduction being mor 11 accession (Figure 4a). Clones showed significant differences in total leaf area at maturity in WW plants (higher in 1084 12 disappeared under WD conditions. The T/CO2/RH regime also interacted with the irrigation level, and CC conditions si 13 area at mid-veraison and 2 weeks after mid-veraison, compared with CS, only in WW plants (Figure 4b). No difference 14 regimes were observed at maturity (Figure 4b). 15 Climate change conditions increased total leaf dry mass at maturity, when considering all the clones as a whole, but 16 did not affect either stem or root growth (Figure 5a,b). Clones showed significant differences in the final dry matter pro 17 organs analysed, 1084 having the highest dry mass under WW conditions (Figure 5b). Water deficit reduced the final dr 18 regardless of the T/CO2/RH regime applied. There was a significant interaction between water deficit and clone for leav 19 mass, with clone 1084 being the most negatively affected by drought (Figure 5b). 20 Bunch characteristics 21 No significant interactions among factors were observed for yield and grape characteristics (Table 2). Leaf gas exchange parameters and photosynthetic pigments 5 Water deficit significantly increased the chlorophyll 24 index (PI=0.001) from 2.05 ± 0.05 to 2.25 ± 0.03, WW and WD, respectively. Two weeks after mid-veraison, CC significantly increased the 25 9 SFR_R index compared with CS (2.09 ± 0.05 and 2.21 ± 0.03 relative units, CS and CC, respectively, PT/CO2/RH=0.042) 1 and the irrigation regime. 2 Phenological development 3 Considering the clones altogether, CC conditions significantly shortened the elapsed time between fruitset and maturity 4 especially under WD (up to 13 days), with slight differences among clones (VN31 being less impacted under WW) (Fi 5 interaction was observed between clones and water availability, where WD significantly delayed maturity in RJ43, CL3 6 not in the 1084 accession (Figure 3b). The elapsed time between fruitset and maturity was significantly different among 7 grape developmental period 25.8 days longer on average than that of RJ43, CL306 and VN31 clones (Figure 3b). 8 Total leaf area and vegetative growth 9 A significant interaction between clone and irrigation level was observed for total leaf area measured at mid-veraison, 2 10 and at maturity, as leaf area was significantly reduced by WD throughout the experiment, with this reduction being mo 11 accession (Figure 4a). Clones showed significant differences in total leaf area at maturity in WW plants (higher in 1084 12 disappeared under WD conditions. The T/CO2/RH regime also interacted with the irrigation level, and CC conditions s 13 area at mid-veraison and 2 weeks after mid-veraison, compared with CS, only in WW plants (Figure 4b). No difference 14 regimes were observed at maturity (Figure 4b). 15 Climate change conditions increased total leaf dry mass at maturity, when considering all the clones as a whole, bu 16 did not affect either stem or root growth (Figure 5a,b). Clones showed significant differences in the final dry matter pro 17 organs analysed, 1084 having the highest dry mass under WW conditions (Figure 5b). Water deficit reduced the final d 18 regardless of the T/CO2/RH regime applied. There was a significant interaction between water deficit and clone for leav 19 mass, with clone 1084 being the most negatively affected by drought (Figure 5b). 20 Bunch characteristics 21 No significant interactions among factors were observed for yield and grape characteristics (Table 2). Leaf gas exchange parameters and photosynthetic pigments 5 Oth 23 improvement of whole plant water relations induced by elevated CO2, through more effective water uptake (fin 24 xylem conductivity (Wullschleger et al. 2002), are possible. Also, the differences in air temperature between CC 25 differences observed in Ψleaf. In a recent study, Galat Giorgi et al. (2019) reported higher leaf water potential va 26 treatment had the lowest grape mass values at mid-veraison. The bunch mass, number of berries per bunch and individual berry mass were 1 significantly reduced by WD. Even though there were no significant interactions between factors, the CL306 clone experienced the strongest 2 reduction in bunch mass as a consequence of WD, especially under CC conditions (reduction of 63%), compared with the remainder of the clones 3 (decreases of 24, 14 and 25% in RJ43, VN31 and 1084, respectively). 4 Phenology, gas exchange characteristics and vegetative and reproductive growth parameters were analysed by principal component analysis. The 6 first two principal components (PC) explained more than 75% of the total variability (Figure 6). Differences between water availability were 7 clearly observed along PC1, and they were mainly associated with lower values of vegetative growth (dry mass and total leaf area), leaf water 8 potential, and gas exchange parameters (An, T and gs), as well as with higher WUE in the WD treatment. Under WW conditions, the 1084 9 accession was separated from the rest of the clones along PC2, regardless of the T/CO2/RH regime, due to a longer fruitset to maturity period and 10 a lower bunch size (fresh mass and number of berries). In contrast, these differences among clones were less evident under water deficit 11 conditions. 12 Discussion 13 It has been proposed that elevated CO2 can improve 18 water use efficiency under drought conditions, through the reduction of gs and, therefore, T values (Tyree and Alexander 1993, Wullschleger et 19 al. 2002). Even though CC conditions reduced gs under well-watered conditions, gs was similar in CC and CS treatments under water deficit. 20 Also, leaf transpiration was even higher in CC (significant at mid-veraison), likely due to a higher vapour pressure deficit (VPD) in this treatment 21 (VPD of 1.51 vs 1.19 kPa, CC and CS, respectively) associated with higher temperature and lower RH values. The higher Ψleaf in CC/WD plants 22 compared with CS/WD was not likely related to differences in root development between these treatments. Other factors, such as the 23 improvement of whole plant water relations induced by elevated CO2, through more effective water uptake (fine-root proliferation) or increased 24 xylem conductivity (Wullschleger et al. 2002), are possible. Also, the differences in air temperature between CC and CS may be behind the 25 differences observed in Ψleaf. In a recent study, Galat Giorgi et al. (2019) reported higher leaf water potential values in grapevine plants exposed 26 The water status of all the clones studied was affected in a similar manner by WD both at mid-veraison and maturity, droughted plants reaching 15 lower Ψleaf (more negative) than their respective WW counterparts. Values of Ψleaf were in the range of previous experiments using fruit-bearing 16 cuttings and similar water deficit levels (Leibar et al. 2015). Climate change conditions did not significantly affect Ψleaf of WW plants, but in the 17 case of WD, plants grown in CS conditions showed lower Ψleaf compared with CC plants. It has been proposed that elevated CO2 can improve 18 water use efficiency under drought conditions, through the reduction of gs and, therefore, T values (Tyree and Alexander 1993, Wullschleger et 19 al. 2002). Even though CC conditions reduced gs under well-watered conditions, gs was similar in CC and CS treatments under water deficit. 20 Also, leaf transpiration was even higher in CC (significant at mid-veraison), likely due to a higher vapour pressure deficit (VPD) in this treatment 21 (VPD of 1.51 vs 1.19 kPa, CC and CS, respectively) associated with higher temperature and lower RH values. The higher Ψleaf in CC/WD plants 22 compared with CS/WD was not likely related to differences in root development between these treatments. Leaf gas exchange parameters and photosynthetic pigments 5 Climate change conditions did not affect either grape yield or yield components at maturity, although this 24 10 11 treatment had the lowest grape mass values at mid-veraison. The bunch mass, number of berries per bunch and 1 significantly reduced by WD. Even though there were no significant interactions between factors, the CL306 cl 2 reduction in bunch mass as a consequence of WD, especially under CC conditions (reduction of 63%), compare 3 (decreases of 24, 14 and 25% in RJ43, VN31 and 1084, respectively). 4 Principal component analysis 5 Phenology, gas exchange characteristics and vegetative and reproductive growth parameters were analysed by p 6 first two principal components (PC) explained more than 75% of the total variability (Figure 6). Differences bet 7 clearly observed along PC1, and they were mainly associated with lower values of vegetative growth (dry mass 8 potential, and gas exchange parameters (An, T and gs), as well as with higher WUE in the WD treatment. Under 9 accession was separated from the rest of the clones along PC2, regardless of the T/CO2/RH regime, due to a lon 10 a lower bunch size (fresh mass and number of berries). In contrast, these differences among clones were less ev 11 conditions. 12 Discussion 13 Response of leaf water potential and leaf gas exchange traits of Tempranillo clones to changes in the T/CO2/RH 14 The water status of all the clones studied was affected in a similar manner by WD both at mid-veraison and mat 15 lower Ψleaf (more negative) than their respective WW counterparts. Values of Ψleaf were in the range of previous 16 cuttings and similar water deficit levels (Leibar et al. 2015). Climate change conditions did not significantly aff 17 case of WD, plants grown in CS conditions showed lower Ψleaf compared with CC plants. It has been proposed 18 water use efficiency under drought conditions, through the reduction of gs and, therefore, T values (Tyree and A 19 al. 2002). Even though CC conditions reduced gs under well-watered conditions, gs was similar in CC and CS tr 20 Also, leaf transpiration was even higher in CC (significant at mid-veraison), likely due to a higher vapour press 21 (VPD of 1.51 vs 1.19 kPa, CC and CS, respectively) associated with higher temperature and lower RH values. T 22 compared with CS/WD was not likely related to differences in root development between these treatments. Discussion 13 Response of leaf water potential and leaf gas exchange traits of Tempranillo clones to changes in the T/CO2/RH conditions and water availability 14 The water status of all the clones studied was affected in a similar manner by WD both at mid-veraison and maturity, droughted plants reaching 15 lower Ψleaf (more negative) than their respective WW counterparts. Values of Ψleaf were in the range of previous experiments using fruit-bearing 16 cuttings and similar water deficit levels (Leibar et al. 2015). Climate change conditions did not significantly affect Ψleaf of WW plants, but in the 17 case of WD, plants grown in CS conditions showed lower Ψleaf compared with CC plants. It has been proposed that elevated CO2 can improve 18 water use efficiency under drought conditions, through the reduction of gs and, therefore, T values (Tyree and Alexander 1993, Wullschleger et 19 al. 2002). Even though CC conditions reduced gs under well-watered conditions, gs was similar in CC and CS treatments under water deficit. 20 Also, leaf transpiration was even higher in CC (significant at mid-veraison), likely due to a higher vapour pressure deficit (VPD) in this treatment 21 (VPD of 1.51 vs 1.19 kPa, CC and CS, respectively) associated with higher temperature and lower RH values. The higher Ψleaf in CC/WD plants 22 compared with CS/WD was not likely related to differences in root development between these treatments. Other factors, such as the 23 improvement of whole plant water relations induced by elevated CO2, through more effective water uptake (fine-root proliferation) or increased 24 xylem conductivity (Wullschleger et al. 2002), are possible. Also, the differences in air temperature between CC and CS may be behind the 25 differences observed in Ψleaf. In a recent study, Galat Giorgi et al. (2019) reported higher leaf water potential values in grapevine plants exposed 26 The water status of all the clones studied was affected in a similar manner by WD both at mid-veraison and maturity, droughted plants reaching 15 lower Ψleaf (more negative) than their respective WW counterparts. Values of Ψleaf were in the range of previous experiments using fruit-bearing 16 cuttings and similar water deficit levels (Leibar et al. 2015). Climate change conditions did not significantly affect Ψleaf of WW plants, but in the 17 case of WD, plants grown in CS conditions showed lower Ψleaf compared with CC plants. Discussion 13 Other factors, such as the 23 improvement of whole plant water relations induced by elevated CO2, through more effective water uptake (fine-root proliferation) or increased 24 xylem conductivity (Wullschleger et al. 2002), are possible. Also, the differences in air temperature between CC and CS may be behind the 25 differences observed in Ψleaf. In a recent study, Galat Giorgi et al. (2019) reported higher leaf water potential values in grapevine plants exposed 26 11 to elevated temperature (45/22°C) and water deficit, compared with their control temperature counterparts (35/20°C). The authors suggested that 1 the control treatment may have kept their stomata open longer during the day, leading to a greater decrease in water potential. 2 Clone 1084 had the lowest An values among the four clones studied. The 1084 plants grown under CS/WW conditions had a significantly 3 lower An and gs compared with other clones under the same T/CO2/RH regime and water availability conditions, 2 weeks after mid-veraison (70.0 4 ± 1.5 days on average after fruitset, that is, from the beginning of the treatments). These differences were not associated with the concentration of 5 leaf chlorophyll. Leaf photosynthesis in grapevine depends upon demand for assimilates and it is regulated by the source: sink relationship (Iland 6 et al. 2011). Reduced stomatal conductance has been proposed as a regulatory mechanism in such relationships in different plant species, 7 including grapevine (Quereix et al. 2001, Blanke 2009). The low fruit load (bunch mass) accompanied by a high leaf development of 1084 may 8 explain such lower photosynthetic activity. Variability for An among grapevine cultivars has been previously reported (Bota et al. 2001, Tomás et 9 al. 2014, Greer 2018). Greer (2018) attributed, in part, such variability to differences in stomatal conductance, as in the present study, rather than 10 to biochemical factors, such as RUBP carboxylation and regeneration. Potential mesophyll diffusion limitations may also explain these 11 differences, although they remain largely unexplored in Tempranillo clones (Salazar-Parra et al. 2012). 12 including grapevine (Quereix et al. 2001, Blanke 2009). The low fruit load (bunch mass) accompanied by a high leaf development of 1084 may 8 explain such lower photosynthetic activity. Variability for An among grapevine cultivars has been previously reported (Bota et al. 2001, Tomás et 9 al. 2014, Greer 2018). Discussion 13 Despite the absence of significant interaction between clone identity and T/CO2/RH 27 12 regime, it is important to note that, under WW conditions the 1084 accession exhibited a more pronounced increase in An in response to CC 1 compared with the remaining clones studied, both at mid-veraison and 2 weeks later. These results may reflect some differences among clones in 2 their photosynthetic response to the projected environmental conditions. 3 Within the context of the IPCC predictions for a decrease in water availability, improved crop water use efficiency has become a priority 4 in basic and applied research in recent years (Tortosa et al. 2019). Although clonal variability of the WUEi has been reported for cv. Tempranillo 5 (Tortosa et al. 2016, 2019), considering all the T/CO2/RH and irrigation regimes, we did not observe significant differences in WUEi and WUEinst 6 among the clones considered in the present work. The results agree with Tortosa et al. (2016, 2019), who also did not observe differences among 7 RJ43, VN31 and 1084 either under field or pot conditions. Focusing on drought conditions, however, CL306 and VN31 showed higher values of 8 WUEinst than the other accessions (CS and CC, respectively) 2 weeks after veraison. The result suggests that under long-term water deficit, and 9 depending on the T/CO2/RH regime, these Tempranillo accessions may exhibit improved WUE. 10 Considering all the clones as a whole, both WD and CC significantly increased WUEi, which was associated with a drop in gs values in the 11 first case, and with a higher photosynthetic capacity and lower gs, in the second case. The improvement in WUEi was especially remarkable when 12 these two environmental conditions were combined (CC/WD), suggesting that, in a future environment with high CO2 and elevated temperature, 13 grapevine WUEi may be improved under drought conditions. When leaf transpiration, however, was used to calculate WUEinst, the gain in water 14 use efficiency of plants under CC conditions disappeared. That is because the reduced gs observed under CC was not accompanied by low 15 transpiration, likely due to the higher vapour pressure deficit under these conditions. Therefore, the increase in water vapour concentration 16 difference between leaf and air, as a consequence of the projected air temperature and RH conditions, would largely offset the potential gain in 17 WUE produced by elevated CO2 (Kaminski et al. 2014). Discussion 13 These results highlight the importance of studying the combined effect of CO2 with 18 other climate change factors (e.g. changes in temperature or precipitation), which may modulate the photosynthetic and water use efficiency 19 response of plants to CO2. In addition, the WUEinst appears to be a more suitable parameter to estimate the photosynthetic water use efficiency 20 under environmental conditions that modify vapour pressure deficit. 21 Discussion 13 Greer (2018) attributed, in part, such variability to differences in stomatal conductance, as in the present study, rather than 10 to biochemical factors, such as RUBP carboxylation and regeneration. Potential mesophyll diffusion limitations may also explain these 11 differences, although they remain largely unexplored in Tempranillo clones (Salazar-Parra et al. 2012). 12 The impact of water deficit on grapevine photosynthesis performance has been extensively studied (Flexas et al. 1998, Medrano et al. 13 2003, Chaves et al. 2010). In the present study, water availability was the factor that most affected gas exchange parameters, reducing An values 14 in all the clones studied, and overshadowed the differences among clones observed in WW plants. Such reduction in carbon assimilation was 15 presumably related to a decrease in CO2 availability when plants closed stomata to prevent water loss, as supported by the reduction in gs. Under 16 mild to moderate water deficits, stomata closure is among the earliest plant responses, restricting water loss and carbon fixation (Chaves et al. 17 2003) 18 Considering all the clones, CC conditions increased An at mid-veraison, regardless of the water availability, thus partially compensating 19 the impact of WD. Unfortunately, we cannot attribute this effect either to CO2 or temperature, but it is likely that CO2 was the main factor 20 affecting photosynthetic rates under the present experimental conditions. Grapevine photosynthesis, as in other C3 species, is limited by CO2, and 21 therefore high CO2 has been reported to increase An (Moutinho-Pereira et al. 2009, Salazar-Parra et al. 2015). Such increase has been recently 22 associated with the up-regulation of Rubisco small chain and Rubisco activase proteins (Zhao et al. 2019). In addition, previous studies in 23 different grapevine cultivars, grown in both natural and controlled environments, show that changes in temperature in the range of the present 24 work (24–28°C) did not significantly affect grapevine photosynthesis (Greer 2018). Two weeks after mid-veraison, a significant interaction 25 between T/CO2/RH regime and irrigation regime was observed, and the positive effect of CC in WW plants was completely diminished by 26 drought, in agreement with the studies of Leibar et al. (2015). ogical response of Tempranillo clones to changes in the T/CO2/RH conditions and wate This is in accordance with previous studies under controlled and semi-controlled conditions (Leibar et al. 2015, Martínez- 9 Lüscher et al. 2016). From mid-veraison onwards, the majority of photoassimilates is directed to berry maturation (Lebon et al. 2008). Therefore, 10 the advancement of ripening in plants grown under CC conditions indicates faster sugar accumulation in grapes, associated with the higher 11 photosynthetic rates measured in CC compared with CS (both in WW and WD at mid-veraison, and in WW 2 weeks later). 12 (Duchêne 2016), especially the early phenological events. For later phenological events, however, the level of complexity increases and other 1 environmental factors may also influence the timing of phenophases (Martínez-Lüscher et al. 2016). In the present study, water availability was 2 the factor that most strongly affected grape development. The severe reduction in An and total leaf area in WD plants probably limited carbon 3 availability, thus slowing down grape ripening in these treatments. Whereas mild water deficit has been proven to enhance ripening, severe water 4 deficit has been reported to reduce carbon fixation, and consequently, to impair berry ripening (Chaves et al. 2010, Martínez-Lüscher et al. 5 2015a). In addition, the interaction between clone and irrigation reveals a differential response of the studied genotypes to water deficit, 1084 6 being the least responsive clone. This result may be explained by the lower difference in the An between WW and WD conditions observed in this 7 accession. In contrast to WD, CC advanced grape maturity when all the clones were taken into consideration, and compensated for the delaying 8 effect due to drought. This is in accordance with previous studies under controlled and semi-controlled conditions (Leibar et al. 2015, Martínez- 9 Lüscher et al. 2016). From mid-veraison onwards, the majority of photoassimilates is directed to berry maturation (Lebon et al. 2008). Therefore, 10 the advancement of ripening in plants grown under CC conditions indicates faster sugar accumulation in grapes, associated with the higher 11 photosynthetic rates measured in CC compared with CS (both in WW and WD at mid-veraison, and in WW 2 weeks later). 12 Vegetative and reproductive response of Tempranillo clones to changes in the T/CO2/RH conditions and water availability 13 The higher photosynthetic rates of plants grown under CC conditions were associated with increased leaf dry mass (only under WW conditions), 14 with minor effects on reproductive growth. Kizildeniz et al. ogical response of Tempranillo clones to changes in the T/CO2/RH conditions and wate Differences in the timing of maturity among Tempranillo clones have been reported in previous studies, the 1084 accession being characterised as 23 having a long phenological development (fruitset–maturity period 22.7 days longer than the average of the clones assessed) (Arrizabalaga et al. 24 2018). Such behaviour, however, could not be completely explained in the present study by a lower photosynthetic activity, although the 25 differences observed may have partially contributed to the delayed maturity in 1084. Grapevine phenology is greatly influenced by temperature 26 13 (Duchêne 2016), especially the early phenological events. For later phenological events, however, the level of complexity increa 1 environmental factors may also influence the timing of phenophases (Martínez-Lüscher et al. 2016). In the present study, water 2 the factor that most strongly affected grape development. The severe reduction in An and total leaf area in WD plants probably li 3 availability, thus slowing down grape ripening in these treatments. Whereas mild water deficit has been proven to enhance ripen 4 deficit has been reported to reduce carbon fixation, and consequently, to impair berry ripening (Chaves et al. 2010, Martínez-Lü 5 2015a). In addition, the interaction between clone and irrigation reveals a differential response of the studied genotypes to water 6 being the least responsive clone. This result may be explained by the lower difference in the An between WW and WD condition 7 accession. In contrast to WD, CC advanced grape maturity when all the clones were taken into consideration, and compensated f 8 effect due to drought. This is in accordance with previous studies under controlled and semi-controlled conditions (Leibar et al. 9 Lüscher et al. 2016). From mid-veraison onwards, the majority of photoassimilates is directed to berry maturation (Lebon et al. 2 10 the advancement of ripening in plants grown under CC conditions indicates faster sugar accumulation in grapes, associated with 11 photosynthetic rates measured in CC compared with CS (both in WW and WD at mid-veraison, and in WW 2 weeks later). 12 Vegetative and reproductive response of Tempranillo clones to changes in the T/CO2/RH conditions and water availability 13 The higher photosynthetic rates of plants grown under CC conditions were associated with increased leaf dry mass (only under W 14 with minor effects on reproductive growth. Kizildeniz et al. (2018) presented similar results in two Tempranillo cultivars, with a 15 elevated CO2 on vegetative than on reproductive growth. ogical response of Tempranillo clones to changes in the T/CO2/RH conditions and wate In contrast, previous studies have reported a positive effect of elevated 16 size (Bindi et al. 2001, Goncalves et al. 2009, Wohlfahrt et al. 2018), especially after consecutive years of CO2 enrichment treatm 17 CO2 effect on bunch mass and number of berries in the present study may be related to the fact that initiation of inflorescence pr 18 place in the previous season, and in our case, plants were not exposed to elevated CO2 during this process (Wohlfahrt et al. 2018 19 Water deficit was the most limiting factor for plant growth and yield. Its impact on C assimilation, previously discussed, 20 reflected at a plant level, reducing leaf area and dry matter production of all the vegetative organs analysed, regardless of the clo 21 T/CO2/RH regime. Roots, however, were less affected than the above-ground part, as was also reported by Kizildeniz et al. (201 22 significant interaction between clone and irrigation regime reveals a certain degree of intra-cultivar diversity in the growth respo 23 Tempranillo to WD, 1084 being one of the accessions more negatively affected. Probably, its longer cycle, and consequently, lo 24 WD contributed to exacerbate the differences between WW and WD plants at maturity for this clone. 25 (Duchêne 2016), especially the early phenological events. For later phenological events, however, the level of complexity increases and other 1 environmental factors may also influence the timing of phenophases (Martínez-Lüscher et al. 2016). In the present study, water availability was 2 the factor that most strongly affected grape development. The severe reduction in An and total leaf area in WD plants probably limited carbon 3 availability, thus slowing down grape ripening in these treatments. Whereas mild water deficit has been proven to enhance ripening, severe water 4 deficit has been reported to reduce carbon fixation, and consequently, to impair berry ripening (Chaves et al. 2010, Martínez-Lüscher et al. 5 2015a). In addition, the interaction between clone and irrigation reveals a differential response of the studied genotypes to water deficit, 1084 6 being the least responsive clone. This result may be explained by the lower difference in the An between WW and WD conditions observed in this 7 accession. In contrast to WD, CC advanced grape maturity when all the clones were taken into consideration, and compensated for the delaying 8 effect due to drought. ogical response of Tempranillo clones to changes in the T/CO2/RH conditions and wate Consequently, we cannot directly extrapol 9 where grapevine plants can be exposed to other abiotic and biotic factors. This is the first step, however, 10 on grapevine physiology prior to validation under field conditions. 11 Conclusion 12 Simulated temperature, CO2, and RH conditions expected with CC by the end of the century, under contr 13 phenological development and increased leaf biomass of the four clones of Tempranillo studied, and was 14 photosynthetic rate. Such effects, however, were overshadowed by water deficit, which was the factor th 15 vegetative and reproductive growth. Climate change increased WUEi, especially when combined with dro 16 however, probably due to the higher vapour pressure deficit induced by the environmental conditions in t 17 clones showed, in general, a similar behaviour under the simulated CC conditions, some degree of variab 18 T/CO2/RH regime was observed for An and gs, as well as in the response to water deficit for gas exchange 19 and reproductive growth. The differences among clones observed in terms of phenological development 20 environmental conditions on vegetative growth, especially that of water deficit. 21 22 23 24 Water deficit also reduced reproductive growth. In a 10-year study of the effect of water availability on two Spanish grapevine cultivars, 1 Medrano et al. (2003) concluded that there was a close link between water availability and grape yield, through water stress effects on 2 photosynthesis. The reduction in bunch mass in the present study was explained by a lower berry size, but also to a reduced number of berries per 3 bunch, thus suggesting a loss of berries produced by a severe water deficit. The reduction in average berry mass for WD plants was already 4 evident at mid-veraison (56.0 ± 1.5 days on average after fruitset), and differences between the WW and WD treatments were maintained 5 constant thereafter until maturity, thus reflecting a higher sensitivity of berry growth to water limitations imposed before mid-veraison. Similarly, 6 McCarthy (1997) and Roby and Matthews (2008) reported that berry size is more sensitive to water deficit before mid-veraison, whereas water 7 deficit after mid-veraison had only minor effects on berry mass at maturity. 8 This study was performed under controlled conditions. Consequently, we cannot directly extrapolate these results to natural conditions, 9 where grapevine plants can be exposed to other abiotic and biotic factors. ogical response of Tempranillo clones to changes in the T/CO2/RH conditions and wate This is the first step, however, to explore the impact of multi-stresses 10 on grapevine physiology prior to validation under field conditions. 11 ogical response of Tempranillo clones to changes in the T/CO2/RH conditions and wate (2018) presented similar results in two Tempranillo cultivars, with a greater effect of 15 elevated CO2 on vegetative than on reproductive growth. In contrast, previous studies have reported a positive effect of elevated CO2 on bunch 16 size (Bindi et al. 2001, Goncalves et al. 2009, Wohlfahrt et al. 2018), especially after consecutive years of CO2 enrichment treatment. The lack of 17 CO2 effect on bunch mass and number of berries in the present study may be related to the fact that initiation of inflorescence primordia takes 18 place in the previous season, and in our case, plants were not exposed to elevated CO2 during this process (Wohlfahrt et al. 2018). 19 Water deficit was the most limiting factor for plant growth and yield. Its impact on C assimilation, previously discussed, was clearly 20 reflected at a plant level, reducing leaf area and dry matter production of all the vegetative organs analysed, regardless of the clone and the 21 T/CO2/RH regime. Roots, however, were less affected than the above-ground part, as was also reported by Kizildeniz et al. (2015, 2018). The 22 significant interaction between clone and irrigation regime reveals a certain degree of intra-cultivar diversity in the growth response of 23 Tempranillo to WD, 1084 being one of the accessions more negatively affected. Probably, its longer cycle, and consequently, longer exposure to 24 WD contributed to exacerbate the differences between WW and WD plants at maturity for this clone. 25 14 15 Water deficit also reduced reproductive growth. In a 10-year study of the effect of water availabil 1 Medrano et al. (2003) concluded that there was a close link between water availability and grape yield, th 2 photosynthesis. The reduction in bunch mass in the present study was explained by a lower berry size, bu 3 bunch, thus suggesting a loss of berries produced by a severe water deficit. The reduction in average berr 4 evident at mid-veraison (56.0 ± 1.5 days on average after fruitset), and differences between the WW and 5 constant thereafter until maturity, thus reflecting a higher sensitivity of berry growth to water limitations 6 McCarthy (1997) and Roby and Matthews (2008) reported that berry size is more sensitive to water defic 7 deficit after mid-veraison had only minor effects on berry mass at maturity. 8 This study was performed under controlled conditions. Conclusion 12 Simulated temperature, CO2, and RH conditions expected with CC by the end of the century, under controlled environment, hastened grape 13 phenological development and increased leaf biomass of the four clones of Tempranillo studied, and was associated with an increase in their 14 photosynthetic rate. Such effects, however, were overshadowed by water deficit, which was the factor that most strongly affected gas exchange, 15 vegetative and reproductive growth. Climate change increased WUEi, especially when combined with drought. It did not modify WUEint, 16 however, probably due to the higher vapour pressure deficit induced by the environmental conditions in the CC treatment. Although the studied 17 clones showed, in general, a similar behaviour under the simulated CC conditions, some degree of variability in their response to changes in the 18 T/CO2/RH regime was observed for An and gs, as well as in the response to water deficit for gas exchange parameters, phenology and vegetative 19 and reproductive growth. The differences among clones observed in terms of phenological development appeared to condition the impact of the 20 environmental conditions on vegetative growth, especially that of water deficit. 21 24 15 Acknowledgements 1 This work was supported by the Ministerio de Economía y Competitividad of Spain [AGL2014-56075-C2-1-R]; Fundación Universitaria de 2 Navarra [PIUNA 2018] and Asociación de Amigos de la Universidad de Navarra (grant to Marta Arrizabalaga-Arriazu). Special thanks to Ms 3 Mónica Oyarzun, Mr Amadeo Urdiain and Dr Hector Santesteban for their excellent technical assistance and Dr Enrique García-Escudero, Dr 4 José Miguel Martínez-Zapater, Ms Edurne Baroja (ICVV), Mr José Félix Cibrain (EVENA) and Mr Rafael García (Vitis Navarra) for the 5 selection of clones and for providing the plant material to do the experiments. 6 Chaves, M.M., Maroco, J.P. and Pereira, J.S. (2003) Understanding plant responses to drought - from genes to the whole plant. 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Zhao, X., Li, W.F., Wang, Y., Ma, Z.H., Yang, S.J., Zhou, Q., Mao, J. and Chen, B.H. (2019) Elevated CO2 concentration promotes 11 photosynthesis of grape (Vitis vinifera L. cv. ’Pinot noir’) plantlet in vitro by regulating RbcS and Rca revealed by proteomic and 12 transcriptomic profiles. BMC Plant Biology 19, 1–16. 13 15 23 Table 1. Effect of current climatic conditions and climate change conditions combined with two irrigation regimes on the intrinsic water use 1 efficiency and instantaneous water use efficiency of four Vitis vinifera cv. Tempranillo clones. ( ) Results (values are means ± SE) are shown according to clone identity (n = 21-32), T/CO2/RH condition (n = 52-60), irrigation regime (n = 39-61) and the 1 three factors together (n = 3-8). Means with letters in common within the same parameter, stage and factor (clone, T/CO2/RH, irrigation regime, or their 2 interaction) are not significantly different according to LSD test (P > 0.05). Probability values (P) for the main effects of clone, P(CL); T/CO2/RH regime 3 P(T/CO2/RH); irrigation regime, P(I); and their interactions, P(CL x T/CO2/RH), P(CL x I), P(T/CO2/RH x I) and P(CL x T/CO2/RH x I). *, P < 0.001; , P < 0.01; , P < 0.05; n.s., 4 not significant. CS, current climatic conditions (T/CO2/RH), 24°C/14°C, 400 µmol/mol and 45%/65 % RH; CC, climate change conditions (T/CO2/RH) CC 5 28°C/18°C, 700 µmol/mol and 33%/53% RH; WW, well-watered; WD, water deficit at mid-veraison and 2 weeks after mid-veraison; WUEi, intrinsic water 6 use efficiency; WUEinst, instantaneous water use efficiency. 7 References 8 2 WUEi (µmol CO2 /mol H2O) WUEinst (µmol CO2 /mmol H2O) Clone/treatment Mid-veraison 2 Weeks after mid-veraison Mid-veraison 2 Weeks after mid-veraison RJ43 104.0±12.2a 130.2±14.4a 4.93±0.40a 5.16±0.24a CL306 119.5±14.6a 123.2±16.8a 4.85±0.28a 4.93±0.39a VN31 104.5±9.9a 109.7±10.3a 4.50±0.38a 5.42±0.37a 1084 115.2±11.7a 123.4±13.3a 4.50±0.25a 4.63±0.24a CS 78.5±5.8b 83.5±5.9b 4.52±0.24a 4.81±0.20a CC 140.6±8.4a 165.5±9.7a 4.86±0.23a 5.27±0.24a WW 87.5±5.2b 94.0±5.8b 4.15±0.11b 4.60±0.13b WD 147.2±10.9a 157.2±11.7a 5.45±0.34a 5.49±0.28a RJ43 CS WW 62.9±4.4fg 64.0±8.5d 4.21±0.17bcde 4.73±0.34bcd WD 107.7±18.4bcde 96.8±18.7c 6.25±1.21a 5.64±0.56bc CC WW 132.9±12.4cde 130.4±11.1bc 4.35±0.44bcde 4.78±0.23bcd WD 158.1±30.6abc 224.2±28.2a 5.58±1.35abc 5.54±0.70bc CL306 CS WW 50.7±2.9fg 63.0±11.1d 3.91±0.26cde 4.47±0.28bcd WD 115.8±15.8bcde 99.3±17.0cd 4.90±0.87abcde 6.23±1.15ab CC WW 133.2±9.1bcd 134.7±21.0bc 4.92±0.13abcde 4.87±0.65bcd WD 200.4±45.9a 235.0±39.0a 5.84±0.71ab 4.51±0.97bcd VN31 CS WW 55.6±5.2g 58.9±2.7d 3.55±0.23e 4.12±0.30cd WD 118.1±10.8bcd 126.8±25.2c 5.37±1.07abcd 5.25±0.53bcd CC WW 111.2±9.4def 141.6±9.2c 3.83±0.30de 5.16±0.37bcd WD 161.4±44.3ab 231.3±42.1ab 5.98±1.09ab 7.42±1.20a 1084 CS WW 43.4±2.9efg 50.8±5.0d 3.75±0.31de 3.85±0.48d WD 129.2±17.1cdef 115.7±8.4cd 5.37±0.60abcde 4.66±0.57bcd CC WW 105.9±7.5bcd 114.6±9.4c 4.65±0.35abcde 4.88±0.20bcd WD 165.8±14.5abc 186.4±30.1a 4.68±0.71abcde 5.11±0.50bcd P(CL) n.s. n.s. n.s. n.s. P(T/CO2/RH) * n.s. n.s. P(I) * * * P(CL x T/CO2/RH) n.s. n.s. n.s. n.s. P(CL x I) n.s. n.s. n.s. n.s. P(T/CO2/RH x I) n.s. * n.s. n.s. 24 n.s. n.s. n.s. n.s. ( ) Results (values are means ± SE) are shown according to clone identity (n = 21-32), T/CO2/RH condition (n = 52-60), irrigation regime (n = 39-61) and the 1 three factors together (n = 3-8). Means with letters in common within the same parameter, stage and factor (clone, T/CO2/RH, irrigation regime, or their 2 interaction) are not significantly different according to LSD test (P > 0.05). Probability values (P) for the main effects of clone, P(CL); T/CO2/RH regime 3 P(T/CO2/RH); irrigation regime, P(I); and their interactions, P(CL x T/CO2/RH), P(CL x I), P(T/CO2/RH x I) and P(CL x T/CO2/RH x I). *, P < 0.001; , P < 0.01; , P < 0.05; n.s., 4 not significant. CS, current climatic conditions (T/CO2/RH), 24°C/14°C, 400 µmol/mol and 45%/65 % RH; CC, climate change conditions (T/CO2/RH) CC 5 28°C/18°C, 700 µmol/mol and 33%/53% RH; WW, well-watered; WD, water deficit at mid-veraison and 2 weeks after mid-veraison; WUEi, intrinsic water 6 use efficiency; WUEinst, instantaneous water use efficiency. 7 8 25 Table 2. Results (values are means ± SE) are shown according to clone identity (n = 26–32), T/CO2/RH condition (n = 59-62), irrigation regime (n = 58-62) and the three factors 4 together (n = 5–8); means with letters in common within the same parameter, stage and factor (clone, temperature and CO2, irrigation regime, or their interaction) are not 5 significantly different according to LSD test (P > 0.05). All probability values for the interactions of factors [P(CL x T/CO2/RH), P(CLxI), P(T/CO2/RH x I) and P(CL x T/CO2/RH x I)] were 6 statistically not significant (P > 0.05). CS, current climatic conditions (T/CO2/RH), 24°C/14°C, 400 µmol/mol and 45%/65 % RH; CC, climate change conditions (T/CO2/RH) 7 CC 28°C/18°C, 700 µmol/mol and 33%/53% RH; FM, fresh mass; WW, well-watered; WD, water deficit at mid-veraison and 2 weeks after mid-veraison. 8 Figure legends Figure 1. Pre-dawn leaf water potential at mid-veraison and 2 weeks after mid-veraison of four Vitis vinifera cv. Tempranillo clones, RJ43, CL306, VN31 and 1084, grown under two T/CO2/RH conditions: CS, current climatic conditions (24°C/14°C, 400 μmol/mol and 45%/65% RH); and CC, climate change conditions (28°C/18°C, 700 μmol/mol and 33%/53% RH), combined with two irrigation regimes: WW, well- watered (□); and WD, water deficit (■). Results (values are means ± SE) are represented according to the T/CO2/RH and irrigation regimes (n = 14–19). Means with letters in common within the same stage are not significantly different according to LSD test (P > 0.05). Probability values (P) for the main effects of clone, P(CL) mid-veraison (MV), n.s., 2 weeks after mid-veraison (2WAMV), n.s..; T/CO2/RH regime, P(T/CO2/RH) MV, n.s., 2WAMV, n.s.; irrigation regime, P(I) MV, , 2WAMV, ; and their interactions, P(CL x T/CO2/RH) MV, n.s., 2WAMV, n.s., P(CL x I) MV, n.s., 2WAMV, n.s., and P(T/CO2/RH x I) MV, , 2WAMV, n.s.;, P < 0.001; , P < 0.01; n.s., not significant. Interaction of all factors P(CL x T/CO2/RH x I) was statistically not significant (P > 0.05). Figure 2. Net photosynthesis (An), transpiration (T) and stomatal conductance (gs) of the four Vitis vinifera cv. Tempranillo clones, RJ43, CL306, VN31 and 1084, grown under two T/CO2/RH conditions: CS, current climatic conditions (24°C/14°C, 400 μmol/mol, and 45%/65% RH) and CC, climate change conditions (28°C/18°C, 700 μmol/mol and 33%/53% RH), combined with two irrigation regimes: WW, (□) well-watered; and WD (■) water deficit, at mid-veraison and 2 weeks after mid-veraison. Results (values are means ± SE) are represented according to (a) the clones, T/CO2/RH and irrigation regimes (n = 5–8) and (b) to T/CO2/RH and irrigation regimes, considering the clones altogether (n = 18–31). Means with letters in common within the same parameter and stage are not significantly different according to LSD test (P > 0.05). Probability values (P) for the main effects of clone, P(CL); T/CO2/RH regime, P(T/CO2/RH); irrigation regime, P(I); and their interactions, P(CL x T/CO2/RH), P(CL x I) and P(T/CO2/RH x I); *, P < 0.001; , P < 0.01; , P < 0.05; n.s., not significant. Interaction of all factors P(CL x T/CO2/RH x I) was statistically not significant (P > 0.05) in all the cases. Figure 3. Number of days between fruitset and maturity (ca. 23°Brix) of the four Vitis vinifera cv. References 8 Effect of current climatic conditions and climate change conditions combined with two irrigation regimes on the bunch mass, number of berries per bunch and individual berry mass (throughout berry development) of four Vitis vinifera cv. Tempranillo clones. Clone/treatment Bunch mass (g FM) No. of berries/bunch Mass of individual berries (g FM) Mid-veraison 1 Week after mid- veraison 2 Weeks after mid- veraison Maturity RJ43 154.5±12.10a 152.9±9.9a 0.85±0.04b 1.02±0.05ab 1.16±0.04a 0.95±0.04ab CL306 98.0±11.29b 103.3±10.6b 0.76±0.04bc 0.97±0.04b 1.11±0.04ab 0.99±0.05a VN31 98.3±8.94b 114.2±9.0b 0.74±0.04c 0.92±0.04b 1.02±0.04b 0.85±0.04b 1084 97.0±8.89b 91.2±6.4b 1.02±0.05a 1.10±0.05a 1.19±0.05a 0.99±0.06a CS 114.5±8.30a 116.8±7.0a 0.93±0.03a 1.04±0.03a 1.15±0.03a 0.93±0.03a CC 110.3±7.53a 115.6±7.1a 0.76±0.03b 0.97±0.03a 1.09±0.03a 0.96±0.03a WW 133.8±8.36a 125.8±6.9a 0.94±0.03a 1.14±0.03a 1.24±0.03a 1.01±0.04a WD 91.1±6.40b 106.1±7.0b 0.74±0.03b 0.86±0.03b 1.00±0.03b 0.87±0.03b RJ43 CS WW 184.9±39.5a 159.8±28.6ab 0.99±0.10bc 1.21±0.11ab 1.28±0.09abc 1.03±0.11ab WD 126.2±17.8bcd 130.1±21.0abc 0.90±0.05bcd 0.94±0.04cdef 1.13±0.03abcde 0.93±0.05abc CC WW 174.6±12.6ab 158.5±11.6ab 0.88±0.03bcde 1.14±0.06abc 1.30±0.06ab 1.06±0.07ab WD 132.2±13.6abc 163.3±15.5a 0.63±0.07fg 0.76±0.06f 0.93±0.06efg 0.77±0.07c CL306 CS WW 129.2±17.4abcd 112.0±15.5bcd 1.00±0.04bc 1.14±0.03abc 1.29±0.06abc 1.17±0.13a WD 95.0±16.8cdef 106.1±23.4cd 0.73±0.07defg 0.89±0.05def 1.08±0.07cdefg 0.87±0.07bc CC WW 122.5±26.4bcde 113.1±24.1bcd 0.77±0.06cdef 1.07±0.08abcd 1.19±0.07abcd 1.01±0.08abc WD 45.3±16.9f 73.4±21.9d 0.53±0.05g 0.78±0.08ef 0.89±0.09fg 0.85±0.09bc VN31 CS WW 117.3±17.5cde 134.4±17.7abc 0.92±0.04bcd 1.06±0.07abcd 1.13±0.03abcde 0.87±0.05bc WD 74.8±9.4def 92.9±12.7cd 0.73±0.07defg 0.80±0.07ef 0.94±0.07efg 0.75±0.04c CC WW 108.3±20.7cde 114.9±21.6abcd 0.68±0.10efg 1.02±0.10bcd 1.14±0.10abcde 0.93±0.12abc WD 92.8±21.1cdef 114.9±19.2abcd 0.62±0.07fg 0.79±0.05ef 0.87±0.06g 0.86±0.04bc 1084 CS WW 116.8±18.8cde 113.8±12.9bcd 1.23±0.10a 1.27±0.10a 1.33±0.10a 0.92±0.12bc WD 71.4±17.3ef 78.7±11.8d 0.90±0.09bcde 0.93±0.13cdef 1.00±0.10defg 0.89±0.10bc CC WW 114.3±17.6cde 96.4±11.1cd 1.06±0.12ab 1.22±0.08ab 1.29±0.07abc 1.10±0.12ab WD 85.5±15.1cdef 74.5±12.3d 0.88±0.07bcde 0.98±0.07cde 1.11±0.11bcdef 1.04±0.11ab Results (values are means ± SE) are shown according to clone identity (n = 26–32), T/CO2/RH condition (n = 59-62), irrigation regime (n = 58-62) and the three ogether (n = 5–8); means with letters in common within the same parameter, stage and factor (clone, temperature and CO2, irrigation regime, or their interaction) ignificantly different according to LSD test (P > 0.05). All probability values for the interactions of factors [P(CL x T/CO2/RH), P(CLxI), P(T/CO2/RH x I) and P(CL x T/CO2/RH x tatistically not significant (P > 0.05). CS, current climatic conditions (T/CO2/RH), 24°C/14°C, 400 µmol/mol and 45%/65 % RH; CC, climate change conditions (T/C CC 28°C/18°C, 700 µmol/mol and 33%/53% RH; FM, fresh mass; WW, well-watered; WD, water deficit at mid-veraison and 2 weeks after mid-v 26 Figure legends Tempranillo clones, RJ43, CL306, VN31 and 1084, grown under two T/CO2/RH conditions: CC, (□) current climatic conditions (24°C/14°C, 400 μmol/mol and 45%/65% RH) and CC, (■) climate change (28°C/18°C, 700 μmol/mol and 33%/53% RH), combined with two irrigation regimes: WW, well-watered; and WD water deficit. Data (values are means ± SE) are presented according to: (a) the T/CO2/RH and irrigation regimes considering the clones altogether (n = 28–31) and (b) the clones, T/CO2/RH and irrigation regimes (n = 6–8). Means with letters in common within the same chart, (a) or (b), are not significantly different according to LSD test (P > 0.05). Probability values (P) for the main effects of clone, P(CL), ; T/CO2/RH regime, P(T/CO2/RH), ; irrigation regime, P(I), ; and their interactions, P(CL x T/CO2/RH), n.s. and P(CL x I), and P(T/CO2/RH x I), n.s.; *, P < 0.001; , P < 0.01; n.s., not 27 significant. Interaction of all factors P(CL x T/CO2/RH x I) was statistically not significant (P > 0.05). significant. Interaction of all factors P(CL x T/CO2/RH x I) was statistically not significant (P > 0.05). Figure 4. Total leaf area at mid-veraison, 2 weeks after mid-veraison and maturity of the Vitis vinifera cv. Tempranillo clones, RJ43 (∆,▲), CL306 (□,■), VN31 (∇,▼) and 1084 (○,●), grown under two T/CO2/RH conditions: CS, current climatic conditions (24°C/14°C, 400 μmol/mol and 45%/65% RH); and CC, climate change (28°C/18°C, 700 μmol/mol and 33%/53% RH), combined with two irrigation regimes: WW, well- watered (∆, □,∇,○): and WD, water deficit (▲,■,▼, ●). Data (values are means ± SE) are represented according to (b) the clones, and irrigation regimes (n = 14–16) and to (b) the T/CO2/RH and irrigation regimes (n = 31). Means with letters in common within the same stage in chart (b) are not significantly different according to LSD test (P > 0.05). Probability values (P) for the main effects of clone, P(CL) mid-veraison (MV), n.s., 2 weeks after mid-veraison (2WAMV), n.s., maturity (M), **; T/CO2/RH regime, P(T/CO2/RH) MV, n.s., 2WAMV, , M, n.s.; irrigation regime, P(I) MV, *, 2WAMV, , M, ; and their interactions, P(CL x T/CO2/RH) MV, n.s., 2WAMV, n.s., M, n.s., P(CL x I) MV, , 2WAMV, , M, * and P(T/CO2/RH x I) MV, , 2WAMV, , M, n.s.;, P < 0.001; , P < 0.01; , P < 0.05; n.s., not significant. Figure legends Interaction of all factors P(CL x T/CO2/RH x I) was statistically not significant (P > 0.05). Figure 5. Leaf, root, stem and total dry mass of the Vitis vinifera cv. Tempranillo clones, RJ43, CL306, VN31 and 1084, grown under two T/CO2/RH conditions: CS, current climatic conditions (24°C/14°C, 400 μmol/mol and 45%/65% RH); CC, and climate change (28°C/18°C, 700 μmol/mol and 33%/53% RH), combined with two irrigation regimes: WW, well-watered (□); and WD, water deficit (■). Data (values are means ± SE) are represented according to (a) the T/CO2/RH and irrigation regimes (n = 28–31) and (b) the clones, T/CO2/RH and irrigation regimes (n = 5–8). Means with letters in common within the same chart, (a) or (b) and organ are not significantly different according to LSD test (P > 0.05). Probability values (P) for the main effects of clone, P(CL); T/CO2/RH regime, P(T/CO2/RH); irrigation regime, P(I); and their interactions, P(CL x T/CO2/RH) and P(CL x I) and P(T/CO2/RH x I); , P < 0.001; , P < 0.01; *, P < 0.05; n.s., not significant. Interaction of all factors P(CL x T/CO2/RH x I) was statistically not significant (P > 0.05). Figure 6. Principal component analysis of pre-dawn water potential, phenology, gas exchange and dry mass production parameters: (a) score and (b) loading plot. CS, current climatic conditions (24°C/14°C, 400 μmol/mol and 45%/65% RH); CC, climate change conditions (28°C/18°C, 700 μmol/mol and 33%/53% RH); WW, well-watered (○); WD, water deficit (●). FM, fresh mass; DM, dry mass; An, net photosynthesis; T, leaf transpiration; gs, stomatal conductance; WUEi, intrinsic water use efficiency; WUEinst, instantaneous water use efficiency; Ψleaf, pre-dawn leaf water potential; fruitset–maturity, number of days between fruitset and maturity. 28 29 30 31 31 31 32 32 33 33 33 34 34
https://openalex.org/W1788048136	https://bmcpharma.biomedcentral.com/counter/pdf/10.1186/1471-2210-6-6	English	null	Comparison of the monoamine transporters from human and mouse in their sensitivities to psychostimulant drugs	BMC pharmacology	2,006	cc-by	5,919	BioMed Central BioMed Central BMC Pharmacology Open Access Received: 03 November 2005 Accepted: 03 March 2006 This article is available from: http://www.biomedcentral.com/1471-2210/6/6 © 2006Han and Gu; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Page 1 of 7 (page number not for citation purposes) Open Research article Comparison of the monoamine transporters from human and mouse in their sensitivities to psychostimulant drugs Dawn D Han1 and Howard H Gu1,2 ddress: 1Department of Pharmacology, The Ohio State University College of Medicine, USA and 2Department of Psy niversity College of Medicine, 333 West 10th Avenue, Columbus, Ohio 43210, USA Email: Dawn D Han - han.195@osu.edu; Howard H Gu - gu.37@osu.edu * Corresponding author Email: Dawn D Han - han.195@osu.edu; Howard H Gu* - gu.37@osu.edu * Corresponding author * Corresponding author Received: 03 November 2005 Accepted: 03 March 2006 Background The potencies of different psychostimulant drugs at the monoamine transporters have been studied and reported by different laboratories [9,10,13-15]. However, there are significant discrepancies among the reported data and the differences are up to 60-fold. For instance, the inhibition constant (KI) for amphetamine to inhibit DA uptake in rat synaptosomes was reported to be 0.034 µM in one study [16], while it was reported to be 2.3 µM in cultured cells expressing rat DAT [9]. The MDMA KI value to inhibit rat SERT was determined to be 0.24 µM [15] and 2.6 µM [13]. The KI values for cocaine to inhibit rat DAT in vitro ranged from 0.33 µM to 2.0 µM [9,17]. These differences are likely due to different experimental procedures employed by each laboratory, the different expression systems or tis- sue preparation methods used, and different qualities of drugs used. g Drug abuse is a serious problem in the United States and around the world that places tremendous social and eco- nomical burdens on individuals and on the whole society [1]. Psychostimulants are a group of drugs that stimulate the activity of the central nervous system and produce a series of effects in humans, such as increasing heart rate and respiration, improving alertness, elevating mood and self-confidence, and producing euphoria [2]. Common psychostimulant drugs include: cocaine, methylphenidate (Ritalin), amphetamine, methamphetamine, and MDMA [2]. Some of these psychostimulants are useful medica- tions that have long been used for treating various disor- ders such as attention deficit hyperactivity disorder (ADHD), narcolepsy, and obesity, while they are also addictive substances that could cause serious adverse effects when abused [2]. Psychostimulant abuse is a major public health problem in the United States. According to the 2003 National Survey on Drug Use and Health [3], cocaine and amphetamine are two of the most abused drugs while methamphetamine abuse has become a grow- ing concern and 12.3 million Americans age twelve and older had tried methamphetamine at least once in their lifetimes. Methylphenidate is commonly prescribed as Ritalin to treat ADHD. As the number of Ritalin prescrip- tion increases, drug and law enforcement agencies are see- ing an increase in Ritalin drug dealing and the illicit use of Ritalin as a recreational drug [3]. Background The abuse of MDMA, also known as Ecstasy, has also spread to a wide range of settings and demographic subgroups and more than 10 million people have tried MDMA at least once [3]. Therefore, it is important to compare DAT, NET and SERT in their responses to psychostimulants in the same cellu- lar background, using a uniform protocol and drugs. One of our earlier studies compared the cloned DAT, NET and SERT transporters stably expressed in LLC-PK1 cells, but the available transporter cDNAs were from different spe- cies [10]. In another study, human clones of the DAT, NET and SERT transporters were stably expressed in HEK293 cells and the uptake inhibition by selected drugs including some psychostimulants were compared [14]. Several other studies examined rat transporters using synapto- somes prepared from rat brains [13,15,18,19]. So far, there is no study focusing on the comparison of psychos- timulant potencies among the mouse monoamine trans- porters and how they compare to the human transporters. With the ever wider use of genetically modified mouse models in recent years, experimental data that compare drug effects between human and mouse monoamine transporters are becoming increasingly important. Plasma membrane neurotransmitter transporters termi- nate neurotransmissions by the reuptake and recycling of the released neurotransmitters [4,5]. The transporters for the monoamines dopamine, norepinephrine, and serot- onin (DAT, NET, and SERT) belong to a family of Na+/Cl- -dependent neurotransmitter transporters, which are intrinsic membrane proteins containing 12 putative trans- membrane domains [6,7]. Psychostimulants, such as cocaine, methylphenidate, and amphetamine related compounds interrupt the reuptake process by DAT, NET and SERT [8-11]. Consequently, neurotransmissions are prolonged and the extracellular concentrations of these amine transmitters are elevated, resulting in complex neu- rochemical changes and profound psychiatric effects [12]. In order to understand the effects of these psychostimu- lant drugs, it is critical to determine which transporter or neurotransmitter systems are most affected at low, medium, or high drug doses. A thorough understanding of the pharmacological profile for each psychostimulant drug would be helpful for the development of treatment protocols for stimulant overdose and dependence. In the present study, we compared the potencies of five psychostimulant drugs in inhibiting human and mouse monoamine transporters in the same background and using the same procedure. The drugs examined were cocaine, methylphenidate, amphetamine, methampheta- mine, and MDMA. Abstract Background: The plasma membrane neurotransmitter transporters terminate neurotransmissions by the reuptake of the released neurotransmitters. The transporters for the monoamines dopamine, norepinephrine, and serotonin (DAT, NET, and SERT) are targets for several popular psychostimulant drugs of abuse. The potencies of the psychostimulant on the monoamine transporters have been studied by several laboratories. However, there are significant discrepancies in the reported data with differences up to 60-fold. In addition, the drug potencies of the 3 monoamine transporters from mouse have not been compared in the same experiments or along side the human transporters. Further studies and systematic comparisons are needed. Results: In this study, we compared the potencies of five psychostimulant drugs to inhibit human and mouse DAT, SERT and NET in the same cellular background. The KI values of cocaine to inhibit the 3 transporters are within a narrow range of 0.2 to 0.7 µM. In comparison, methylphenidate inhibited DAT and NET at around 0.1 µM, while it inhibited SERT at around 100 µM. The order of amphetamine potencies was NET (KI = 0.07–0.1 µM), DAT (KI ≈ 0.6 µM), and SERT (KI between 20 to 40 µM). The results for methamphetamine were similar to those for amphetamine. In contrast, another amphetamine derivative, MDMA (3–4 methylenedioxymethamphetamine), exhibited higher potency at SERT than at DAT. The human and mouse transporters were similar in their sensitivities to each of the tested drugs (KI values are within 4-fold). Conclusion: The current and previous studies support the following conclusions: 1) cocaine blocks all 3 monoamine transporters at similar concentrations; 2) methylphenidate inhibits DAT and NET well but a 1000-fold higher concentration of the drug is required to inhibit SERT; 3) Amphetamine and methamphetamine are most potent at NET, while being 5- to 9-fold less potent at DAT, and 200- to 500-fold less potent at SERT; 4) MDMA has moderately higher apparent affinity for SERT and NET than for DAT. The relative potencies of a drug to inhibit DAT, NET and SERT suggest which neurotransmitter systems are disrupted the most by each of these stimulants and thus the likely primary mechanism of drug action. Page 1 of 7 (page number not for citation purposes) Page 1 of 7 (page number not for citation purposes) BMC Pharmacology 2006, 6:6 http://www.biomedcentral.com/1471-2210/6/6 http://www.biomedcentral.com/1471-2210/6/6 http://www.biomedcentral.com/1471-2210/6/6 http://www.biomedcentral.com/1471-2210/6/6 Background Our results provide new information and confirm most of the published data while differ from some of the previous results. This study, combined with results from other studies, provides very useful informa- tion about which neurotransmission pathways are likely to be affected the most by each of the drugs, which gives insight into primary mechanisms of drug actions. Page 2 of 7 (page number not for citation purposes) Results and discussion Transiently transfected cells are used to determine the KI values for drugs inhibiting wild type and mutant monoamine transporters in our laboratory and other lab- oratories [20-26]. Fluctuations in transporter expression Page 2 of 7 (page number not for citation purposes) Page 2 of 7 (page number not for citation purposes) BMC Pharmacology 2006, 6:6 http://www.biomedcentral.com/1471-2210/6/6 Drug inhibition profiles of mouse and human monoamine transporters by psychostimulants igure 1 Drug inhibition profiles of mouse and human monoamine transporters by psychostimulants. Intestine 407 cells were transfected with human or mouse DAT, SERT, or NET cDNAs. Twenty to 24 hours after transfection, cells were incu- ated with [3H] labeled substrate in PBS/Mg/Ca buffer for 10 min in the absence or presence of increasing concentrations of a sychostimulant drug as indicated. Uptake was terminated by two successive washes with PBS/Mg/Ca. The amounts of [3H] abeled substrate accumulated in the cells were determined by scintillation counting. The uptake activities are presented as ractional activities relative to those in the absence of drugs. The experiments were performed in triplicates. Each data point is xpressed as mean ± SEM. The five drugs tested are: A) Cocaine; B) Methylphenidate (Ritalin); C) Amphetamine; D) Metham- hetamine; and E) MDMA. Results and discussion 0.0 0.2 0.4 0.6 0.8 1.0 1.2 -9 -8 -7 -6 -5 -4 -3 Log[drug], M 1B Methylphenidate (Ritalin) mDAT hDAT mNET hNET mSERT hSERT 0.0 0.2 0.4 0.6 0.8 1.0 1.2 -9 -8 -7 -6 -5 -4 -3 Log[drug], M 1C Amphetamine Transport Activity mDAT hDAT mNET hNET mSERT hSERT 0.0 0.2 0.4 0.6 0.8 1.0 1.2 -9 -8 -7 -6 -5 -4 -3 Log[drug], M 1A Cocaine Transport Activity mDAT hDAT mNET hNET mSERT hSERT 0.0 0.2 0.4 0.6 0.8 1.0 1.2 -9 -8 -7 -6 -5 -4 -3 Log[drug], M 1D Methamphetamine mDAT hDAT mNET hNET mSERT hSERT 0.0 0.2 0.4 0.6 0.8 1.0 1.2 -9 -8 -7 -6 -5 -4 -3 Log[drug], M 1E MDMA % Transport Activity mDAT phDAT mNET phNET mSERT phSERT 0.0 0.2 0.4 0.6 0.8 1.0 1.2 -9 -8 -7 -6 -5 -4 -3 Log[drug], M 1B Methylphenidate (Ritalin) mDAT hDAT mNET hNET mSERT hSERT 0.0 0.2 0.4 0.6 0.8 1.0 1.2 -9 -8 -7 -6 -5 -4 -3 Log[drug] M 1A Cocaine Transport Activity mDAT hDAT mNET hNET mSERT hSERT Methylphenidate (Ritalin) 6 5 Log[drug], M Log[drug], M 0.0 0.2 0.4 0.6 0.8 1.0 1.2 -9 -8 -7 -6 -5 -4 -3 Log[drug], M 1C Amphetamine Transport Activity mDAT hDAT mNET hNET mSERT hSERT Log[drug], M 0.0 0.2 0.4 0.6 0.8 1.0 1.2 -9 -8 -7 -6 -5 -4 -3 Log[drug], M 1D Methamphetamine mDAT hDAT mNET hNET mSERT hSERT Methamphetamine Log[drug], M Log 0.0 0.2 0.4 0.6 0.8 1.0 1.2 -9 -8 -7 -6 -5 -4 -3 Log[drug], M 1E MDMA % Transport Activity mDAT phDAT mNET phNET mSERT phSERT Log[drug], M Log[drug], M 0.0 0.2 0.4 0.6 0.8 1.0 1.2 -9 -8 -7 -6 -5 -4 -3 Log[drug], M 1E MDMA % Transport Activity mDAT phDAT mNET phNET mSERT phSERT g p p y p y g Drug inhibition profiles of mouse and human monoamine transporters by psychostimulants. Intestine 407 cells were transfected with human or mouse DAT, SERT, or NET cDNAs. Twenty to 24 hours after transfection, cells were incu- bated with [3H] labeled substrate in PBS/Mg/Ca buffer for 10 min in the absence or presence of increasing concentrations of a psychostimulant drug as indicated. Uptake was terminated by two successive washes with PBS/Mg/Ca. The amounts of [3H] labeled substrate accumulated in the cells were determined by scintillation counting. Results and discussion The uptake activities are presented as fractional activities relative to those in the absence of drugs. The experiments were performed in triplicates. Each data point is expressed as mean ± SEM. The five drugs tested are: A) Cocaine; B) Methylphenidate (Ritalin); C) Amphetamine; D) Metham- phetamine; and E) MDMA. Page 3 of 7 (page number not for citation purposes) Page 3 of 7 (page number not for citation purposes) http://www.biomedcentral.com/1471-2210/6/6 BMC Pharmacology 2006, 6:6 Table 1: Comparison of the KI values of 5 psychostimulants to inhibit human and mouse monoamine transporters. Drug Cocaine Methylphenidate Amphetamine Methamphetamine MDMA Human hDAT 0.23 ± 0.03 0.06 ± 0.01 0.64 ± 0.14 0.46 ± 0.06 8.29 ± 1.67 hNET 0.48 ± 0.05 0.10 ± 0.01 0.07 ± 0.01 0.11 ± 0.01 1.19 ± 0.13 hSERT 0.74 ± 0.03 132.43 ± 10.71 38.46 ± 3.84 31.74 ± 2.40 2.41 ± 0.73 Ratio 1 3.2 2207 549 288 7.0 Mouse mDAT 0.49 ± 0.04 2 0.26 ± 0.03 0.56 ± 0.11 0.47 ± 0.08 4.87 ± 0.65 mNET 0.46 ± 0.06 2 0.17 ± 0.03 0.12 ± 0.02 0.19 ± 0.05 1.75 ± 0.51 mSERT 0.73 ± 0.12 114.37 ± 7.61 23.82 ± 1.71 9.28 ± 0.86 0.64 ± 0.05 Ratio 1 1.6 672 199 49 7.6 The KI values (in µM) were determined as illustrated in Fig. 1. They are expressed as mean ± S.E.M. of four to seven experiments. 1 The ratios of highest KI values over the lowest are shown to highlight the differences. 2 The difference between these two KI values was not statistically significant (ANOVA post hoc Bonferroni test, p > 0.05); all other values were statistically different (ANOVA comparing the three transporters within the same species and for the same drug). ues of 5 psychostimulants to inhibit human and mouse monoamine transporters. Table 1: Comparison of the KI values of 5 psychostimulants to inhibit human and mouse monoamine transporters. mparison of the KI values of 5 psychostimulants to inhibit human and mouse monoamine transporters. The KI values (in µM) were determined as illustrated in Fig. 1. They are expressed as mean ± S.E.M. of four to seven experiments. 1 The ratios of highest KI values over the lowest are shown to highlight the differences. Methylphenidate (Ritalin) As shown in Fig. 1B and Table 1, methylphenidate was a very potent inhibitor of hDAT and hNET (KI = 0.06 µM and 0.10 µM), while it was not a potent inhibitor of hSERT (KI = 132 µM). Therefore, hSERT is over 2000 fold less sensitive to methylphenidate than hDAT. Compari- son between the two species revealed that methylpheni- date was 4-fold more potent to inhibit hDAT than mDAT, while it had similar effects on NET or SERT from the two species. Our data show trends similar to those previously reported for human transporters expressed in HEK cells [14] and for rat transporters studied with rat brain synap- tosomes [13]. For instance, the methylphenidate KIvalues determined by Eshleman et al are 0.19 µM for hDAT, 0.038 µM for hNET and 55 µM for hSERT [14]. The com- mon conclusion from all these studies is that SERT is much less sensitive to methylphenidate than DAT and NET. Results and discussion 2 The difference between these two KI values was not statistically significant (ANOVA post hoc Bonferroni test, p > 0.05); all other values were statistically different (ANOVA comparing the three transporters within the same species and for the same drug). levels have little impact on KI measurement except when expression levels are very low resulting in unacceptable signal to noise ratio. In this study, human and mouse DAT, NET, and SERT cDNAs were transiently expressed in cultured Intestine 407 cells. Transport activities by the transfected cells were measured in the presence of increas- ing concentrations of drugs. The KI values were then deter- mined. Five psychostimulant drugs were studied: cocaine, methylphenidate, amphetamine, methamphetamine, and MDMA. For each of these drugs, the transporters were studied in the same experiments for more precise compar- ison. Fig. 1 shows representative results for each of the 5 drugs. The results are summarized in Table 1 displaying the average KI values from 4–7 experiments. The ratios of highest KI values over the lowest were calculated to high- light the differences. The results and conclusions from this study were obtained using Intestine 407 cells, which may not apply to other cell lines or in vivo systems. porters expressed in cultured cells [14,15] suggest that SERT is slightly more sensitive to cocaine than NET. Despite the small differences, all studies show that cocaine inhibits DAT, NET and SERT within a narrow con- centration range, suggesting that modulation of all three neurotransmitter systems are likely to contribute to the biochemical and behavioural effects of cocaine. Page 4 of 7 (page number not for citation purposes) MDMA As its name indicated, 3,4-methylenedioxymethampheta- mine is a compound with a methylenedioxy group added to methamphetamine. The chemical modification sub- stantially increases MDMA's potency to inhibit SERT while reducing its potencies to inhibit DAT and NET com- pared to methamphetamine. This brings the KI values for all three monoamine transporters to a close range (7- fold). In mouse, the order of potencies for MDMA was SERT (KI = 0.64 µM), NET (KI = 1.75 µM), and DAT (KI = 4.87 µM). For human transporters, the order was NET (KI = 1.19 µM), SERT (KI = 2.41 µM), and DAT (KI = 8.29 µM). In previous studies, Rothman, et al. has reported that among the rat transporters, MDMA is most potent at inhibiting rSERT (KI = 0.238 µM), followed by rNET (KI = 0.462 µM) and rDAT (KI = 1.572 µM) [15]. However, another study has reported that MDMA is more potent at inhibiting rDAT (KI = 1.53 µM) than rSERT (KI = 2.6 µM) [13]. Our data for the mouse and human transporters are similar to the results by Rothman et al for rat transporters [15]. Our results indicate that MDMA is more potent in inhibiting SERT than DAT, which is in contrary to the other amphetamine derivatives. Cocaine Fig. 1A shows that cocaine inhibited DAT, SERT, and NET from human or mouse within a narrow range of concen- trations. The KI values were from 0.2 to 0.7 µM. Among the human monoamine transporters, cocaine was a slightly more potent inhibitor of hDAT (KI = 0.23 µm) than hSERT (KI = 0.74 µm) with hNET in the middle (KI = 0.48 µm). The cocaine potencies that inhibit mouse trans- porters were very similar to those for the human trans- porters except that the KI for mDAT (0.49 µm) was about twice the value for hDAT. Compared to earlier studies, our KI value (0.23 µM) for hDAT was similar to the KI value (0.278 µM) reported by Eshleman, et al. for hDAT stably expressed in HEK293 cells [14] but 4 times the value (KI = 0.058 µM) reported by Giros, et al. for hDAT expressed in mouse fibroblast Ltk-cells [27]. For both human and mouse NET and SERT, our data indicated that NET is slightly more sensitive to cocaine than SERT, while two previous studies using rat synaptosomes or human trans- Methylphenidate is marketed as Ritalin and is prescribed to treat ADHD particularly for children. This prescription drug is also addictive and has been abused. It has been reported that cocaine and methylphenidate accumulate in the same regions in the human brain and have similar effectiveness in blocking DAT in vitro and in vivo [28]. However, methylphenidate abuse by humans is much less frequent than that for cocaine [29]. The major difference between methylphenidate and cocaine is that SERT is not significantly inhibited by methylphenidate with doses that completely block DAT and NET while cocaine blocks Page 4 of 7 (page number not for citation purposes) Page 4 of 7 (page number not for citation purposes) http://www.biomedcentral.com/1471-2210/6/6 http://www.biomedcentral.com/1471-2210/6/6 BMC Pharmacology 2006, 6:6 all three transporters equally well. This difference likely contributes to the different effects produced by the two drugs. Another likely contributing factor is the different pharmacokinetics for the two drugs with cocaine reaching its site of action much more rapidly than methylpheni- date. releases. The results show that amphetamine and meth- amphetamine are most potent in inhibiting NET and much less potent in inhibiting SERT in both human and mouse. Amphetamine and methamphetamine Among human transporters, amphetamine was most potent at inhibiting hNET (KI = 0.07 µM). Compared to the amount of amphetamine required to inhibit hNET, 9- fold and over 500-fold more amphetamine was required to inhibit hDAT (KI = 0.64 µM) and hSERT (KI = 38 µM) respectively. The potencies of methamphetamine (Fig. 1D) to inhibit human and mouse monoamine transport- ers are similar to those of amphetamine (Fig. 1C). We compared the human and mouse monoamine transport- ers and found that the transporters from the two spices responded to amphetamine and methamphetamine in a similar fashion (Fig. 1C, 1D, and Table 1). Our results are not the same as those from previous studies but show a similar trend. In one study with rat synaptosomes, the amphetamine KI values are 0.034 µM for rDAT, 0.039 µM for rNET, and 3.8 µM for rSERT [15]. In another study using cultured cells expressing human transporters, the methamphetamine KI values are 0.082 µM for hDAT, 0.0013 µM for rNET, and 20.7 µM for hSERT. Therefore, amphetamine and methamphetamine are at least 100- fold less potent at inhibiting SERT than DAT or NET. Conclusion Amphetamine is a synthetic drug. While prescribed for treating ADHD, amphetamine has also been frequently diverted from prescription to recreational use. Metham- phetamine is an amphetamine analogue which also has some limited therapeutic uses, primarily in the treatment of ADHD and obesity. In recent years, the abuse of meth- amphetamine becomes an extremely serious and growing problem. Amphetamine and its analogues are also sub- strates of the monoamine transporters and the vesicular monoamine transporter. They compete with and exchange with monoamines at the plasma membrane transporters and also at the vesicular monoamine trans- porter, disrupting the reuptake process and causing the release of monoamines [7,30]. Amphetamine induced monoamine release is usually studied using animal brain preparations which contain both the plasma membrane and vesicular monoamine transporters. Cultured cells expressing only plasma membrane transporters are usu- ally used to measure the KI values of uptake inhibition which reflect the apparent affinities of the drugs to each transporter. The KI value includes the effect of ampheta- mine induced substrate release and it is not exactly the same as the dissociation constant KD, a true measurement of drug affinity. In this study, we focused on comparing KI values of the transporters and did not study drug induced There are significant discrepancies in previous studies on the potencies of psychostimulant drugs at monoamine transporters, likely due to differences in experimental set- ups, expression systems, tissue preparations, and/or drug qualities. In this study, we compared the potencies of five commonly abused psychostimulants at the human and mouse DAT, SERT and NET in the same cellular back- ground. Cocaine blocked the 3 monoamine transporters at similar concentrations (KI = 0.2–0.7 µM). In compari- son, methylphenidate inhibited DAT and NET around 0.1 µM, while inhibited SERT at 1000 fold higher concentra- tion (around 100 µM). Amphetamine and methampheta- mine were most potent for NET (KI around 0.1 µM), less potent for DAT (KI around 0.5 µM), and much less potent for SERT (KI between10 to 40 µM). In contrast, MDMA, another amphetamine derivative, exhibited higher potency at SERT than at DAT. The human and mouse transporters were similar in their sensitivities to each of the tested drugs (KI values within 4 folds). The relative potencies of a drug in inhibiting DAT, NET and SERT sug- gest the neurotransmitter systems that are disrupted the most and thus the primary mechanism of drug action. Page 5 of 7 (page number not for citation purposes) Methods Materials amount of accumulated [3H]-labelled substrate in the cells were determined by counting in scintillation fluid (MicroScint-20, PerkinElmer Life Sciences, Boston, MA) using a Packard TopCount, a microplate scintillation and luminescence counter. All experiments were performed in triplicates. Cocaine, D-amphetamine, and methylphenidate were kindly provided by National Institute on Drug Abuse through its Drug Supply Program. D-methamphetamine and MDMA were purchased from Sigma (St Louis, MO). [3H]-labelled dopamine (23.5 Ci/mmole) and [3H]- labelled serotonin (27.1 Ci/mmole) were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). The human dopamine, norepinephrine, and serotonin transporter cDNAs used in the experiments were described previously [10,31,32]. The cloning of mouse DAT cDNA was reported in an earlier publication [33]. The mouse NET and SERT cDNAs were amplified with nested PCR using mouse brain cDNAs as the template. The forward primers for mNET are: mNETf1 (CAGCCG- CACCCATGCTTCT) and mNETf2 (AAAAGGTACCAC- CATGCTTCTGGCGCGAAT); the reverse primers are mNETr1 (TCCTCCACATTGCCAGGTTCAGA) and mNETr2 (AAAATCTAGAGGTTCAGAT- GGCCAGCCAGTG). The forward primers for mSERT are mSTf1 (AGCTAGTCAGGGTCCTTGGCAGATG) and mSTf2 (ATATCCATGGAGACCACACCTTTGAATTCTC), and reverse primers are mSTr1 (TGGGGCTTTTCAGAGAT- GAGGAGTC) and mSTr2 (TAATCTCGAGCCATGTC- CTCTCCCTCAGTGTGTTAC). Restriction enzyme sites were incorporated in the primers for insertion into plas- mid vectors. Oligonucleotide primers were synthesized by commercial DNA synthesis services. The correct sequences of mNET and mSERT were confirmed by sequence deter- mination. Cocaine, D-amphetamine, and methylphenidate were kindly provided by National Institute on Drug Abuse through its Drug Supply Program. D-methamphetamine and MDMA were purchased from Sigma (St Louis, MO). [3H]-labelled dopamine (23.5 Ci/mmole) and [3H]- labelled serotonin (27.1 Ci/mmole) were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). The human dopamine, norepinephrine, and serotonin transporter cDNAs used in the experiments were described previously [10,31,32]. The cloning of mouse DAT cDNA was reported in an earlier publication [33]. The mouse NET and SERT cDNAs were amplified with nested PCR using mouse brain cDNAs as the template. The forward primers for mNET are: mNETf1 (CAGCCG- CACCCATGCTTCT) and mNETf2 (AAAAGGTACCAC- CATGCTTCTGGCGCGAAT); the reverse primers are mNETr1 (TCCTCCACATTGCCAGGTTCAGA) and mNETr2 (AAAATCTAGAGGTTCAGAT- f f Authors' contributions DDH carried out most of the experiments and data analy- ses, and drafted the manuscript. HHG conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. ( GGCCAGCCAGTG). The forward primers for mSERT are mSTf1 (AGCTAGTCAGGGTCCTTGGCAGATG) and mSTf2 (ATATCCATGGAGACCACACCTTTGAATTCTC), and reverse primers are mSTr1 (TGGGGCTTTTCAGAGAT- GAGGAGTC) and mSTr2 (TAATCTCGAGCCATGTC- CTCTCCCTCAGTGTGTTAC). Restriction enzyme sites were incorporated in the primers for insertion into plas- mid vectors. Oligonucleotide primers were synthesized by commercial DNA synthesis services. The correct sequences of mNET and mSERT were confirmed by sequence deter- mination. Data analysis The IC50 values were determined by nonlinear regression of experimental data for each experiment according to a hyperbolic model using the computer program Origin (MicroCal Software, Northampton, MA). The KI values were then calculated from the IC50 values using the equa- tion KI = IC50/(1 + [S]/KM). Data are presented as arithme- tic mean ± SEM of four to seven independent experiments. 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Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 7 of 7 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge 28. Volkow ND, Wang GJ, Fowler JS, Fischman M, Foltin R, Abumrad NN, Gatley SJ, Logan J, Wong C, Gifford A, Ding YS, Hitzemann R, Pappas N: Methylphenidate and cocaine have a similar in vivo potency to block dopamine transporters in the human brain. Life Sci 1999, 65:PL7-12. 29. Parran TVJ, Jasinski DR: Intravenous methylphenidate abuse. Prototype for prescription drug abuse. Arch Intern Med 1991, 151:781-783. 30. 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https://openalex.org/W2907251702	https://dergipark.org.tr/tr/download/article-file/610935	Turkish	null	Compensation of time delay effect in vehicle yaw stability control systems by using communication disturbance observer	Mühendislik bilimleri dergisi/Mühendislik bilimleri dergisi	2,018	cc-by	6,301	doi: 10.5505/pajes.2018.66492 Özel Sayı Makalesi/Special Issue Article Geliş Tarihi/Received: 28.01.2018, Kabul Tarihi/Accepted: 30.07.2018 * Yazışılan yazar/Corresponding author Öz Abstract Time delay is a control system design factor, which reduces the performance of the system and makes the system unstable in the worst case. Many methods have been proposed in the literature for time delay compensation and the Smith predictor is the most widely used. Although the Smith predictor is easy to implement, the delay compensation performance degrades as the time delay model used by the predictor differs from the actual time delay in the system. Communication disturbance observer has been proposed as an alternative method to the Smith predictor and it has been applied to the bilateral teleoperation systems, robotic manipulators and network-based gait rehabilitation systems. This paper deals with the application of communication disturbance observer to the vehicle yaw stability control. The performance of communication disturbance observer is tested for time varying delays by using several simulations and the results are compared with the Smith predictor results. Zaman gecikmesi kontrol sistemleri tasarımında dikkate alınması gereken sistemin performansını azaltan ve en kötü durumda da sistemi kararsız yapan etkenlerden birisidir. Zaman gecikmesi telafisi için literatürde pek çok yöntem önerilmiştir. Bunlardan en yaygın olarak kullanılanı Smith tahmincisidir. Smith tahmincisi kolaylıkla uygulanabilse de tahmincinin kullandığı zaman gecikmesi modeli ile sistemdeki gerçek zaman gecikmesi farklılaştıkça gecikme telafisi performansı düşmektedir. Bu metodun alternatifi olarak zaman gecikmesi gözleyicisi önerilmiş ve literatürde daha önce bilateral teleoperasyon sistemlerine, robotik manipülatörlere ve iletişim ağı tabanlı yürüyüş şekli rehabilitasyon cihazlarına uygulanmıştır. Bu çalışmada ise zaman gecikmesi gözleyicisinin taşıt savrulma dinamiği kontrolüne uygulanması ele alınmıştır. Zaman gecikmesi gözleyicisinin performansı zamanla değişen gecikmeler için çeşitli simülasyon çalışmalarıyla test edilmiş, ayrıca sonuçlar Smith tahmincisiyle karşılaştırılmıştır. Keywords: Time delay, Vehicle dynamics control, Smith predictor, Disturbance observer, Parameter space approach Anahtar kelimeler: Zaman gecikmesi, Taşıt dinamiği kontrolü, Smith tahmincisi, Bozucu gözleyicisi, Parametre uzayı yaklaşımı değişen gecikme durumlarında da iyi sonuçlar vermesidir. Yöntem daha sonra tek ve iki serbestlik dereceli döner manipülatörlere [6],[7] ve iletişim ağı tabanlı yürüyüş şekli rehabilitasyon cihazlarına [8], kablosuz hareket kontrol sistemlerine [9],[10] ve TCP/IP protokolü yoluyla birbirine bağlı teleoperasyon sistemlerinin kontrolüne [11] uygulanmıştır. Taşıt savrulma dinamiği kontrol sistemlerinde zaman gecikmesi etkisinin zaman gecikmesi gözleyicisi kullanılarak telafi edilmesi Taşıt savrulma dinamiği kontrol sistemlerinde zaman gecikmesi etkisinin zaman gecikmesi gözleyicisi kullanılarak telafi edilmesi Compensation of time delay effect in vehicle yaw stability control systems by using communication disturbance observer Mümin Tolga EMİRLER1* , Bilin AKSUN GÜVENÇ2 , Levent GÜVENÇ3 1Makina Mühendisliği Bölümü, Mühendislik Fakültesi, İstanbul Okan Üniversitesi, İstanbul, Türkiye. tolgaemirler@yahoo.com 2Otomotiv Araştırma Merkezi, Otonom Sürüş Laboratuvarı, Ohio State Üniversitesi, Columbus, OH, USA. aksunguvenc.1@osu.edu 3Elektrik ve Bilgisayar Mühendisliği Bölümü, Ohio State Üniversitesi, Columbus, OH, USA. guvenc.1@osu.edu doi: 10.5505/pajes.2018.66492 Özel Sayı Makalesi/Special Issue Article Pamukkale Univ Muh Bilim Derg, 24(8), 1450-1457, 2018 (TOK’2017 – Otomatik Kontrol Türk Milli Komitesi Ulusal Toplantısı Özel Sayısı) Pamukkale Üniversitesi Mühendislik Bilimleri Dergisi Pamukkale University Journal of Engineering Sciences Taşıt savrulma dinamiği kontrol sistemlerinde zaman gecikmesi etkisinin zaman gecikmesi gözleyicisi kullanılarak telafi edilmesi Compensation of time delay effect in vehicle yaw stability control systems by using communication disturbance observer Mümin Tolga EMİRLER1* , Bilin AKSUN GÜVENÇ2 , Levent GÜVENÇ3 1Makina Mühendisliği Bölümü, Mühendislik Fakültesi, İstanbul Okan Üniversitesi, İstanbul, Türkiye. tolgaemirler@yahoo.com 2Otomotiv Araştırma Merkezi, Otonom Sürüş Laboratuvarı, Ohio State Üniversitesi, Columbus, OH, USA. aksunguvenc.1@osu.edu 3Elektrik ve Bilgisayar Mühendisliği Bölümü, Ohio State Üniversitesi, Columbus, OH, USA. guvenc.1@osu.edu Pamukkale Univ Muh Bilim Derg, 24(8), 1450-1457, 2018 (TOK’2017 – Otomatik Kontrol Türk Milli Komitesi Ulusal Toplantısı Özel Sayısı) Pamukkale Üniversitesi Mühendislik Bilimleri Dergisi Pamukkale University Journal of Engineering Sciences Taşıt savrulma dinamiği kontrol sistemlerinde zaman gecikmesi etkisinin zaman gecikmesi gözleyicisi kullanılarak telafi edilmesi Compensation of time delay effect in vehicle yaw stability control systems by using communication disturbance observer Mümin Tolga EMİRLER1* , Bilin AKSUN GÜVENÇ2 , Levent GÜVENÇ3 1Makina Mühendisliği Bölümü, Mühendislik Fakültesi, İstanbul Okan Üniversitesi, İstanbul, Türkiye. tolgaemirler@yahoo.com 2Otomotiv Araştırma Merkezi, Otonom Sürüş Laboratuvarı, Ohio State Üniversitesi, Columbus, OH, USA. aksunguvenc.1@osu.edu 3Elektrik ve Bilgisayar Mühendisliği Bölümü, Ohio State Üniversitesi, Columbus, OH, USA. guvenc.1@osu.edu Pamukkale Univ Muh Bilim Derg, 24(8), 1450-1457, 2018 (TOK’2017 – Otomatik Kontrol Türk Milli Komitesi Ulusal Toplantısı Özel Sayısı) Pamukkale Üniversitesi Mühendislik Bilimleri Dergisi Pamukkale University Journal of Engineering Sciences 1 Giriş 𝑑̂ = 𝑄(𝑢−𝐺𝑛−1𝑦) (3) (3) burada Q filtresi alçak geçiren bir filtredir. Nominal sistemin derecesine göre Q/Gn oranını nedensel kılacak şekilde Q alçak geçiren filtresinin derecesi belirlenir. Alçak geçiren Q filtresi yardımıyla belli bir kesim frekansına kadar tahminin yapılması sağlanmaktadır. Bu frekanstan sonra tahmin yapılmaya çalışılmamakta böylece dayanıklı kararlılık gözetilmektedir (Bkz. [22]). Şekil 1: Taşıtlarda kullanılan iletişim ağı kontrol sistemlerinin temel bileşenleri.  n G s  U s  Y s + -  D s  Q s +-  ˆD s   n Q s G s Şekil 2: Bozucu gözleyici yapısı, [4]. Bu çalışmada CAN tabanlı taşıt savrulma dinamiği kontrolünde zaman gecikmesinin etkisi ZGG (Zaman Gecikmesi Gözleyicisi) kullanılarak telafi edilmeye çalışılmıştır. Zamanla değişen gecikmeler için ZGG’siz ve ZGG eklenmiş sistemin cevabı çeşitli simülasyonlar yapılarak incelenmiştir. Zaman gecikmesi telafisi için Smith tahmincisi ile ZGG’nin performansı karşılaştırılmıştır. Ayrıca zaman gecikmesi marjinine göre parametre uzayı tabanlı kontrol sistemi tasarım denklemleri elde edilmiş ve PI kontrol sistemi tasarımı bu yolla gerçekleştirilmiştir. Bu çalışma yazarların önceki çalışmasının [19] genişletilmiş halidir. Çalışmanın bundan sonraki bölümleri şu şekilde düzenlenmiştir. Bölüm 2’de zaman gecikmesi gözleyicisi tanıtılmıştır. Bölüm 3’te taşıt savrulma dinamiği kontrolü problemi ve kullanılacak sistem modeli kısaca anlatılmıştır. Bölüm 4’te ZGG taşıt savrulma dinamiği kontrolüne uygulanmıştır. Zaman gecikmesi marjinine göre kontrol sistemi tasarımı ve ZGG için Q filtresinin kesim frekansının seçilmesi bu bölümde anlatılmıştır. Bölüm 5’te dört farklı simülasyonla ZGG test edilmiş ve performansı Smith tahmincisi ile karşılaştırılmıştır. Bildiri Bölüm 6’de verilen sonuçlarla sonlandırılmıştır. Şekil 2: Bozucu gözleyici yapısı, [4]. ZGG’nin yapısı bozucu etken tanımı ve zaman gecikmesi telafisi haricinde BG’ye benzemektedir. Zaman gecikmeli sistemdeki (𝐺𝑛(𝑠)𝑒−𝑇𝑠) zaman gecikmesi, sisteme etkiyen bozucunun bir parçasıymış gibi Şekil 3’teki gibi gösterilebilir. Görüldüğü gibi sistem bu şekilde yazıldığında yine aynı çıkış elde edilmektedir (𝑌(𝑠) = 𝐺𝑛(𝑠)𝑒−𝑇𝑠𝑈(𝑠)). Burada gösterilen bozucu, iletişim ağı bozucusu adıyla anılır ve zaman tanımlı bölgesinde alttaki gibi yazılabilir: 1 Giriş Zaman gecikmesi, sistemlerin frekans cevabına faz gecikmesi ekleyerek kararlılıklarını azaltan ve en kötü durumda sistemi kararsızlığa götüren kontrol sistemleri tasarımında dikkate alınması gereken önemli bir etkendir. Literatürde Smith tahmincisi gibi oldukça iyi bilinen metotlarla zaman gecikmesi telafisi üzerine çalışmalar bulunmaktadır [1]-[3]. Smith tahmincisi ve bu yöntem üzerine geliştirilen yöntemler zaman gecikmesi telafisinde oldukça güçlü, anlaşılması ve uygulanması kolay yöntemlerdir. Bu yöntemlerin en büyük dezavantajı, yöntemin düzgün çalışabilmesi için tam (kesin) zaman gecikmesi modeline ihtiyaç duyulmasıdır. Model ile gerçek zaman gecikmesi birbirinden farklılaştıkça tahmincinin performansı düşmektedir [4]. Zaman gecikmesi pek çok otomotiv kontrol sisteminde görülmektedir. Bunlara örnek olarak rölanti devri kontrolü [12]-[14], anti-jerk kontrolü [15], kooperatif adaptif seyir kontrolü [16],[17] ve CAN (Controller Area Network) tabanlı aktif güvenlik sistemleri (ABS (Antilock Brake System), TCS (Traction Control System) ve ESC (Electronic Stability Control)) verilebilir. CAN tabanlı aktif güvenlik sistemleri iletişim ağı kontrol sistemlerinin iyi bir örneğidir. İletişim ağı kontrol sistemleri sağladıkları güvenlik, esneklik, uygun maliyet ve kolay yönetilebilirlikle otomobiller ve uçaklar gibi karmaşık sistemlerde kullanılmaktadır. Ancak kapalı çevrim kontrollü sistemlere iletişim ağlarının entegre edilmesi, iletişim ağı kaynaklı zaman gecikmelerinden ötürü sistemde Smith tahmincisine alternatif olarak geliştirilen yöntemlerden bir tanesi zaman gecikmesi gözleyicisidir. Bu yöntem ilk kez 2004 yılında Natori vd. tarafından bilateral teleoperasyon sistemlerinde zaman gecikmesini telafi etmek için önerilmiştir [5]. Yöntemin önemli özelliklerinden biri tam (kesin) zaman gecikmesi modeline ihtiyaç duymaması ve hatta zamanla 1450 Denklem (1) d’ye göre alttaki gibi yazılabilir: Denklem (1) d’ye göre alttaki gibi yazılabilir: performans kaybına ve bazen kararsızlığa sebep olabilmektedir [18]. 𝑑= 𝑢−𝐺𝑛−1𝑦 (2) (2) Şekil 1’de taşıtlarda kullanılan iletişim ağı kontrol sisteminin temel bileşenleri verilmiştir. İletişim ağı kaynaklı iki zaman gecikmesi bulunmaktadır: bunlar kontrolcü ile eyleyici arasındaki gecikme Tke ve sensör ile kontrolcü arasındaki gecikme Tsk’dır. Gerçek fiziksel sistemlerde, sistem çıkışları nedenselliği (causality) sağlayacak şekilde sadece önceki ve/veya şuan ki girişlere dayalı olmalıdır. Denklem (2)’de olduğu gibi nominal sistemin tersi alındığı zaman bu durum problem yaratabilir. Bunun önüne geçebilmek için tahmin edilen bozucu 𝑑̂ alttaki gibi ifade edilebilir: Gerçek fiziksel sistemlerde, sistem çıkışları nedenselliği (causality) sağlayacak şekilde sadece önceki ve/veya şuan ki girişlere dayalı olmalıdır. Denklem (2)’de olduğu gibi nominal sistemin tersi alındığı zaman bu durum problem yaratabilir. Bunun önüne geçebilmek için tahmin edilen bozucu 𝑑̂ alttaki gibi ifade edilebilir: g Kontrolcü r İletişim Ağı (CAN) Eyleyici Taşıt Sensör u y Kontrolcü- Eyleyici Gecikmesi (Tke) Sensör - Eyleyici Gecikmesi (Tsk) Şekil 1: Taşıtlarda kullanılan iletişim ağı kontrol sistemlerinin temel bileşenleri. 3 Taşıt savrulma dinamiği kontrolü Taşıt şasesinde beklenmedik savrulma momenti değişimleri taşıtın tehlikeli yanal hareketlerine sebep olabilir. Bu ani savrulma momenti değişimleri yol sürtünmesinin değişmesinden, agresif sürüş şeklinden, ani yan rüzgar alımından vb. nedenlerden kaynaklanabilir. Bu tip durumlarda taşıt kararlılığının artırılması taşıt savrulma dinamiği kontrolünün araştırma alanına girmektedir. Taşıt kararlılığını artırabilmek için çeşitli yaklaşımlar mevcuttur. Bunlara örnek olarak tekil frenleme, aktif ön tekerlek kontrolü ve aktif arka tekerlek kontrolü verilebilir. Konuyla ilgili daha fazla bilgi [23], [24] nolu kaynaklarda ve bu kaynaklardaki diğer yayınlarda bulunabilir. Bu çalışmada kablosuz direksiyon vasıtasıyla aktif ön tekerlek kontrolü göz önüne alınmıştır. 𝐺𝑛= 𝐾𝑛 𝜏𝑛𝑠+ 1 (8) (8) burada 𝜏𝑛 nominal taşıt savrulma dinamiğini yansıtacak şekilde seçilen zaman sabitini ve 𝐾𝑛= 𝑏0 𝑎0 ise nominal statik kazancı göstermektedir. 2 Zaman gecikmesi gözleyicisinin yapısı ZGG, zaman gecikmesi tahmini ve telafisi haricinde bozucu gözleyiciye benzer bir yapıya sahiptir. BG (Bozucu gözleyici), kontrol sistemleri alanında iyi bilinen modelleme hatalarının etkilerini azaltan ve bozucu etkiyi gidermede kullanılan bir yaklaşımdır. Bozucu gözleyici ilk kez 1987 yılında Ohnishi tarafından önerilmiş ve 1991 yılında Umeno ve Hori tarafından iyileştirilmiştir [20],[21]. 𝑑(𝑡) = 𝑢(𝑡) −𝑢(𝑡−T) (4) (4) Benzer şekilde Laplace formunda alttaki gibi yazılabilir: Benzer şekilde Laplace formunda alttaki gibi yazılabilir: 𝐷(𝑠) = 𝑈(𝑠) −𝑈(𝑠)𝑒−T𝑠 (5) (5) burada u sistem girişini, 𝑇 zaman gecikmesini, d ise iletişim ağı bozucusunu göstermektedir. BG yaklaşımında, nominal sistem modelinin tersi Şekil 2’de görüldüğü gibi bozucuyu tahmin için kullanılmaktadır. Bu tahmin edilen sinyal, sistemde bozucunun etkisini gidermek için kullanılabilir.    Ts D s U s U s e    n G s  U s    Ts D s U s U s e   +-    Ts n Y s G s e U s    n G s  U s    Ts D s U s U s e   +-    Ts n Y s G s e U s   Şekil 3: İletişim ağı bozucusu. Bozucu tahmini denklemleri alttaki gibi çıkarılabilir. Nominal sistem çıkışı y, nominal sistem modeli Gn, sistem girişi u ve giriş bozucusu d’ye göre alttaki gibi yazılabilir. 𝑦= 𝐺𝑛(𝑢−𝑑) (1) (1) Şekil 3: İletişim ağı bozucusu. 1451 Pamukkale Univ Muh Bilim Derg, 24(8), 1450-1457, 2018 r  f  f  r  CG fl rl x y V rF fF rV f V Şekil 5: Tek izli taşıt modeli. r  f  f  r  CG fl rl x y V rF fF rV f V Sekil 4’te görüldüğü gibi ZGG iki kısımdan oluşmaktadır. Bu kısımlar iletişim ağı bozucusu tahmini ve zaman gecikmesi telafisidir. İletişim ağı bozucusu BG’de olduğu gibi Denklem (3) kullanılarak tahmin edilebilir. Daha sonra bu tahmin geri besleme sinyalindeki zaman gecikmesi etkisini telafi etmek için kullanılır. Sekil 4’te görüldüğü gibi ZGG iki kısımdan oluşmaktadır. Bu kısımlar iletişim ağı bozucusu tahmini ve zaman gecikmesi telafisidir. İletişim ağı bozucusu BG’de olduğu gibi Denklem (3) kullanılarak tahmin edilebilir. Daha sonra bu tahmin geri besleme sinyalindeki zaman gecikmesi etkisini telafi etmek için kullanılır. 2 Zaman gecikmesi gözleyicisinin yapısı  Ts G s e  U s    Ts Y s G s U s e   ˆD s  Q s +-   n Q s G s  C s  n G s  E s  R s +- + + Zaman Gecikmesi Telafisi İletişim Ağı Gecikmesi Tahmini      ˆ Ts D s Q s U s Q s U s e     ˆ n D s G s Şekil 4: Zaman gecikmesi gözleyicisinin yapısı, [4]. Şekil 5: Tek izli taşıt modeli. buradaki katsayılar şu şekilde tanımlanabilir: 𝑏1 = 𝜇𝐶𝑓𝑙𝑓𝑚𝑉2, 𝑏0 = 𝜇2𝐶𝑓𝐶𝑟(𝑙𝑓+ 𝑙𝑟)𝑉, 𝑎2 = 𝐽𝑚𝑉2 𝑎1 = 𝜇(𝐶𝑓( 𝐽+ 𝑙𝑓 2𝑚) + 𝐶𝑟( 𝐽+ 𝑙𝑟2𝑚)) 𝑉 𝑎0 = 𝜇2𝐶𝑓𝐶𝑟(𝑙𝑓+ 𝑙𝑟) 2 + 𝜇(𝐶𝑟𝑙𝑟−𝐶𝑓𝑙𝑓)𝑚𝑉2 Şekil 4: Zaman gecikmesi gözleyicisinin yapısı, [4]. Şekil 4: Zaman gecikmesi gözleyicisinin yapısı, [4]. Şekil 4’te görülen referans girişi r ile sistem çıkışı y arasındaki kapalı çevrim transfer fonksiyonu alttaki gibi yazılabilir: Burada, 𝜇 tekerlek-yol sürtünme katsayısını, 𝑚 taşıt kütlesini, 𝐽 taşıt atalet momentini, V taşıt hızını, 𝐶𝑓 ve 𝐶𝑟 sırasıyla ön ve arka tekerlekler için toplam dönüş katılığını, 𝑙𝑓 taşıt ön aksı ile taşıt ağırlık merkezi arasındaki mesafeyi, 𝑙𝑟 taşıt arka aksı ile taşıt ağırlık merkezi arasındaki mesafeyi göstermektedir. Parametrelerin sayısal değerleri şu şekildedir: 𝜇= 1, 𝑚= 1296 𝑘𝑔, 𝐽= 1759 𝑘𝑔𝑚2, 𝑙𝑓= 1.25 𝑚, 𝑙𝑟= 1.32 𝑚, 𝐶𝑓= 84000 𝑁/𝑟𝑎𝑑, 𝐶𝑟= 96000 𝑁/𝑟𝑎𝑑. 𝐺𝑦𝑟= 𝐶𝐺𝑒−𝑇𝑠 1 + 𝐶𝐺𝑛𝑄+ 𝐶𝐺𝑒−𝑇𝑠(1 −𝑄) (6) (6) burada G belirsizlikte içeren sistem modelini, Gn ise nominal (istenen) sistem modelini ve C kontrol sistemini göstermektedir. Denklem (6)’da görüldüğü gibi birim kazançlı alçak geçiren Q filtresi kullanılarak belirlenen frekans değerine kadar paydadaki (sistemin karakteristik denklemindeki) zaman gecikmesi etkisi giderilebilir. Taşıt savrulma açısal hızı ile direksiyon açısı arasında istenen değişim göz önüne alınarak zaman gecikmesi telafisinde kullanılacak nominal transfer fonksiyonu 𝐺𝑛, birinci mertebeden transfer fonksiyonu olarak alttaki gibi seçilebilir: 4 Zaman gecikmesi gözleyicisinin taşıt savrulma dinamiği kontrolüne uygulanması Şekil 6’da ZGG’siz ve ZGG’li taşıt savrulma dinamiği kontrol sistemi blok diyagramı gösterilmiştir. Taşıt savrulma dinamiği kontrolünde amaç taşıt savrulma açısal hızını referans (istenen) değere göre kontrol etmektir. Referans savrulma açısal hızı nominal model 𝐺𝑛 kullanılarak hesaplanabilir. Referans ve ölçülen değer farkı olarak hesaplanan savrulma açısal hızı hata değeri 𝑒 kontrolcüden geçirilerek taşıt savrulma hareketini kararlı hale getiren kontrol sinyali 𝑢 elde edilir. Bu bildiride kontrolcü 𝐶 bir PI kontrolcü olarak tasarlanmıştır. Tasarlanan PI kontrol sistemi üzerine ZGG tasarımı yapılarak eklenmiştir. Taşıt savrulma dinamiğinin modellenmesinde ve kontrol sistemi tasarımında bisiklet modeli adı da verilen tek izli taşıt modeli kullanılabilir. Tek izli taşıt modelinde ön ve arka tekerlekler tek bir tekerleğe indirgenerek ifade edilmektedir. Bu model tekerlek davranışlarının lineer bir değişim gösterdiği günlük sürüş koşullarında taşıt yanal dinamiğini iyi bir şekilde yansıtmaktadır [25]. Şekil 5’te tek izli taşıt modeli geometrisi, parametreleri ve değişkenleri gösterilmektedir. Ön tekerlek açısı 𝛿𝑓 ile savrulma açısal hızı r arasındaki transfer fonksiyonu G, tek izli taşıt modelinin doğrusallaştırılmasıyla alttaki gibi yazılabilir [25]: 4.2 ZGG için Q filtresi kesim frekansının seçilmesi 4.2 ZGG için Q filtresi kesim frekansının seçilmesi Q filtresinin kesim frekansının seçimi model regülasyonu ve bozucu etkisinin engellenmesi istenen frekans bandına, yüksek frekanstaki sensör gürültüsü etkisinin engellenmesine, dayanıklı kararlılığa ve eyleyici bant genişliğine bağlı bir tasarım parametresidir [22]. Kazanç geçiş frekansında (𝜔𝑔𝑐), çevrim kazanç transfer fonksiyonu 𝐿(𝑠) alttaki gibi yazılabilir: 𝐿(𝑗𝜔𝑔𝑐) = \|𝐿(𝑗𝜔𝑔𝑐)\|ej∠𝐿(𝑗𝜔𝑔𝑐) (10) (10) ZGG için dayanıklı kararlılığı gözönüne alan Nyquist kararlılık kriteri tabanlı bir koşul [4] nolu kaynakta geliştirilmiştir. Bu koşul alttaki biçimde ifade edilebilir: Kazanç marjininin ve faz marjininin tanımı kullanılarak sırasıyla \|𝐿(𝑗𝜔𝑔𝑐)\| = 1 ve ∠𝐿(𝑗𝜔𝑔𝑐) = −𝜋+ 𝑃𝑀 olarak yazılabilir. Bu bilgiler kullanılarak denklem (10) Kazanç marjininin ve faz marjininin tanımı kullanılarak sırasıyla \|𝐿(𝑗𝜔𝑔𝑐)\| = 1 ve ∠𝐿(𝑗𝜔𝑔𝑐) = −𝜋+ 𝑃𝑀 olarak yazılabilir. Bu bilgiler kullanılarak denklem (10) \|CGn(1 −Q) 1 + CGn \| < \| 1 ∆𝑚 \| , ∀ω (15) 𝐿(𝑗𝜔𝑔𝑐) = ej(−𝜋+𝑃𝑀) (11) (15) (11) olarak ifade edilebilir. Euler formülü (𝑒𝑗𝜃= 𝑐𝑜𝑠𝜃+ 𝑗𝑠𝑖𝑛𝜃) ve denklem (9) kullanılarak çevrim kazanç transfer fonksiyonu reel ve imajiner kısımlardan oluşacak şekilde alttaki gibi ifade edilebilir: burada 𝐶 kontrol sistemini, 𝐺𝑛 nominal sistem modelini, 𝑄= 𝜔𝑐(𝑠+ 𝜔𝑐) ⁄ alçak geçiren ZGG filtresini ve ∆𝑚 çarpımsal (multiplicative) belirsizliği göstermektedir. Zaman gecikmeli sistem için çarpımsal belirsizlik alttaki gibi ifade edilebilir: burada 𝐶 kontrol sistemini, 𝐺𝑛 nominal sistem modelini, 𝑄= 𝜔𝑐(𝑠+ 𝜔𝑐) ⁄ alçak geçiren ZGG filtresini ve ∆𝑚 çarpımsal (multiplicative) belirsizliği göstermektedir. Zaman gecikmeli sistem için çarpımsal belirsizlik alttaki gibi ifade edilebilir: 𝐿(𝑗𝜔𝑔𝑐) = −cos(Tmaksωgc) −jsin(Tmaksωgc) (12) (12) ∆m= Ge−Tmakss −Gn Gn (16) (16) Çevrim kazancı transfer fonksiyonunun reel ve imajiner kısımları denklem (12)’nin sağ tarafına eşitlenerek alttaki tasarım denklemleri elde edilebilir: Denklem (15)’teki koşulu sağlayacak kapalı çevrim sistemi dayanıklı kararlı yapacak şekilde alçak geçiren 𝑄 filtresinin kesim frekansı seçilebilir. Önceki bölümde bahsedilen iki farklı PI kontrol sistemi içeren ZGG’li sistemler için bu koşul uygulanarak 𝑄 filtresinin kesim frekansı sırasıyla 600 rad/s ve 150 rad/s olarak seçilmiştir. Şekil 8 ve 9’da seçilen kesim frekanslarıyla denklem (15)’teki koşulun sağlandığı diğer bir ifadeyle eğrilerin kesişmeyerek denklem (15)’teki dayanıklı kararlılık eşitsizliğinin sağlandığı görülmektedir. ZGG’nin gerçek sistemlere uygulanmasında 𝑄 filtresinin bant genişliği kullanılan eyleyicinin bant genişliğiyle de sınırlanmaktadır. Uygulamada bu durum da dikkate alınmalıdır. Denklem (15)’teki koşulu sağlayacak kapalı çevrim sistemi dayanıklı kararlı yapacak şekilde alçak geçiren 𝑄 filtresinin kesim frekansı seçilebilir. Önceki bölümde bahsedilen iki farklı PI kontrol sistemi içeren ZGG’li sistemler için bu koşul uygulanarak 𝑄 filtresinin kesim frekansı sırasıyla 600 rad/s ve 150 rad/s olarak seçilmiştir. 4.1 Zaman gecikmesi marjinine göre parametre uzayında kontrol sistemi tasarımı PI kontrol sisteminin tasarımı için zaman gecikmesi marjinini dikkate alan parametre uzayı tabanlı bir yaklaşım önerilmiştir. Parametre uzayı yöntemiyle tasarım konusunda daha fazla bilgi [22] ve [25] nolu kaynaklarda bulunabilir. 𝐺(𝑠) = r(s) 𝛿𝑓(𝑠) = 𝑏1𝑠+ 𝑏0 𝑎2𝑠2 + 𝑎1𝑠+ 𝑎0 (7) (7) 1452 Pamukkale Univ Muh Bilim Derg, 24(8), 1450-1457, 2018 (TOK’2017 – Otomatik Kontrol Türk Milli Komitesi Ulusal Toplantısı Özel Sayısı) M.T. Emirler, B.A. Güvenç, L. Güvenç u y ist r +- C İstenen değerin hesabı f  Ts Ge e gecikmesini tolere edebilecek (𝑘𝑝, 𝑘𝑖) değerlerini göstermektedir. Pamukkale Univ Muh Bilim Derg, 24(8), 1450-1457, 2018 (TOK’2017 – Otomatik Kontrol Türk Milli Komitesi Ulusal Toplantısı Özel Sayısı) M.T. Emirler, B.A. Güvenç, L. Güvenç Pamukkale Univ Muh Bilim Derg, 24(8), 1450-1457, 2018 (TOK’2017 – Otomatik Kontrol Türk Milli Komitesi Ulusal Toplantısı Özel Sayısı) M.T. Emirler, B.A. Güvenç, L. Güvenç M.T. Emirler, B Q +- n Q G +- + + (b) u y ist r (a) +- C İstenen değerin hesabı C İstenen değerin hesabı n G u y ist r f  f  Ts Ge Ts Ge e e Şekil 6: Taşıt savrulma dinamiği kontrol sistemi blok diyagramı. (a): ZGG’siz, (b): ZGG’li, [26]. u y ist r +- C İstenen değerin hesabı f  Ts Ge e (𝑘𝑝, 𝑘𝑖) değerlerini gecikmesini tolere edebilecek (𝑘𝑝, 𝑘𝑖) değerlerini göstermektedir. değerlerini ZGG’nin performansını test etmek için iki adet PI kontrol katsayısı seti seçilmiştir. Bunlardan ilki zaman gecikmesini tolere etmeyecek şekilde (𝑘𝑝1, 𝑘𝑖1) = (3, 15), ikincisi ise zaman gecikmesini tolere edecek şekilde (𝑘𝑝2, 𝑘𝑖2) = (0.5, 5) olarak parametre uzayı çözüm bölgesinden seçilmiştir. Şekil 7’de seçilen kontrol katsayıları çarpı ile işaretlenmiştir. İlk seçilen set, zaman gecikmesinin kontrolcü tasarımı sırasında bilinmediği durumu yansıtmaktadır. Q +- n Q G +- + + (b) (a) C İstenen değerin hesabı n G u y ist r f  Ts Ge e (a) Şekil 7: 𝑘𝑝− 𝑘𝑖 parametre uzayı zaman gecikmesi sınırı. (b) (b) Şekil 6: Taşıt savrulma dinamiği kontrol sistemi blok diyagramı. (a): ZGG’siz, (b): ZGG’li, [26]. Zaman gecikmesi marjini alttaki gibi tanımlanabilir [27]: 𝑇𝑚𝑎𝑘𝑠= 𝑃𝑀 𝜔𝑔𝑐 (9) (9) Şekil 7: 𝑘𝑝− 𝑘𝑖 parametre uzayı zaman gecikmesi sınırı. burada 𝑇𝑚𝑎𝑘𝑠 sistem kararsız hale geçmeden tolere edilebilecek zaman gecikmesini, 𝑃𝑀 faz marjinini ve 𝜔𝑔𝑐 kazanç geçiş frekansını göstermektedir. 5 Simülasyon sonuçları ve performans analizi CAN hattında bulunan zamanla değişen iletişim ağı kaynaklı gecikme, kontrolcü-eyleyici arasındaki gecikme Tke ile sensör- kontrolcü arasındaki gecikmenin Tsk toplamı olarak ifade edilebilir. CAN hattı kaynaklı zamanla değişen iletişim ağı gecikmesi 6 ms ile 30 ms arasında alınabilir [18]. Şekil 10’da bu zamanla değişen sınırlı gecikme görülmektedir. Şekil 11: Zamanla değişen gecikme durumunda savrulma açısal hızı sonuçları. Şekil 12: Zamanla değişen gecikme durumunda kontrol işareti değişimleri. Şekil 10: CAN iletişiminde zamanla değişen gecikme T(t). Şekil 12: Zamanla değişen gecikme durumunda kontrol işareti değişimleri. Şekil 10: CAN iletişiminde zamanla değişen gecikme T(t). 5.2 Zaman gecikmesini tolere eden PI kontrol sistemiyle birlikte ZGG’nin kullanımı Bu çalışmada, ZGG’nin performansını test etmek için çeşitli simülasyon çalışmaları yapılmıştır. İlk iki simülasyonda farklı PI kontrol katsayıları için Şekil 10’da görülen zamanla değişen gecikme için istenen, ZGG’siz ve ZGG’li savrulma açısal hızı değerleri karşılaştırılmıştır. Üçüncü simülasyonda taşıt savrulma dinamiği problemi için Smith tahmincili ve ZGG’li sisteminin sonuçları kararlılık ve performans açısından karşılaştırılmıştır. Dördüncü simülasyonda ise sinüzoidal ön tekerlek açısı girişi için ZGG’siz ve ZGG’li PI kontrol sistemi kullanılarak taşıt savrulma açısal hızı değerleri ve kontrol işaretleri karşılaştırılmıştır. Bu ikinci simülasyon çalışmasında, önceki simülasyona benzer bir çalışma yapılmıştır. Daha az agresif bir manevra olacak şekilde taşıt ön tekerlek açısı girişi (𝛿𝑓) 4 derece olarak verilmiştir. Taşıt hızı 10 m/s olarak alınmıştır. PI kontrol katsayıları CAN hattı zaman gecikmesini tolere edecek şekilde (𝑘𝑝2, 𝑘𝑖2) = (0.5, 5) olarak Şekil 7’de gösterilen parametre uzayı çözümünden seçilmiştir. Şekil 13’te görülen sonuçlara göre zaman gecikmesini tolere edecek şekilde parametre uzayında tasarlanmış PI kontrol sistemiyle gecikme etkisi giderilebilmekte ve kapalı çevrim kontrollü sistem kararlı kılınabilmektedir. ZGG’nin sisteme eklendiği durumda geçici rejim bölgesinde istenen değerlere daha iyi bir yakınsama sağlanabilmektedir. 4.2 ZGG için Q filtresi kesim frekansının seçilmesi Şekil 8: (𝑘𝑝1, 𝑘𝑖1) = (3, 15) ve seçilen 600 rad/s kesim frekanslı 𝑄 filtresi için dayanıklı kararlılık durumu. Şekil 8: (𝑘𝑝1, 𝑘𝑖1) = (3, 15) ve seçilen 600 rad/s kesim frekanslı 𝑄 filtresi için dayanıklı kararlılık durumu. Şekil 8: (𝑘𝑝1, 𝑘𝑖1) = (3, 15) ve seçilen 600 rad/s kesim frekanslı 𝑄 filtresi için dayanıklı kararlılık durumu. k l (𝑘 𝑘) ( 5 5) l 5 d/ k Şekil 11: Zamanla değişen gecikme durumunda savrulma açısal hızı sonuçları. Şekil 9: (𝑘𝑝2, 𝑘𝑖2) = (0.5, 5) ve seçilen 150 rad/s kesim Şekil 9: (𝑘𝑝2, 𝑘𝑖2) = (0.5, 5) ve seçilen 150 rad/s kesim frekanslı 𝑄 filtresi için dayanıklı kararlılık durumu. Ş ( 𝑝2, 𝑖2) ( , ) ç / frekanslı 𝑄 filtresi için dayanıklı kararlılık durumu. 𝑝 frekanslı 𝑄 filtresi için dayanıklı kararlılık durumu. 5 Simülasyon sonuçları ve performans analizi 4.2 ZGG için Q filtresi kesim frekansının seçilmesi Şekil 8 ve 9’da seçilen kesim frekanslarıyla denklem (15)’teki koşulun sağlandığı diğer bir ifadeyle eğrilerin kesişmeyerek denklem (15)’teki dayanıklı kararlılık eşitsizliğinin sağlandığı görülmektedir. ZGG’nin gerçek sistemlere uygulanmasında 𝑄 filtresinin bant genişliği kullanılan eyleyicinin bant genişliğiyle de sınırlanmaktadır. Uygulamada bu durum da dikkate alınmalıdır. 𝑅𝑒{𝐿(𝑗𝜔, 𝑘𝑝, 𝑘𝑖)} = −cos(Tmaksω) (13) 𝐼𝑚{𝐿(𝑗𝜔, 𝑘𝑝, 𝑘𝑖)} = −sin(Tmaksω) (14) (13) (14) Denklem (13) ve (14), 𝜔 frekansına bağlı olarak iki serbest parametre olarak seçilen kontrol katsayıları 𝑘𝑝 ve 𝑘𝑖 için çözülebilir. 𝜔 frekansı taranarak istenen maksimum zaman gecikmesini tolere edebilecek kontrol katsayısı değerleri iki boyutlu parametre düzleminde gösterilebilir. Denklem (13) ve (14), 𝜔 frekansına bağlı olarak iki serbest parametre olarak seçilen kontrol katsayıları 𝑘𝑝 ve 𝑘𝑖 için çözülebilir. 𝜔 frekansı taranarak istenen maksimum zaman gecikmesini tolere edebilecek kontrol katsayısı değerleri iki boyutlu parametre düzleminde gösterilebilir. CAN hattı zaman gecikmesinin maksimum değeri 30 ms [18] alınarak pozitif kontrol katsayısı değerleri için hesaplanan parametre uzayı çözüm bölgesi Şekil 7’de gösterilmiştir. Burada yeşil bölge sistem kararsız olmadan 30 ms zaman 1453 Pamukkale Univ Muh Bilim Derg, 24(8), 1450-1457, 2018 (TOK’2017 – Otomatik Kontrol Türk Milli Komitesi Ulusal Toplantısı Özel Sayısı) M.T. Emirler, B.A. Güvenç, L. Güvenç Pamukkale Univ Muh Bilim Derg, 24(8), 1450-1457, 2018 (TOK’2017 – Otomatik Kontrol Türk Milli Komitesi Ulusal Toplantısı Özel Sayısı) M.T. Emirler, B.A. Güvenç, L. Güvenç Şekil 8: (𝑘𝑝1, 𝑘𝑖1) = (3, 15) ve seçilen 600 rad/s kesim frekanslı 𝑄 filtresi için dayanıklı kararlılık durumu. Şekil 9: (𝑘𝑝2, 𝑘𝑖2) = (0.5, 5) ve seçilen 150 rad/s kesim frekanslı 𝑄 filtresi için dayanıklı kararlılık durumu. sistemleri için elde edilmiştir. CAN hattında Şekil 10’da gösterilen 6 ms ile 30 ms arasında zamanla değişen gecikme söz konusudur. Taşıta ön tekerlek açısı olarak (𝛿𝑓) 8 derece verilmiştir. Taşıt hızı 30 m/s olarak alınmıştır. Burada PI kontrol katsayıları CAN hattı zaman gecikmesini tolere edemeyecek şekilde (𝑘𝑝1, 𝑘𝑖1) = (3, 15) olarak seçilmiştir. Bu seçimdeki amaç, kontrol sistemi tasarımı sırasında zaman gecikmesinin bilinmediği durumu yansıtarak en kötü durumda ZGG’nin performansını test etmektir. Şekil 8: (𝑘𝑝1, 𝑘𝑖1) = (3, 15) ve seçilen 600 rad/s kesim frekanslı 𝑄 filtresi için dayanıklı kararlılık durumu. Şekil 11’de görülen sonuçlara göre zamanla değişen gecikme durumunda sadece PI kontrolcü kullanıldığında sistem kararsızlığa gitmektedir. ZGG’nin eklendiği durumdaysa sistem kararlı kalmakta ve istenen modele yakınsamaktadır. Şekil 12’de sadece PI kontrolcü kullanıldığında ve ZGG’li PI kontrollü sistemdeki kontrol işareti değişimi görülmektedir. ZGG’nin eklenmediği durumda kontrol işareti belli bir değere oturamamaktadır. 5.3 Smith tahminci ile ZGG’nin karşılaştırılması Sekil 15’te yapısı gösterilen Smith tahmincisi zaman gecikmesi telafisinde kullanılan en yaygın metotlardan biridir. Daha önce belirtildiği gibi Smith tahmincisi zaman gecikmesi modelini kullanmakta ve kullanılan model ile sistemdeki gerçek zaman gecikmesi arasındaki fark artıkça tahmincinin gecikmeyi telafi etme performansı düşmektedir. Şekil 16: Çeşitli zaman gecikmeleri için Smith tahmincili sistemin sonuçları. Sekil 17’de Smith tahmincili sistemde çeşitli zaman gecikmeleri için elde edilen kontrol işareti değişimleri görülmektedir. Sadece Smith tahmincisi modelinde kullanılan gecikme ile sistem gecikmesi aynı olduğu zaman salınımsız bir kontrol işareti oluşmaktadır. Sekil 17’de Smith tahmincili sistemde çeşitli zaman gecikmeleri için elde edilen kontrol işareti değişimleri görülmektedir. Sadece Smith tahmincisi modelinde kullanılan gecikme ile sistem gecikmesi aynı olduğu zaman salınımsız bir kontrol işareti oluşmaktadır.  Ts G s e  U s  Y s  C s  E s  R s +- + + Smith Tahmincisi   1 d T s n G s e  Şekil 15: Smith tahmincisi yapısı.  Ts G s e  U s  Y s  C s  E s  R s +- + + Smith Tahmincisi   1 d T s n G s e  Şekil 15: Smith tahmincisi yapısı.  Ts G s e  U s  Y s  C s  E s  R s +- + + Smith Tahmincisi   1 d T s n G s e  Şekil 15: Smith tahmincisi yapısı. Şekil 17: Çeşitli zaman gecikmeleri için Smith tahmincili sistemin kontrol işareti değişimleri. Şekil 17: Çeşitli zaman gecikmeleri için Smith tahmincili sistemin kontrol işareti değişimleri. Aynı simülasyon, farkı görmek için Smith tahmincisi yerine ZGG kullanılarak da gerçekleştirilmiştir. Şekil 18’de görüldüğü gibi aynı zaman gecikmesi değerleri için ZGG çok daha iyi bir performans göstermiş ve sistem kararlılığı sağlanmıştır. Şekil 15: Smith tahmincisi yapısı. Şekil 15’te gösterilen Smith tahmincisi eklenmiş kontrollü sistem için referans giriş r ile sistem çıkışı y arasındaki transfer fonksiyonu alttaki gibi yazılabilir: 𝐺𝑦𝑟𝑆𝑚𝑖𝑡ℎ= 𝐶𝐺𝑒−𝑇𝑠 1 + 𝐶𝐺𝑛(1 −𝑒−𝑇𝑑𝑠) + 𝐶𝐺𝑒−𝑇𝑠 (17) Şekil 17: Çeşitli zaman gecikmeleri için Smith tahmincili sistemin kontrol işareti değişimleri. (17) Aynı simülasyon, farkı görmek için Smith tahmincisi yerine ZGG kullanılarak da gerçekleştirilmiştir. Şekil 18’de görüldüğü gibi aynı zaman gecikmesi değerleri için ZGG çok daha iyi bir performans göstermiş ve sistem kararlılığı sağlanmıştır. 5.1 Zamanla değişen gecikmede ZGG’nin etkisi Bu simülasyon çalışmasında¸ Sekil 6’da gösterilen ZGG’siz ve ZGG’li taşıt savrulma dinamiği kontrol sistemleri kurulmuş ve taşıt savrulma açısal hızları istenen, ZGG’siz ve ZGG’li kontrol 1454 Pamukkale Univ Muh Bilim Derg, 24(8), 1450-1457, 2018 (TOK’2017 – Otomatik Kontrol Türk Milli Komitesi Ulusal Toplantısı Özel Sayısı) M.T. Emirler, B.A. Güvenç, L. Güvenç Pamukkale Univ Muh Bilim Derg, 24(8), 1450-1457, 2018 (TOK’2017 – Otomatik Kontrol Türk Milli Komitesi Ulusal Toplantısı Özel Sayısı) M.T. Emirler, B.A. Güvenç, L. Güvenç Şekil 13: Zaman gecikmesinin PI kontrol sistemiyle tolere edildiği durumunda savrulma açısal hızı sonuçları. performansını etkilediği görülmektedir. 𝑇𝑑’nin değeri, sistemdeki zaman gecikmesi 𝑇 ile aynı olduğunda karakteristik denklemde zaman gecikmesi elemanı (üstel eleman) kalmamaktadır. Smith tahmincisinin görevini iyi bir şekilde gerçekleştirebilmesi için sistemdeki gecikme değeri 𝑇’nin tasarım sırasında bilinmesi ve 𝑇𝑑’nin buna göre seçilmesi gerekmektedir. Diğer yandan denklem (6)’da gösterilen ZGG’li kapalı çevrim transfer fonksiyonu 𝐺𝑦𝑟 incelendiğinde zaman gecikmesi tahminine ihtiyaç duyulmadığı seçilen 𝑄 filtresiyle zaman gecikmesi elemanının karakteristik denklemden elenebildiği görülmektedir. Zaman gecikmesi tahminine ihtiyaç duyulmaması ZGG’nin zamanla değişen gecikmelerde de kullanımına imkan sağlayarak Smith tahmincisine göre önemli bir avantaj sağlamaktadır. Şekil 13: Zaman gecikmesinin PI kontrol sistemiyle tolere edildiği durumunda savrulma açısal hızı sonuçları. Şekil 14’te bu simülasyon için sadece PI ve ZGG’li PI kontrollü sistemlerin kontrol işareti değişimleri görülmektedir. ZGG’nin eklendiği durumda geçici rejim bölgesinde kontrol işaretinin değeri sadece PI kontrollü sisteme göre biraz daha yüksek kalmakta; ancak, daha düzgün bir kontrol işareti elde edilmektedir. Smith tahmincisinin performansını test etmek için bir simülasyon çalışması yapılmıştır. Smith tahmincisinde kullanılan zaman gecikmesi modelindeki tahmini gecikme (𝑇𝑑) CAN hattında zamanla değişen gecikmelerin (6 ms, 30 ms) ortalama değeri olarak 18 ms alınmıştır. Daha sonra sistemdeki gecikme (𝑇)’nin farklı değerleri için simülasyonlar yapılmıştır. Burada PI kontrol katsayıları (𝑘𝑝1, 𝑘𝑖1) = (3, 15) olarak alınmıştır. Şekil 14: Zaman gecikmesinin PI kontrol sistemiyle tolere edildiği durumunda kontrol işareti değişimleri. Şekil 16’da gösterilen sonuçlara göre zaman gecikmesi modelinde kullanılan tahmini gecikme (𝑇𝑑) ile sistem gecikmesi (𝑇) aynı olduğu zaman Smith tahmincisi zaman gecikmesini iyi bir şekilde telafi etmiştir. Ancak model ile gerçek gecikme arasındaki fark artıkça telafi kötüleşmiş ve hatta 30 ms gecikme için sistem kararsız davranış göstermiştir. Şekil 16: Çeşitli zaman gecikmeleri için Smith tahmincili sistemin sonuçları. Şekil 14: Zaman gecikmesinin PI kontrol sistemiyle tolere edildiği durumunda kontrol işareti değişimleri. 5.3 Smith tahminci ile ZGG’nin karşılaştırılması Denklem (17) ile gösterilen kapalı çevrim transfer fonksiyonu 𝐺𝑦𝑟𝑆𝑚𝑖𝑡ℎ incelediğinde ve sistem modeli ile nominal modelin aynı olduğu varsayıldığında (𝐺= 𝐺𝑛), Smith tahmincisinde kullanılan tahmini gecikme 𝑇𝑑’nin sistem Aynı simülasyon, farkı görmek için Smith tahmincisi yerine ZGG kullanılarak da gerçekleştirilmiştir. Şekil 18’de görüldüğü gibi aynı zaman gecikmesi değerleri için ZGG çok daha iyi bir performans göstermiş ve sistem kararlılığı sağlanmıştır. 1455 Pamukkale Univ Muh Bilim Derg, 24(8), 1450-1457, 2018 (TOK’2017 – Otomatik Kontrol Türk Milli Komitesi Ulusal Toplantısı Özel Sayısı) M.T. Emirler, B.A. Güvenç, L. Güvenç Şekil 18: Çeşitli zaman gecikmeleri için ZGG’li sistemin sonuçları. Şekil 21: Sinüzoidal ön tekerlek açısı girişi durumunda savrulma açısal hızı sonuçları. Ş kil 22’d i id l k l k i i i i l i i Pamukkale Univ Muh Bilim Derg, 24(8), 1450-1457, 2018 (TOK’2017 – Otomatik Kontrol Türk Milli Komitesi Ulusal Toplantısı Özel Sayısı) M.T. Emirler, B.A. Güvenç, L. Güvenç Pamukkale Univ Muh Bilim Derg, 24(8), 1450-1457, 2018 (TOK’2017 – Otomatik Kontrol Türk Milli Komitesi Ulusal Toplantısı Özel Sayısı) M.T. Emirler, B.A. Güvenç, L. Güvenç Pamukkale Univ Muh Bilim Derg, 24(8), 1450-1457, 2018 (TOK’2017 – Otomatik Kontrol Türk Milli Komitesi Ulusal Toplantısı Özel Sayısı) M.T. Emirler, B.A. Güvenç, L. Güvenç Şekil 18: Çeşitli zaman gecikmeleri için ZGG’li sistemin sonuçları. Şekil 21: Sinüzoidal ön tekerlek açısı girişi durumunda savrulma açısal hızı sonuçları. Şekil 18: Çeşitli zaman gecikmeleri için ZGG’li sistemin sonuçları. Şekil 21: Sinüzoidal ön tekerlek açısı girişi durumunda savrulma açısal hızı sonuçları. Şekil 18: Çeşitli zaman gecikmeleri için ZGG’li sistemin sonuçları. Şekil 22’de sinüzoidal ön tekerlek açısı girişi simülasyonu için kontrol işareti değişimi ZGG eklenmemiş ve ZGG eklenmiş PI kontrol sistemi için verilmiştir. Sadece PI kontrol sistemi kullanıldığı durumda kararsızlığa giden kontrol sinyali görülmektedir. Şekil 22’de sinüzoidal ön tekerlek açısı girişi simülasyonu için kontrol işareti değişimi ZGG eklenmemiş ve ZGG eklenmiş PI kontrol sistemi için verilmiştir. Sadece PI kontrol sistemi kullanıldığı durumda kararsızlığa giden kontrol sinyali görülmektedir. Şekil 19’da ise ZGG’li sistemde farklı zaman gecikmelerinde elde edilen kontrol işareti değişimleri görülmektedir. Sadece geçici rejim bölgesinde kontrol işareti değişimi farklılık göstermiş daha sonra kontrol işareti tüm gecikme değerleri için aynı değere oturmuştur. Şekil 19: Çeşitli zaman gecikmeleri için ZGG’li sistemde kontrol işareti değişimleri. Şekil 22: Sinüzoidal ön tekerlek açısı girişi durumunda kontrol işareti değişimleri. Şekil 19: Çeşitli zaman gecikmeleri için ZGG’li sistemde kontrol işareti değişimleri. Şekil 22: Sinüzoidal ön tekerlek açısı girişi durumunda kontrol işareti değişimleri. 6 Sonuçlar Bu dördüncü simülasyon çalışmasında sinüzoidal ön tekerlek açısı girişi için ZGG’siz ve ZGG’li kontrol sistemleri için sonuçlar elde edilmiştir. Taşıta ön tekerlek açısı (𝛿𝑓) olarak genliği 10 derece olan 0.4 Hz frekanslı sinüzoidal giriş uygulanmıştır. Bu giriş radyan cinsinden Şekil 20’de gösterilmiştir. Simülasyonda taşıt hızı 30 m/s olarak alınmıştır. CAN hattında 6 ms ile 30 ms arasında zamanla değişen gecikme dikkate alınmıştır. Burada PI kontrol katsayıları (𝑘𝑝1, 𝑘𝑖1) = (3, 15) olarak kullanılmıştır. Bu çalışmada CAN tabanlı taşıt savrulma dinamiği kontrolünde zaman gecikmesi etkisini telafi edecek zaman gecikmesi gözleyicisi önerilmiştir. ZGG’siz ve ZGG’li taşıt savrulma dinamiği kontrolü sistemlerinin cevapları elde edilerek karşılaştırılmıştır. ZGG’li sistemin zamanla değişen gecikme etkisini çeşitli girişler için telafi ederek sistemi kararlı kıldığı görülmüştür. ZGG, literatürde zaman gecikmesi telafisi için sıklıkla kullanılan Smith tahmincisi ile karşılaştırılmıştır. Elde edilen sonuçlara göre ZGG’nin zamanla değişen gecikmeyi başarıyla telafi etmesi ve tam (kesin) zaman gecikmesi modeline ihtiyaç duymaması yüzünden Smith tahmincisine göre daha iyi performans gösterdiği görülmüştür. Ayrıca zaman gecikmesi marjinine göre parametre uzayında kontrol sistemi tasarımı için analitik denklemler elde edilmiş ve PI kontrol sistemi tasarımında kullanılmıştır. ZGG sadece CAN tabanlı taşıt savrulma dinamiği kontrolünde değil, kooperatif adaptif seyir kontrolü gibi zaman gecikmesinin önem kazandığı başka taşıt dinamiği kontrolü (otomotiv kontrolü) problemlerinde de uygulanabilir. Bu konuların üzerinde ilerleyen çalışmalarda durulacaktır. Şekil 20: Sinüzoidal ön tekerlek açısı girişi. Şekil 21’de görülen savrulma açısal hızı sonuçlarına göre, sadece PI kontrolcü kullanıldığında sistem zaman gecikmesinin etkisiyle kararsızlığa gitmektedir. ZGG’nin eklendiği durumdaysa sistem kararlı olmaktadır ve taşıt savrulma açısal hızı istenen savrulma açısal hızı değerlerine yakınsamaktadır. Şekil 20: Sinüzoidal ön tekerlek açısı girişi. Şekil 20: Sinüzoidal ön tekerlek açısı girişi. 7 Kaynaklar Şekil 21’de görülen savrulma açısal hızı sonuçlarına göre, sadece PI kontrolcü kullanıldığında sistem zaman gecikmesinin etkisiyle kararsızlığa gitmektedir. ZGG’nin eklendiği durumdaysa sistem kararlı olmaktadır ve taşıt savrulma açısal hızı istenen savrulma açısal hızı değerlerine yakınsamaktadır. Şekil 21’de görülen savrulma açısal hızı sonuçlarına göre, sadece PI kontrolcü kullanıldığında sistem zaman gecikmesinin etkisiyle kararsızlığa gitmektedir. ZGG’nin eklendiği durumdaysa sistem kararlı olmaktadır ve taşıt savrulma açısal hızı istenen savrulma açısal hızı değerlerine yakınsamaktadır. [1] Smith OJM. “A controller to overcome dead time”. ISA Journal, 6(2), 28-33, 1959. [2] Rao AS, Chidambaram M. “Enhanced Smith predictor for unstable processes with time delay”. Industrial & Engineering Chemistry Research, 44(22), 8291-8299, 2004. 1456 Pamukkale Univ Muh Bilim Derg, 24(8), 1450-1457, 2018 (TOK’2017 – Otomatik Kontrol Türk Milli Komitesi Ulusal Toplantısı Özel Sayısı) M.T. Emirler, B.A. Güvenç, L. Güvenç [3] Normey-Rico JE, Camacho EF. “Unified approach for robust dead-time compensator design”. Journal of Process Control, 19(1), 38-47, 2009. [15] Baumann J, Torkzadeh DD, Ramstein A, Kiencke U, Schlegl T. “Model-based predictive anti-jerk control”. Control Engineering Practice, 14(3), 259-266, 2006. 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https://openalex.org/W4239199842	https://www.scielo.br/j/bjmbr/a/zrBkgpM4ShZDyh6pBdDzJqq/?lang=en&format=pdf	English	null	TNNI3K is a novel mediator of myofilament function and phosphorylates cardiac troponin I	Brazilian Journal of Medical and Biological Research	2,013	cc-by	7,349	Brazilian Journal of Medical and Biological Research (2013) 46: 128-137, http://dx.doi.org/10.1590/1414-431X20122515 ISSN 1414-431X Brazilian Journal of Medical and Biological Research (2013) 46: 128-137, http://dx.doi.org/10.1590/1414-431X20122515 ISSN 1414-431X Brazilian Journal of Medical and Biological Research (2013) 46: 128-137, http://dx.doi.org/10.1590/1414-431X20122515 ISSN 1414 431X Abstract The phosphorylation of cardiac troponin I (cTnI) plays an important role in the contractile dysfunction associated with heart failure. Human cardiac troponin I-interacting kinase (TNNI3K) is a novel cardiac-specific functional kinase that can bind to cTnI in a yeast two-hybrid screen. The purpose of this study was to investigate whether TNNI3K can phosphorylate cTnI at specific sites and to examine whether the phosphorylation of cTnI caused by TNNI3K can regulate cardiac myofilament contractile function. Co-immunoprecipitation was performed to confirm that TNNI3K could interact with cTnI. Kinase assays further indicated that TNNI3K did not phosphorylate cTnI at Ser23/24 and Ser44, but directly phosphorylated Ser43 and Thr143 in vitro. The results obtained for adult rat cardiomyocytes also indicated that enhanced phosphorylation of cTnI at Ser43 and Thr143 correlated with rTNNI3K (rat TNNI3K) overexpression, and phosphorylation was reduced when rTNNI3K was knocked down. To determine the contractile function modulated by TNNI3K-mediated phosphorylation of cTnI, cardiomyocyte contraction was studied in adult rat ventricular myocytes. The contraction of cardiomyocytes increased with rTNNI3K overexpression and decreased with rTNNI3K knockdown. We conclude that TNNI3K may be a novel mediator of cTnI phosphorylation and contribute to the regulation of cardiac myofilament contraction function. Key words: TNNI3K; cTnI; Phosphorylation; Contraction function TNNI3K is a novel mediator of myofilament function and phosphorylates cardiac troponin I Hui Wang, Lin Wang, Li Song, Yan-Wan Zhang, Jue Ye, Rui-Xia Xu, Na Shi and Xian-Min Meng Hui Wang, Lin Wang, Li Song, Yan-Wan Zhang, Jue Ye, Rui-Xia Xu, Na Shi and Xian-Min Meng Core Laboratory, Fu Wai Hospital and Cardiovascular Institute, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China Core Laboratory, Fu Wai Hospital and Cardiovascular Institute, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China Correspondence: Xian-Min Meng, Core Laboratory, Fu Wai Hospital and Cardiovascular Institute, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. Fax: +86-010-6833-1748. E-mail: xmmengcn@163.com Correspondence: Xian-Min Meng, Core Laboratory, Fu Wai Hospital and Cardiovascular Institute, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. Fax: +86-010-6833-1748. E-mail: xmmengcn@163.com These authors contributed equally to this study. Received August 28, 2012. Accepted October 22, 2012. First published online February 1, 2013. Received August 28, 2012. Accepted October 22, 2012. First published online February 1, 2013. Introduction To prepare total cell lysates, cells from a 60-mm dish were lysed in 0.5 mL cold immunoprecipitation buffer. Total protein (80 mg) was separated on 12% SDS-PAGE and immunoblotting was performed with the anti-Xpress, anti-Flag, and anti- GAPDH antibodies (Zhongshan Goldenbridge, China). causally contribute to the contractile dysfunction asso- ciated with heart failure (10-12). In this study, we performed co-immunoprecipitation analysis to further confirm the in vivo interaction between TNNI3K and cTnI. We focused on TNNI3K-mediated phosphorylation of cTnI and identified the pertinent phosphorylation sites by kinase analysis, site-directed mutagenesis, and Western blot. It is of great interest to determine the functional consequences of TNNI3K- mediated cTnI phosphorylation in rat left ventricular myocytes. Introduction The human cardiac troponin I-interacting kinase (TNNI3K) gene expresses a novel cardiac-specific func- tional kinase. It was first cloned from an adult heart cDNA library based on large-scale expressed sequence tag sequencing (1). The TNNI3K protein contains three domains, including seven N-terminal ankyrin repeats, a protein kinase (PK) domain that contains motifs con- served in both serine/threonine and tyrosine PKs, and a C-terminal Ser-rich domain. TNNI3K belongs to a new family of kinases, called the mixed lineage kinase family in the tyrosine kinase-like group, based on a sequence comparison of the catalytic domain together with knowl- edge of sequence similarity and domain structures outside the catalytic domain (2). Numerous studies have demonstrated that TNNI3K can phosphorylate several substrates and undergo autophosphorylation (3,4). The TNNI3K C-terminal Ser-rich domain was previously used as bait to perform a yeast two-hybrid screen of a human cardiac library, and cardiac troponin I (cTnI) was identified as a TNNI3K-interacting protein (1). Troponin, in conjunction with tropomyosin, functions as a molecular switch and regulates muscle contraction in response to changes in the intracellular Ca2+ concentra- tion. Troponin consists of three subunits, including the Ca2+-binding subunit troponin C, the tropomyosin-binding subunit T, and the inhibitory subunit troponin I (5). cTnI plays a key role in the regulation of cardiac muscle contraction (6,7). As a major physiological mechanism for altering myofilament properties, the phosphorylation of cTnI stimulates a conformational change of troponin and regulates cardiomyocyte contractility (8,9). In addition to phosphorylation at specific serine and threonine residues by several different kinases, cTnI has a major role in the dynamic modulation of contractile function, which may Braz J Med Biol Res 46(2) 2013 www.bjournal.com.br 129 TNNI3K-mediated myofilament function Cellular debris was pelleted by centrifugation at 12,000 g for 15 min, and the lysate was incubated with the corresponding antisera for 1-2 h at 4 ˚C under rotation. The lysates were then incubated with 2 mg anti-Flag antibody (Cell Signaling Technology, USA) for 6 h at 4 ˚C and then mixed with 20 mL protein A-agarose beads (Vigorous Biotechnology Beijing Co., Ltd., China) for an additional 3 h. Immunoprecipitates were collected by centrifugation and washed twice in the immunoprecipita- tion buffer to remove unbound protein, boiled in sample buffer, centrifuged, and then removed from the beads. Immunoprecipitated proteins were subjected to 12% SDS- PAGE, and immunoblotting was performed with the anti- Xpress antibody (Invitrogen). Plasmids and adenovirus vectors The pcDNA4-Xpress/TNNI3Kmut, pcDNA6-Flag/cTnI, and pcDNA4-Xpress/TNNI3K expression vectors were prepared as previously described (1). The AdEasy System was used to prepare adenoviruses with constitutively active rat TNNI3K (Ad.Flag-rTNNI3K). Ad.EGFP was purchased from Vector Gene Technology Co., Ltd. (China) and used as a control. The small hairpin RNA (shRNA), which targeted rat TNNI3K (NCBI Accession NM_181769.1), was designed and cloned into RNA interference (RNAi) adenovirus vectors (Ad.rTNNI3KRNAi) by Genechem Co., Ltd., (China). The adenovirus vector containing the rat TNNI3K targeting shRNA was named Ad.rTNNI3KRNAi, and a negative control adenovirus vector containing negative control shRNA (Ad.rTNNI3Knc) was constructed by Genechem. Isolation of adult rat ventricular myocytes y y Isolated left ventricular myocytes were prepared from adult rat hearts as previously described (13). Sprague- Dawley rats weighing approximately 180 g were obtained from the Department of Laboratory Animals, Hebei Medical University. All experimental protocols using animals were approved by the Animal Ethics Review Committee at FuWai Hospital and Cardiovascular Institute. All experiments conformed to the Guide for the Care and Use of Laboratory Animals published by the US In vitro kinase assay For the kinase assay, 293T cells were transfected with expression plasmids. After 72 h, cells from a 60-mm dish were washed twice with cold PBS and then lysed in 0.5 mL cold lysis buffer (1% NP-40, 0.25% deoxycholate, 1 mM EGTA, 1 mM EDTA, 150 mM NaCl, 50 mM Tris- HCl, pH 7.5, 1 mM Na3VO4, 100 mM NaF, 1 mM b- glycerophosphate, and 5 g/L Protease Inhibitor Cocktail Tablets). Xpress-tagged TNNI3K and Flag-tagged cTnI were co-immunoprecipitated using the anti-Flag antibody and protein A-agarose beads and then washed three times with lysis buffer and two times with kinase reaction buffer (25 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM MnCl2, 5 mM b-glycerophosphate, and 2 mM DTT). To initiate the reactions, 20 mL phosphorylation mix contain- ing ATP was added to the samples and the mixture was incubated at 37 ˚C for 30 min. Reactions were terminated by adding SDS-PAGE sample buffer and boiling for 10 min. After boiling, the samples were centrifuged and the beads were removed. Samples were separated on 12% SDS-PAGE. Western blots were performed using anti-p-cTnI (Cell Signaling Technology), anti-p-cTnI (Thr143; cat: ab58546, Abcam, UK), and anti-p-cTnI (Ser43; cat: ab59420, Abcam) antibodies. Total cell lysates were prepared as described above. www.bjournal.com.br Cell culture and transient transfection The human embryonic kidney cell line HEK293T was obtained from the Cell Resource Center, IBMS, CAMS/ PUMC and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified incubator at 37 ˚C, 5% CO2, and 95% O2. Transient transfection of plasmids was performed using the LipofectamineTM 2000 reagent (Invitrogen, USA) according to manufacturer protocol. For co-immunopreci- pitation, 2 6 107 cells at approximately 70% confluence were co-transfected with 8 mg each of pcDNA4-Xpress- TNNI3K and pcDNA6-Flag or pcDNA6-Flag-cTnI. For the kinase assay, 2 6 107 cells at approximately 70% confluence were co-transfected with pcDNA6-cTnI and pcDNA4-Xpress, pcDNA4-Xpress-TNNI3K, or pcDNA4- Xpress-TNNI3Kmut (8 mg of each vector). Functional studies Cardiomyocyte contractility was measured using a video-based edge detection system (Ion Optix Co., USA). Myocytes were placed on the stage of an inverted microscope, superfused with Tyrode solution containing a Ca2+-concentration of 1.8 mM at a flow rate of 1.8 mL/ min, and electrically stimulated at 1 Hz at room tempera- ture. Cell length was monitored from the bright-field image by an optical edge-tracking method. The contraction amplitude was measured as the percentage of shortening of cell length. Co-immunoprecipitation analysis Equal volumes of 5X SDS-PAGE loading buffer were added to each sample and boiled at 100 ˚C for 10 min. Total protein (80 mg) was subjected to 12% SDS-PAGE and electrophoretically transferred onto a nitrocellulose membrane. The membranes were probed with mouse monoclonal anti-Flag or rabbit polyclonal anti- p-cTnI (Ser43) or anti-p-cTnI (Thr143) antibodies, all at 1:1000 dilutions, for 2 h at room temperature. After washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary anti-mouse or anti-rabbit antibodies, respectively (1:2000 dilution), for 1 h at room temperature. For loading controls and normalization, the same membranes were re-blocked with 5% nonfat dry milk in Tris-buffer saline containing 0.05% Tween-20 and then washed and incubated with a monoclonal anti-GAPDH antibody (1:1000) overnight at 4 ˚C. The blots were subsequently blotted with an HRP-conjugated anti-mouse secondary antibody. Immunoreactive proteins were visualized using enhanced chemiluminescence detection reagent by exposure to Eastman Kodak X-ray films (Kodak, USA). Evaluation of the expression of specific proteins was performed using Alpha Imager Software (Alpha Innotech, USA) by quanti- fying the pixels of electronic images. National Institutes of Health (NIH publication No. 85-23, revised in 1996). Culture of adult rat cardiac myocytes The entire culture procedure was performed under a class II laminar flow hood. Laminin solution [10 mg/mL mouse laminin (Invitrogen) in PBS] was used to coat the tissue culture-treated dishes and incubated overnight at 37 ˚C. Freshly isolated cardiac myocytes were suspended in the basal culture (CCT) medium, which was modified medium 199 supplemented with 2 mM L-carnitine, 10 mM cytosine-D-arabinofuranoside, and 5 mM taurine (13). After the myocytes were pelleted by gravity for 10 min, the supernatant was aspirated and the myocytes were washed two more times using the same protocol. The myocytes were then plated at a density of 1 6 105 cells per 35 mm dish in CCT medium containing 1% penicillin- streptomycin. After 2-h incubation in a 5% CO2 incubator at 37 ˚C, the medium was changed to FBS-free CCT medium. RNAi and adenoviral infection For RNAi, adult rat cardiac myocytes were transfected with adenovirus rTNNI3KRNAi and incubated for 24 h. The Ad.rTNNI3Knc, which contained a negative control shRNA, was used as a negative control. Nontransfected cells were used as a blank control. Cardiomyocytes were infected with a control adenovirus (Ad.EGFP), Ad.Flag- rTNNI3K, or Ad.rTNNI3KRNAi for 24 h. Adenovirus- directed gene transfer was initiated after 2 h of culture. The culture medium was aspirated together with unat- tached myocytes, and a half-volume (e.g., 1 mL for a 35- mm Petri dish) of the FBS-free CCT medium containing an appropriate titer of gene-carrying adenovirus was added to the dish. Cells were incubated in the FBS-free medium in the presence of Ad.Flag-rTNNI3K (20, 40, 60, 80, or 100 multiplicity of infection [MOI]) virus or Ad.rTNNI3KRNAi virus (40, 60, 80, or 100 MOI) or Ad- EGFP control virus (100 MOI) for 6 h. Subsequently, an additional half-volume of FBS-free CCT was added. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) analysis Quantitative real-time RT-PCR analysis was used to determine the level of rTNNI3K mRNA after cells had been transfected with RNAi against rTNNI3K for 24 h. Total RNA was extracted using Trizol solution, quantified by spectrophotometry, and then reverse-transcribed using SuperScript III (Invitrogen). Relative gene expression levels were determined from threshold cycle values and normalized to GAPDH. Quantification of specific RNA transcripts was performed by SYBR Green real-time PCR using Opticon 2 (Bio-Rad, USA). The primer sequences for rTNNI3K and GAPDH were as follows: rTNNI3K forward primer: 59-CACCTTCCTCTTCTTCCGATT-39, rTNNI3K reverse primer: 59-CTGTCCTCAAAGTTGCT- GTCG-39; GAPDH forward primer: 59-CAACGACCCCTT- CATTGACCT-39, GAPDH reverse primer: 59-CAGTAGA- CTCCACGACATACTC-39. Co-immunoprecipitation analysis For co-immunoprecipitation experiments, 293T cells were harvested 72 h after transfection by first washing the cells grown on a 60-mm dish twice with cold PBS and then lysing the cells with 0.5 mL cold immunoprecipitation buffer [1% NP-40, 0.25% deoxycholate, 2 mM EGTA, 1 mM EDTA, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, and 5 g/L protease inhibitor cocktail tablets (Roche, USA)]. Braz J Med Biol Res 46(2) 2013 130 Hui Wang et al. (Thr143) antibodies were used for Western blot analysis. Cardiomyocytes infected with Ad.rTNNI3K on a 60-mm dish were washed twice with cold PBS and lysed in RAPI buffer containing 1 mM PMSF, 1 mM Na3VO4, and 100 mM NaF. Cell debris was pelleted by centrifugation at 12,000 g for 15 min and the supernatant was then collected. Protein concentrations of each sample were measured using the bicinchoninic acid method (BCA kit, Applygen, China). Equal volumes of 5X SDS-PAGE loading buffer were added to each sample and boiled at 100 ˚C for 10 min. Total protein (80 mg) was subjected to 12% SDS-PAGE and electrophoretically transferred onto a nitrocellulose membrane. The membranes were probed with mouse monoclonal anti-Flag or rabbit polyclonal anti- p-cTnI (Ser43) or anti-p-cTnI (Thr143) antibodies, all at 1:1000 dilutions, for 2 h at room temperature. After washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary anti-mouse or anti-rabbit antibodies, respectively (1:2000 dilution), for 1 h at room temperature. For loading controls and normalization, the same membranes were re-blocked with 5% nonfat dry milk in Tris-buffer saline containing 0.05% Tween-20 and then washed and incubated with a monoclonal anti-GAPDH antibody (1:1000) overnight at 4 ˚C. The blots were subsequently blotted with an HRP-conjugated anti-mouse secondary antibody. Immunoreactive proteins were visualized using enhanced chemiluminescence detection reagent by exposure to Eastman Kodak X-ray films (Kodak, USA). Evaluation of the expression of specific proteins was performed using Alpha Imager Software (Alpha Innotech, USA) by quanti- fying the pixels of electronic images. (Thr143) antibodies were used for Western blot analysis. Cardiomyocytes infected with Ad.rTNNI3K on a 60-mm dish were washed twice with cold PBS and lysed in RAPI buffer containing 1 mM PMSF, 1 mM Na3VO4, and 100 mM NaF. Cell debris was pelleted by centrifugation at 12,000 g for 15 min and the supernatant was then collected. Protein concentrations of each sample were measured using the bicinchoninic acid method (BCA kit, Applygen, China). Identification of the interaction between cTnI and TNNI3K Identification of the interaction between cTnI and TNNI3K The TNNI3K C-terminal Ser-rich domain was used as bait to perform a yeast two-hybrid screen of a human cardiac library, which previously identified cTnI as one of TNNI3K-interacting proteins (1). To further confirm the interaction between cTnI and TNNI3K, we co-transfected HEK293T cells with Xpress-tagged TNNI3K and Flag- tagged cTnI or Flag-tagged empty control vector, incu- bated the cells for 72 h, and then lysed the cells. The lysates were co-immunoprecipitated with anti-Xpress antibodies and analyzed by immunoblotting using an anti-Flag antibody. In addition, anti-Flag immunoprecipi- tates were analyzed by immunoblotting with an anti- Xpress antibody. The results showed that TNNI3K co- immunoprecipitated cTnI, while no significant bands were detected in the control (Figure 1). Thus, we found that TNNI3K interacts with cTnI in vivo, which is consistent with the results of two-hybrid binding in yeast cells. Our previous results demonstrated that TNNI3K localized in the nucleus and cytoplasm of fetal and adult cardiac myocytes (1). Moreover, cTnI is a sarcomere protein that localizes in the cytoplasm of cardiac myocytes. Therefore, It is known that different phosphorylation sites on cTnI have different roles in the regulation of myofilament function (11,16). Thus, we examined phosphorylation sites on cTnI that were targeted by TNNI3K. We performed Western blots to detect the phosphorylation site on cTnI after a 30-min incubation with TNNI3K. As show in Figure 2C and D, the anti-p-cTnI (Ser43) and anti- p-cTnI (Thr143) antibodies both reacted with a 24-kDa band. These results demonstrated that TNNI3K directly phosphorylates in vitro cTnI at Ser43 and Thr143, which are commonly referred to as the PKC sites (11,17). Results TNNI3K-mediated phosphorylation of cTnI in vitro TNNI3K is a cardiac-specific functional PK, but its substrates in vivo have not been elucidated. Phosphorylation of cTnI plays an important role in the regulation of myofilament function. Therefore, we were interested in assessing whether TNNI3K could phospho- rylate cTnI. We performed an in vitro kinase assay using wild-type TNNI3K, a TNNI3K mutant where lysine 490 was changed to alanine (TNNI3Kmut), and an empty vector as control. The point mutation of TNNI3Kmut occurs at a conserved lysine residue within the sub- domain II, which plays a crucial role in kinase activity. Therefore, we tested whether a substitution of this lysine residue in TNNI3K, which is reported to block the phosphotransfer reaction in a number of PKs (1,14,15), would lead to the loss of kinase activity in this protein as well. As illustrated in Figure 2A, the expression level of Flag-tagged cTnI (FLAG-cTnI), Xpress-tagged TNNI3K (Xpress-TNNI3K), and Xpress-tagged TNNI3Kmut was similar in each group. As shown in Figure 2B, a 24-kDa band was identified using an anti-p-cTnI antibody, while the phosphorylation of cTnI was sharply reduced with the TNNI3Kmut, and was completely absent in the control group. Therefore, these data show that TNNI3K can phosphorylate cTnI as a functional PK. Western blot analysis of the relationship between TNNI3K protein levels and cTnI phosphorylation levels Data are reported as means ± SE and were compared by one-way analysis of variance (ANOVA) in a two-sided test. Differences were considered to be The anti-Flag, anti-p-cTnI (Ser43), and anti-p-cTnI Braz J Med Biol Res 46(2) 2013 www.bjournal.com.br 131 TNNI3K-mediated myofilament function statistically significant at P , 0.05. All other data were analyzed by SPSS v.13.0 (SPSS, Inc., USA). statistically significant at P , 0.05. All other data were analyzed by SPSS v.13.0 (SPSS, Inc., USA). we inferred that TNNI3K and cTnI can co-localize in the myocyte cytoplasm. www.bjournal.com.br Braz J Med Biol Res 46(2) 2013 TNNI3K-mediated phosphorylation of cTnI in adult rat cardiac myocytes We next tested whether changes in the expression of rTNNI3K could affect cTnI phosphorylation at Ser43 and Thr143 in isolated myocytes. Cardiac myocytes were infected for 24 h with optimal MOIs of 20, 40, 60, 80, or 100 of an adenovirus harboring the rTNNI3K sequence. Phosphorylation of cTnI at Ser43 and Thr143 significantly increased after rTNNI3K was overexpressed in myocytes for 24 h compared to the adenoviral control and blank control (Figure 3A and B; n = 5; P , 0.01). Moreover, the phosphorylation level of cTnI Ser43 and Thr143 sites markedly increased when the MOIs of Ad.Flag-rTNNI3K increased from 20 to 100 (Figure 3A and B; n = 5; P , 0.01). These data indicated that overexpression of TNNI3K can increase the phos- Figure 1. Human cardiac troponin I-interacting kinase (TNNI3K) interacts with cardiac troponin I (cTnI) in vitro. HEK293T cells were co-transfected with Xpress-tagged TNNI3K and Flag- tagged cTnI or a Flag control vector. Cell lysates were immunoprecipitated (IP) with an anti-Xpress monoclonal antibody and immunoblotted (IB) with an anti-Flag polyclonal antibody. Conversely, anti-Flag immunoprecipitates were analyzed by immunoblotting with an anti-Xpress (lower). The expression of cTnI and TNNI3K was confirmed by Western blot (upper). Braz J Med Biol Res 46(2) 2013 132 Hui Wang et al. Figure 2. Human cardiac troponin I-interacting kinase (TNNI3K) phosphorylates cardiac troponin I (cTnI) at Ser43 (S43) and Thr143 (T143). HEK293T cells were transfected with Xpress-tagged TNNI3K, Xpress-tagged TNNI3Kmut, or Flag-tagged cTnI and incubated for 72 h. A, Western blot using anti-Flag and anti-Xpress antibodies (n = 5). B, Western blot using anti-Xpress and anti-p-cTnI antibodies (n = 5). C, Western blot using anti-P-cTnI (S43) and anti-Xpress antibodies (n = 5). D, Western blot using anti-P-cTnI (Thr143) and anti-Xpress antibodies (n = 5). IP = immunoprecipitated; IB = immunoblotted. Data are reported as means ± SE. P , 0.01 vs TNNI3Kmut-mediated phosphorylation (one-way ANOVA). 132 Hui Wang et al. Figure 2. Human cardiac troponin I-interacting kinase (TNNI3K) phosphorylates cardiac troponin I (cTnI) at Ser43 (S43) and Thr143 (T143). HEK293T cells were transfected with Xpress-tagged TNNI3K, Xpress-tagged TNNI3Kmut, or Flag-tagged cTnI and incubated for 72 h. A, Western blot using anti-Flag and anti-Xpress antibodies (n = 5). B, Western blot using anti-Xpress and anti-p-cTnI antibodies (n = 5). C, Western blot using anti-P-cTnI (S43) and anti-Xpress antibodies (n = 5). D, Western blot using anti-P-cTnI (Thr143) and anti-Xpress antibodies (n = 5). Braz J Med Biol Res 46(2) 2013 TNNI3K-mediated phosphorylation of cTnI in adult rat cardiac myocytes IP = immunoprecipitated; IB = immunoblotted. Data are reported as means ± SE. P , 0.01 vs TNNI3Kmut-mediated phosphorylation (one-way ANOVA). phorylation level of cTnI at Ser43 and Thr143 in functional adult rat cardiac myocytes. rTNNI3K sequence. Ad.rTNNI3Knc was used as a control. We evaluated the expression of the rTNNI3K gene by real-time PCR after shRNA-mediated silencing. As illustrated in Figure 4, Ad.rTNNI3KRNAi efficiently reduced the expression of rTNNI3K in adult rat cardiac In a reciprocal experiment, rTNNI3K knockdown was performed in adult rat cardiac myocytes using an adenovirus-mediated delivery of a shRNA targeting the www.bjournal.com.br 133 TNNI3K-mediated myofilament function t i ll h MOI f 60 80 d 100 Ad TNNI3K (100 MOI) i d ild t d lt t Figure 3. The expression of human cardiac troponin I-interacting kinase (TNNI3K) correlates with the phosphorylation of cardiac troponin I (cTnI) at Ser43 (S43) and Thr143 (T143). Rat cardiac myocytes were infected with adenoviral vectors with multiplicity of infections (MOIs) of 20, 40, 60, 80, or 100 for 24 h. Cell lysates were then subjected to Western blot analysis to detect the expression of rTNNI3K as well as the phosphorylation of cTnI at Ser43 and Thr143 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Overexpression of rTNNI3K produces a significant increase in the phosphorylation of cTnI at Ser43 (A) and Thr143 (B). Knockdown of rTNNI3K with Ad.rTNNI3KRNAi significantly reduces phosphorylation at Ser43 (C) and Thr143 (D) of cTnI. Data were from five independent experiments. Data are reported as means ± SE. P , 0.01 vs blank control (without virus infection). #P , 0.01 vs cardiomyocytes infected with Ad.EGFP (one-way ANOVA). Figure 3. The expression of human cardiac troponin I-interacting kinase (TNNI3K) correlates with the phosphorylation of cardiac troponin I (cTnI) at Ser43 (S43) and Thr143 (T143). Rat cardiac myocytes were infected with adenoviral vectors with multiplicity of infections (MOIs) of 20, 40, 60, 80, or 100 for 24 h. Cell lysates were then subjected to Western blot analysis to detect the expression of rTNNI3K as well as the phosphorylation of cTnI at Ser43 and Thr143 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Overexpression of rTNNI3K produces a significant increase in the phosphorylation of cTnI at Ser43 (A) and Thr143 (B). Knockdown of rTNNI3K with Ad.rTNNI3KRNAi significantly reduces phosphorylation at Ser43 (C) and Thr143 (D) of cTnI. Data were from five independent experiments. Data are reported as means ± SE. www.bjournal.com.br TNNI3K-mediated phosphorylation of cTnI in adult rat cardiac myocytes P , 0.01 vs blank control (without virus infection). #P , 0.01 vs cardiomyocytes infected with Ad.EGFP (one-way ANOVA). Ad.rTNNI3Knc (100 MOI) virus and wild-type adult rat cardiac myocytes were used as the adenovirus vector and blank controls, respectively. After 24 h, the cells were lysed and subjected to Western blot analysis. Compared to the controls, addition of the Ad.rTNNI3KRNAi signifi- myocytes, especially when MOIs of 60, 80, and 100 were used. Based on these results, we knocked down the expression of rTNNI3K using Ad.rTNNI3KRNAi with MOIs of 40, 60, 80, and 100 while concomitantly infecting the same cells with Ad.Flag-rTNNI3K (100 MOI) virus. The Braz J Med Biol Res 46(2) 2013 134 Hui Wang et al. Ad.rTNNI3K, Ad.rTNNI3Knc, or Ad.rTNNI3KRNAi and incubated for 24 h. Cell contractility was then measured in the infected cardiomyocytes as described in Material and Methods. As shown in Figure 5A, TNNI3K over- expression induced a significant increase in cell short- ening of cardiomyocytes compared to Ad-GFP-infected cells (2.88 ± 0.087 vs 2.06 ± 0.045%, respectively; n = 20; P , 0.05). In contrast, TNNI3K knockdown caused a decrease in cell shortening compared to control (1.12 ± 0.08 vs 2.13 ± 0.063%, respectively; n = 20; P , 0.05; Figure 5B). Taken together, these data suggest that TNNI3K may regulate cardiomyocyte contraction. cantly reduced cTnI phosphorylation at Ser43 and Thr143 (Figure 3C and D). These results demonstrated that a reduction in TNNI3K expression significantly reduced cTnI phosphorylation at Ser43 and Thr143 in adult rat cardiac myocytes. Based on these experiments, we concluded that rTNNI3K can increase the phosphorylation of cTnI at Ser43 and Thr143 and, most importantly, phosphorylate cTnI in adult rat cardiac myocytes. Functional consequences of TNNI3K-mediated cTnI phosphorylation Because kinase-mediated phosphorylation of cTnI can regulate the contractility function of myofilaments, we next determined whether TNNI3K-mediated phosphorylation of cTnI at Ser43 and Thr143 could change the contractility of adult rat cardiomyocytes. Based on the results cited above, we tested Ad.Flag-rTNNI3K or Ad.rTNNI3KRNAi at 100 MOI. The adult rat cardiac myocytes were directly infected with Ad.Flag-rTNNI3K or Ad.EGFP (as vector control) at 100 MOI. We also knocked down the expression of rTNNI3K in adult rat cardiac myocytes using Ad.rTNNI3KRNAi at 100 MOI with Ad.rTNNI3Knc at 100 MOI as the adenovirus vector control. In both experiments, wild-type adult rat cardiac myocytes were used as a blank control. egu a e e co ac y u c o o yo a e s, e e determined whether TNNI3K-mediated phosphorylation of cTnI at Ser43 and Thr143 could change the contractility of adult rat cardiomyocytes. Based on the results cited above, we tested Ad.Flag-rTNNI3K or Ad.rTNNI3KRNAi at 100 MOI. The adult rat cardiac myocytes were directly infected with Ad.Flag-rTNNI3K or Ad.EGFP (as vector control) at 100 MOI. We also knocked down the expression of rTNNI3K in adult rat cardiac myocytes using Ad.rTNNI3KRNAi at 100 MOI with Ad.rTNNI3Knc at 100 MOI as the adenovirus vector control. In both experiments, wild-type adult rat cardiac myocytes were used as a blank control. To determine the effect of TNNI3K on cardiomyocyte contractility, we examined contractility of the cells after infection with the indicated adenoviruses using a video- based edge detection system. Isolated adult rat ventri- cular cardiomyocytes were infected with Ad.GFP, Figure 4. Knockdown of human cardiac troponin I-interacting kinase (TNNI3K) in isolated rat adult cardiomyocytes. Freshly isolated rat adult cardiomyocytes were infected with an adeno- virus carrying shRNA for silencing TNNI3K or scrambling shRNA for 24 h at the indicated multiplicity of infection (MOI). Samples were analyzed by quantitative real-time reverse transcription polymerase chain reaction and normalized to glyceraldehyde-3- phosphate dehydrogenase (GAPDH). Data are reported as means ± SE (n = 6). P , 0.01 vs control (MOI = 0); #P , 0.01 vs green bar at the same MOI value (one-way ANOVA). Figure 5. Effects of human cardiac troponin I-interacting kinase (TNNI3K) on cardiomyocyte contraction. A, Representative traces of cell shortening from non-infected cardiomyocytes or cardiomyocytes infected with Ad.GFP or Ad.rTNNI3K. B, Representative traces of cell shortening from non-infected cardiomyocytes or cardiomyocytes infected with Ad.TNNI3Knc or Ad.rTNNI3KRNAi. Braz J Med Biol Res 46(2) 2013 Functional consequences of TNNI3K-mediated cTnI phosphorylation Braz J Med Biol Res 46(2) 2013 www bjournal com br To determine the effect of TNNI3K on cardiomyocyte contractility, we examined contractility of the cells after infection with the indicated adenoviruses using a video- based edge detection system. Isolated adult rat ventri- cular cardiomyocytes were infected with Ad.GFP, Figure 4. Knockdown of human cardiac troponin I-interacting kinase (TNNI3K) in isolated rat adult cardiomyocytes. Freshly isolated rat adult cardiomyocytes were infected with an adeno- virus carrying shRNA for silencing TNNI3K or scrambling shRNA for 24 h at the indicated multiplicity of infection (MOI). Samples were analyzed by quantitative real-time reverse transcription polymerase chain reaction and normalized to glyceraldehyde-3- phosphate dehydrogenase (GAPDH). Data are reported as means ± SE (n = 6). P , 0.01 vs control (MOI = 0); #P , 0.01 vs green bar at the same MOI value (one-way ANOVA). Figure 4. Knockdown of human cardiac troponin I-interacting kinase (TNNI3K) in isolated rat adult cardiomyocytes. Freshly isolated rat adult cardiomyocytes were infected with an adeno- virus carrying shRNA for silencing TNNI3K or scrambling shRNA for 24 h at the indicated multiplicity of infection (MOI). Samples were analyzed by quantitative real-time reverse transcription polymerase chain reaction and normalized to glyceraldehyde-3- phosphate dehydrogenase (GAPDH). Data are reported as means ± SE (n = 6). P , 0.01 vs control (MOI = 0); #P , 0.01 vs green bar at the same MOI value (one-way ANOVA). Figure 4. Knockdown of human cardiac troponin I-interacting kinase (TNNI3K) in isolated rat adult cardiomyocytes. Freshly isolated rat adult cardiomyocytes were infected with an adeno- virus carrying shRNA for silencing TNNI3K or scrambling shRNA for 24 h at the indicated multiplicity of infection (MOI). Samples were analyzed by quantitative real-time reverse transcription polymerase chain reaction and normalized to glyceraldehyde-3- phosphate dehydrogenase (GAPDH). Data are reported as means ± SE (n = 6). *P , 0.01 vs control (MOI = 0); #P , 0.01 vs green bar at the same MOI value (one-way ANOVA). Figure 5. Effects of human cardiac troponin I-interacting kinase (TNNI3K) on cardiomyocyte contraction. A, Representative traces of cell shortening from non-infected cardiomyocytes or cardiomyocytes infected with Ad.GFP or Ad.rTNNI3K. B, Representative traces of cell shortening from non-infected cardiomyocytes or cardiomyocytes infected with Ad.TNNI3Knc or Ad.rTNNI3KRNAi. www.bjournal.com.br Braz J Med Biol Res 46(2) 2013 135 TNNI3K-mediated myofilament function Figure 6. Discussion As an important post-translational modification, phos- phorylation of cTnI and other myofilament proteins plays a major role in the dynamic modulation of contractile function and in the regulation of thin filament function in the transition from compensated hypertrophy to heart failure (6,11,18,19). In the present study, we confirmed that TNNI3K, which is a cardiac-specific functional kinase, interacts with cTnI and directly phosphorylates cTnI at Ser43 and Thr143. Furthermore, TNNI3K overexpression not only enhanced cTnI Ser43 and Thr143 phosphoryla- tion, but also increased myocardial contractility. These data also suggest that the effects of TNNI3K are mediated by phosphorylation of cTnI Ser43 and Thr143 and other potential mechanisms, which may participate in the regulation of myocardial contractile function. Adult rat cardiomyocytes provide a useful model for gene delivery and contractility studies. However, it is easy for the cells to lose their native rod shape and contractility in an in vitro culture. We first confirmed that TNNI3K can be efficiently overexpressed or knocked down in adult rat cardiac myocytes by infecting the cells with appropriate adenoviruses for 24 h (Figure 3). We also found that adult rat cardiomyocytes still maintained their rod-shaped appearance and contractility after adenoviral infection for 24 h, but lost those features after 48 h post-transfec- tion (Figure 6 and data not shown). Therefore, we chose 24 h post-infection as the optimal time for experimental assessment. Figure 6. Morphology of rat cardiac myocytes immediately after isolation (0 h) or after 24 h in culture. A and B, Transillumination images. C, D, E, and F, Expression of recombinant adenoviral transgenes in cultured adult rat ventricular myocytes. The rat cardiac myocytes were infected with adenoviral vectors carrying a marker gene, EGFP, at 100 multiplicity of infection after 24 h in culture. The phosphorylation of cTnI and its regulation of cardiac myofilament contraction have been studied in a variety of systems, ranging from reconstituted myofila- ment proteins to myocardial cells or transgenic animals with heterologous expression of modified cTnI proteins (11,20-22). cTnI is also a substrate for protein kinase A (PKA) and protein kinase C (PKC). PKA mainly phos- phorylates Ser23/24 of cTnI in the N-terminus of the protein (rodent sequence position; Ser22/23 in humans) (6,23). PKC mainly phosphorylates cTnI at the Ser43/45 and Thr143 positions (numbered as in the human cTnI sequence) (6,24). Functional consequences of TNNI3K-mediated cTnI phosphorylation Morphology of rat cardiac myocytes immediately after isolation (0 h) or after 24 h in culture. A and B, Transillumination images. C, D, E, and F, Expression of recombinant adenoviral transgenes in cultured adult rat ventricular myocytes. The rat cardiac myocytes were infected with adenoviral vectors carrying a marker gene, EGFP, at 100 multiplicity of infection after 24 h in culture. www.bjournal.com.br Braz J Med Biol Res 46(2) 2013 Discussion More recently, other kinases have been found to produce functional effects through the regulation of cTnI phosphorylation, such as myosin light-chain kinase, protein kinase D, and mammalian sterile 20-like kinase 1 (25-28). In addition, many studies have shown that protein kinase G and p21-activated kinases may also be involved in the modification of cTnI phosphorylation (11,29-31). In the present study, our data demonstrated that TNNI3K specifically targeted cTnI and catalyzed the phosphorylation of cTnI at Ser43 and Thr143 (the PKC sites, numbered as in the human cTnI sequence), which are mostly conserved in cTnI from multiple species, but not at Ser23/24 (the PKA sites; data not shown). Furthermore, we confirmed in adult rat cardiac myocytes that the expression level of TNNI3K positively correlated with the phosphorylation of cTnI at Ser43 and Thr143. These data suggest that active TNNI3K phosphorylates cTnI at Ser43 and Thr143 not only when the substrate protein is used in isolation, but also in isolated myocytes. Thus, cTnI likely represents a physiological substrate for TNNI3K in the myocardium. It has been reported that PKA phosphorylation of cTnI at Ser23/24 in living myocardium contributes to acceler- ated relaxation in diastole and increases the rates of force development in systole (32). However, the precise functional effects mediated by PKC-dependent phospho- rylation of cTnI that are related to whole heart or muscle contractile function are not yet clear. Evidence suggests that PKC phosphorylation of cTnI at Ser43/45 can reduce maximal Ca2+-activated tension and decrease MgATPase activity as well as the crossbridge cycling rate, which can lead to a reduction in energy consumption and impaired relaxation, while phosphorylation of Thr144 would reduce sliding velocity. Based on these findings, it can be postulated that PKC-mediated phosphorylation of cTnI at Ser43/45 can reduce contractility and prolong relaxa- Braz J Med Biol Res 46(2) 2013 136 Hui Wang et al. tion (11). Furthermore, these effects may be relatively beneficial in energetic terms, at least in the non-diseased heart (11). Other studies have shown that there may be significant interdependence between the effects that result from PKC-dependent Ser43/45 phosphorylation and PKA-dependent Ser23/24 phosphorylation. Roman et al. (33) found that phosphorylation of cTnI at Ser23/24 increases in transgenic mice where Ser43/45 is mutated to alanine, which results in enhanced contraction and relaxation under basal conditions (11,33). Discussion To further explore the functional effects of TNNI3K-mediated cTnI phosphorylation, we examined the myocardial contractility of cultured adult rat cardiac myocytes in which TNNI3K was overexpressed or knocked down. Our data suggest that overexpression of TNNI3K can enhance myocardial contractility. Taken together, the data support the hypothesis that the phosphorylation of cTnI mediated by TNNI3K, as a novel mechanism, may regulate the functions of the myofilament. induction of increased shortening might be related to heart physiology-pathology states. We previously found that TNNI3K had a higher expression when it was stimulated by the pathology factor, ET-1 (35), and our data show that the high expression enhanced cardiomyocyte shortening through phosporylation of cTnI. Therefore, the high expression might be a response to compensatory hypertrophy. Moreover, TNNI3K-mediated control of cardiomyocyte shortening through the phosphorylation of cTnI might be a novel mechanism. In conclusion, TNNI3K may be a novel mediator of cTnI phosphorylation and may contribute to the regulation of cardiac myofila- ment contractile function. Studies of TNNI3K open a new area of research with the goal of developing a better understanding of cardiac contractile dysfunction asso- ciated with ischemic heart diseases and heart failure. Acknowledgments We thank Prof. Yongli Wang and Prof. Hailin Zhang (Department of Pharmacology, Hebei Medical University, Shijiazhuang, Hebei Province, China) for isolation and culture of adult rat ventricular myocytes as well as for the functional study. We also sincerely thank Prof. Philip R. Mayeux (Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, USA) for editing the manuscript. Research supported by grants from the National Natural Science Foundation of China (#30871057 and #30940029). Data from this study also indicate that ventricular myocytes infected with Ad-TNNI3K exhibit increased peak shortening. In contrast, ventricular myocytes infected with Ad-TNNI3KRNAi showed decreased peak shortening. 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https://openalex.org/W2063252582	https://bmccomplementmedtherapies.biomedcentral.com/counter/pdf/10.1186/s12906-015-0551-5	English	null	Evaluation of antitumor property of extracts and steroidal alkaloids from the cultivated Bulbus Fritillariae ussuriensis and preliminary investigation of its mechanism of action	BMC complementary and alternative medicine	2,015	cc-by	8,708	RESEARCH ARTICLE Open Access Dongdong Wang1†, Yun Jiang2†, Ke Wu1†, Shu Wang1 and Yitao Wang2 Dongdong Wang1†, Yun Jiang2†, Ke Wu1†, Shu Wang1 and Yitao Wang2 Dongdong Wang1†, Yun Jiang2†, Ke Wu1†, Shu Wang1 and Yitao Wang2 Dongdong Wang1†, Yun Jiang2†, Ke Wu1†, Shu Wang1 and Yitao Wang2 * Correspondence: wddong1988@hotmail.com; huaxiyaoxue3000@gmail.com †Equal contributors 1Department of Pharmacognosy, West China College of Pharmacy, Sichuan University, No. 17, Duan 3, Renmin Nan Road, Chengdu 610041, Peoples’ Republic of China Full list of author information is available at the end of the article Wang et al. BMC Complementary and Alternative Medicine (2015) 15:29 DOI 10.1186/s12906-015-0551-5 Wang et al. BMC Complementary and Alternative Medicine (2015) 15:29 DOI 10.1186/s12906-015-0551-5 Open Access Abstract Background: Cancer is well known as a leading cause of death in the world. At present, it is the very active area to search for anticancer drugs from natural products. In this study, we evaluated the antitumor property of chloroform extract (CE), n-hexane extract (HE), water extract (WE) and steroidal alkaloids from the cultivated Bulbus Fritillariae ussuriensis (BFU) and its preliminary mechanism for its action was investigated. Methods: Firstly, cytotoxicity of the different extracts from BFU against Lewis lung carcinoma cell line (LLC), Human ovarian cancer cell line (A2780), human hepatocellular carcinoma cell line (HepG2), human lung carcinoma cell line (A549) was measured by MTT assay. Then, we identified the compounds from the active extract of BFU by bioassay guided isolation, determined their antitumor activity in vitro, and detected cell cycle distribution using flow cytometry. Moreover, the extract of BFU which showed remarked anti-proliferative activity in vitro was further evaluated using S180 and LLC tumor models. Additionally, a preliminary investigation of the mechanism of the action was carried out by using histological and immunohistochemical staining technique. Results: The results showed that CE and the purified total alkaloids of BFU (TAFU) exhibited stronger cytotoxic activity than the others (WE and HE). We further isolated the four main steroidal alkaloids from TAFU, and found all alkaloids showed significant cytotoxicity, and peimisine induced G0/G1 phase arrest and increased apoptosis. The results showed that TAFU had significant antitumor activity and low toxicity in vivo. Additionally, the immunohistochemical examinations signified that TAFU remarkably increased caspase-3 expression and reduced microvessel density (MVD) in tumor tissues of transplantable S180 and LLC tumor models. Conclusions: These results achieved suggested that the steroidal alkaloids could hold a good potential for use as an antitumor drug. Notably, our finding is the first report on the antitumor activity of extracts and steroidal alkaloids from the cultivated BFU in vitro and in vivo and its mechanisms. Keywords: Bulbus Fritillariae ussuriensis, Alkaloids, Antitumor, Cell cycle, Microvessel density, Caspase-3 * Correspondence: wddong1988@hotmail.com; huaxiyaoxue3000@gmail.com †Equal contributors 1Department of Pharmacognosy, West China College of Pharmacy, Sichuan University, No. 17, Duan 3, Renmin Nan Road, Chengdu 610041, Peoples’ Republic of China Full list of author information is available at the end of the article © 2015 Wang et al.; licensee BioMed Central. Abstract This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. © 2015 Wang et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 11 Page 2 of 11 Page 2 of 11 Wang et al. BMC Complementary and Alternative Medicine (2015) 15:29 Methods Bulbus Fritillariae ussuriensis (BFU) is the well-known food and folk medicine distributed in the Northeast Provinces of China, such as Liaoning, Heilongjiang and Jiling provinces, because it has remarkable antitussive, expectorant and anti- asthmatic activities [1]. Plant materials Th BFU The BFU were purchased in September 2012 from Chengdu International Trade City Hehuachi Chinese Medicinal Herbal Market (Chengdu city, Sichuan province, China) and identified by Prof. Shu Wang (Department of Pharmacogonosy, West China College of Pharmacy, Sichuan University). The sample (W201204001) has been deposited in the pharmacognosy laboratory of West China College of Pharmacy, Sichuan University. Cancer is well known as a leading cause of death in the world. It is predicted that cancer related deaths will in- crease to over 24 million in 2035 [10]. So, it is the very ac- tive area to search for anticancer drugs from natural products. It is worthwhile to note that several recent re- search have indicated that verticine, verticinone, ebeiedine and the crude alkaloid of Fritillaria ebeiensis showed strong antitumor activity in inhibiting the growth of the solid type of hepatoma and Ehrlich ascites carcinoma in mice [11]. Besides, the crude alkaloid extract of bulbs of Fritillaria puqiensis and puqietinone could inhibit the growth of three types of cancers [12]. Moreover, our previ- ous studies also suggested that the crude alkaloid extract of bulbs of Fritillaria Cirrhosae showed remarkable anti- tumor in vitro and in vivo [13,14]. Chemicals and reagents RPMI-1640 medium, Dulbecco’s Modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Hyclone (USA). Trypsin, penicillin, streptomycin, antibodies, propidium iodine (PI) and phosphate buffer solution (PBS) were purchased from Gibco BRL, Life Technologies (USA). 3-(4,5-Dimethylthiazol-2-yl)-diphe- nyl tetrazolium bromide (MTT), dimethyl sulphoxide (DMSO) were purchased from Sigma Aldrich, Inc. (USA). Mitozantrone hydrochloride was purchased from Jiangsu Hansoh Pharmaceutical Co., Ltd (China). Cyclophospha- mide (CTX) was purchased from Jiangsu Hengrui Co. Ltd (China). The ultra pure water was prepared by a Milli-Q water system (China). Sterilized cell culture materials, such as syringe filter, 15 ml and 50 ml tubes, 96-well plates, and pipettes were purchased from Nest Biotech Co., Ltd (China). Other chemicals and reagents used were analytical grade and commercially available. The previous studies have exhibited that the crude al- kaloid extracts of BFU possess remarkable antitussive, expectorant and antiasthmatic activities [2,3]. In vitro previous studies suggested that verticinone, imperialine and peimisine from BFU inhibited angiotensin I con- verting enzyme activity in a dose-dependent manner [4], and in vivo ethylacetate and butanol extracts from BFU significantly lowered the mean arterial pressure, decreased angiotensin converting enzyme and angioten- sin I-induced vasoconstriction, and they increased nitric oxide (NO) and cGMP productions in intact vascular tissue [5]. In addition, verticinone from BFU could in- duce Leukemia HL-60 cells to differentiate toward gran- ulocytes in vitro [6]. Moreover, ethanol extract of BFU inhibited the production of inflammatory cytokine and MAPKs in mast cells [7]. In our previous study, imperia- line, chuanbeinone, verticinone, verticine, imperialine-β- N-oxide, isoverticine and isoverticine-β-N-oxide isolated from genus Fritillaria have significant anti-inflammatory effect in vivo [8,9]. Extraction and isolation h d ff The different extracts of BFU were prepared according to previously described methods [14]. Briefly, the dried powder was immersed in ammonia, and then extracted with EtOH solvent by maceration for 6 hours. After fil- tration, the solvent was evaporated to obtain EtOH ex- tract (EE). The EE was dissolved in 3% HCl and then partitioned with petroleum ether. The pH of the aque- ous solution was readjusted to 10.0 and extracted with chloroform and n-hexane in sequence. Each of these fractions was evaporated to yield petroleum ether extract (PE), chloroform extract (CE), n-hexane extract (HE) and water extract (WE). To the best of our knowledge, there are few publica- tions focusing on the antitumor activity of different ex- tracts of BFU in vitro and in vivo. Therefore, in this study, we prepared different extracts (chloroform extract (CE), n-hexane extract (HE), water extract (WE) and total alkaloids of BFU (TAFU)) from BFU and assessed their antitumor activity in vitro. Furthermore, we identified the compounds from the active extract of BFU by bioassay guided isolation, and determined their antitumor property in vitro. Moreover, the extract of BFU which showed remarked anti-proliferative activity in vitro was further evaluated in vivo. Additionally, preliminary mechanism of its action was investigated. From the previous research, we could conclude that the CE had more total alkaloids [13,14]. The CE which mainly contained total alkaloids showed the highest anti- tumor activity in vitro among three extracts (CE, HE and WE). So, the CE was subjected to further purifica- tion to get total alkaloids of BFU (TAFU). TAFU was prepared by using previous methods [15]. Simply, the CE was dissolved and chromatographed over H-103 resin column, which was eluted with distilled water, 10% alcohol and 90% ethanol, respectively. The fraction eluted with 90% alcohol was collected and lyophilized, Page 3 of 11 Wang et al. BMC Complementary and Alternative Medicine (2015) 15:29 and the resulting powder (TAFU) was subjected to the determination of total alkaloids contents, isolation and pharmacological studies. 200μl DMSO was added to each well. The absorbance was recorded on a Thermo Scientific microplate spectro- photometer (Thermo Fisher Scientific Inc., USA) at the wavelength of 490 nm. The concentration of the extracts which gives 50% growth inhibition is referred to as the IC50 (μg/ml), which was calculated for each extracts from the dose-response curves [13,14]. Measurement of total alkaloids content The total alkaloids content was determined according to previously described method [13]. For alkaloids stand- ard, a stock solution of imperialine was prepared and di- luted to give working standards. For samples, the adequate CE and TAFU were dissolved in chloroform. An aliquot of each sample or standard solution was stained with bromo- cresol green buffer solution to measure total alkaloids content. Absorbance was measured at 412 nm using Alpha-1900PC UV–Vis spectrophotometer (Shanghai Lab-Spectrum Instruments Co., Ltd., China). Data were reported as mean ± standard error of mean (S.E.M.). Changes in cell morphology were checked visually under phase contrast microscope (Carl Zeiss Axiovert 40 CFL, Germany) after incubation with IC50 concentra- tions of TAFU for 24, 72 h, where untreated cells served as control. Quantitative detection of apoptosis and cell cycle distribution The cell cycle was analyzed using flow cytometry as pre- viously described [14,21]. A2780 cells (5.0 × 105 cells/ well) were seeded in 6-well plate and grown until they reached 80% confluence. Then, the cells were treated with peimisine (15 μg/ml) for 24, 48 and 72 h. The cells were harvested, washed thrice with cold PBS and fixed with 1.0 ml of 70% ethanol at 4°C overnight. Then, cells were washed with PBS and incubated in 1.0 ml of PBS containing 100 μg PI, 100 μg RNase A in darkness for 30 min at 37°C, and sorted in a FACScan flow cytometry using Kaluza 1.1 software (Bechman Coulter Inc., USA). Cell cycle distribution and sub-G1 groups were calcu- lated using ModFit LT software. We did not evaluate verticinone for cell cycle distribution because we did not have sufficient amount of verticinone to perform this experiment. Extraction and isolation h d ff The TAFU was separated repeatedly by silica gel col- umn chromatography using petroleum ether-acetone- diethylamine (6:1:1 ~ 1:1:1) of increasing polarity as eluent as reported [8,9]. 15-ml fractions were collected throughout and combined on the basis of Thin Layer Chromatography (TLC). The alkaloids isolated from TAFU were determined by comparing the samples’ melting point and spectral data (1H-, 13C-NMR spectra) with those reported in literature [9,11,16]. Moreover, the serial concentrations of TAFU were used to treat the tumor cells for 24, 48 and 72 h to test the time-dependent cytotoxicity. Furthermore, we mea- sured IC50 of four alkaloids monomers (imperialine, pei- misine, verticine and verticinone) for these tumor cells by MTT assay as mentioned above. All experiments were performed independently in triplicate and data were pre- sented as mean ± S.E.M. Cell lines and cell culture Lewis lung carcinoma cell line (LLC), Human ovarian cancer cell line (A2780), human hepatocellular carcin- oma cell line (HepG2), and human lung carcinoma cell line (A549) were obtained from Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Educa- tion, Sichuan University (Chengdu, China). These cell lines were grown and maintained in a humidified incu- bator at 37°C and in 5% CO2 atmosphere. RPMI-1640 and DMEM (high glucose) medium were supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin. A549 cells were maintained with RPMI1640, A2780, HepG2 and LLC cells were cultured with DMEM. Animals and establishment of tumor model Male ICR mice and C57BL/6 J mice weighing 18–22 g were purchased from Experimental Animal Center of West China College of Pharmacy, Sichuan University (Certificate No. SCXK (Chuan) 2012–09, Chengdu, China). All mice were housed in specific pathogen-free (SPF) level laboratory with a 12 h light–dark cycle, and provided sterile food and water ad libitum [22]. All the procedures were in strict accordance with the Chinese legislation on the use and care of laboratory animals and the guidelines established by Institute for Experimental Animals of Sichuan University and were approved by the Sichuan University Committee on Animal Care and Use. Cytotoxicity assay The cytotoxicity of the different kinds of extracts against the tumor cells was assessed via MTT assay [13,17-20]. HepG2 cells were seeded into six wells at a density of 6×103 cells per well in 96-well microplates, and A2780, A549 and LLC cells were seeded at a density of 8×103 cells per well. Cells were permitted to adhere for 24 h, and then treated with the various concentrations of CE, HE, WE or TAFU for 72 h. DMSO was used to dissolve the extracts, and its final concentration was maintained at 0.5% (v/v). The cultured medium was removed and replaced with 150 μl MTT (0.5 mg/ml) per well before termination at 4 h. After removal of the MTT solution, Page 4 of 11 Wang et al. BMC Complementary and Alternative Medicine (2015) 15:29 Sarcoma 180 (S180) tumor model was established fol- lowing the protocols previously described [9,13,23,24]. S180 cells were maintained in the ascitic form by sequen- tial passages in ICR mice. A mouse bearing a 7-day S180 was sacrificed. The cells were pipetted out and diluted with PBS to reach the final concentration of 1 × 107 cells/ ml. A volume of 0.2 ml S180 cells suspension was subcuta- neously inoculated into the left axilla of ICR mice to estab- lish the S180 tumor model. antigen. Non-specific binding sites were blocked for 1 h in PBS containing 1.5% normal serum. Then, the slides were incubated with primary caspase-3 and CD31 antibody at 4°C overnight, and then incubated with biotinylated sec- ondary antibody for 20 min, followed by incubation with streptavidin-horseradish peroxidase for 20 min. The im- mune reactions were visualized by immersing the slides in 3,3’-diaminobenzidine tetra hydrochloride reagent. The slides were counterstained with hematoxylin and then dehydrated with sequential ethanol for sealing. LLC tumor model was established based on the methods previously described [14,23,25]. LLC cells sus- pension was subcutaneously inoculated into the left axilla of C57BL/6 J mice (2 × 106 cells/mouse). After 7– 10 days, the solid tumor was removed from the mice and single cells were prepared by mincing tumor frag- ments. The cells were adjusted to 1 × 107 cells/ml and a volume of 0.2 ml LLC cells suspension was subcutane- ously inoculated into the left axilla of C57BL/6 J mice to establish the LLC tumor model. We performed immunohistochemical detection of CD31 in tumor sections to determine the antiangiogenesis effects. Statistical analysis The results were expressed as means ± S.E.M.. Statistical analysis was performed by a one-way analysis of variance ANOVA test for multiple group comparison and by Stu- dent’s t-test for comparison of two groups using the SPSS statistics 17.0 software package (LEAD Technolo- gies, Inc., USA). A value of p < 0.05 was considered to be statistically significant. Measurement of xenograft tumour growth inhibition Measurement of xenograft tumour growth inhibition The tumor model animals were randomly divided into five groups of 10 mice each at 24 h after tumor inocula- tion, group 1 (Control, 1% Tween 80 solution, 0.2 ml/ 20 g/day), group 2 (the positive control, CTX, 20 mg/kg/ day), group 3 (the low dose of the TAFU, 20 mg/kg/day), group 4 (the medium dose of the TAFU, 40 mg/kg/day), group 5 (the high dose of the TAFU, 80 mg/kg/day). The dose selection of TAFU is based on the previous experi- ments. The mice were orally administered drugs once a day for 10 days. On the 11th day, the mice were weighed, sacri- ficed, and then tumor, thymus, spleen, heart, liver, lung and kidney were collected to detect inhibition rate of tumor and organ indices. Then, the tumor tissues were fixed in 4% neutral buffered paraformaldehyde immediately for the histological and immunohistochemistry examination. The in vivo antitumor activity of TAFU was expressed as an in- hibition ratio: Inhibition (%) = (Cw −Tw)/Cw × 100, where Cw is the average tumor weight of the control group, and Tw is the average tumor weight of tested group. The organ index was calculated as follows: Organ index (mg/g) = organ weight/(body weight −tumor weight) [13,26,27]. Histological and immunohistochemical examinations Histological and immunohistochemical examinations The histological evaluation was performed as described previously [14,28]. All tumor tissues were randomly cut into 10 histological sections and stained with hematoxylin and eosin (H&E). Histological examinations were carried out by light microscopy (Nikon Eclipse E100). Cytotoxicity assay Tumor microvessel density (MVD) was quantified for high dose group, CTX group and Control group. MVD was determined by counting the number of microvessels per high-power field in the sections as previously de- scribed [14,30]. Immunohistochemistry assessment of caspase-3 expres- sion was conducted to investigate the apoptosis of tumor cells. Immunohistochemically positive cells for casaspe-3 showed brown granules. Integrated optical density (IOD) of brown color of every field of view was quantified using Image-Pro plus software (Media Cybernetics Inc., USA) as previously described [14,31]. Extraction, isolation and content of total alkaloids of the extracts BFU was extracted and fractioned to afford EE (15.27%), PE (0.021%), CE (1.028%), HE (7.692%) and WE (0.660%). Four steroidal alkaloids were obtained from TAFU and identified as imperialine (I), peimisine (II), verticinone (III), and verticine (IV) (Figure 1) based on their physical and spectral data which showed good agreement with published references [9,11,12]. The structures of the four compounds presented in Figure 1. In addition, the total al- kaloids contents in CE and TAFU were determined as 19.67% and 54.37%, respectively. Evaluation of cytotoxicity against tumor cells To evaluate cytotoxicity of the different fractions against tumor cell lines, the MTT assay was used. The cytotoxic effects of HE, WE, CE and TAFU on LLC, A2780, HepG2 and A549 cells were examined at different concentrations (Figure 2A), and their IC50 were determined (Table 1). Immunohistochemical detection of caspase-3 and CD31 was carried out by standard immunohistochemical tech- niques [14,29]. Briefly, these sections were incubated with 1% H2O2 to inactivate endogenous peroxidase and put into 10 mM sodium citrate buffer solution to recover Wang et al. BMC Complementary and Alternative Medicine (2015) 15:29 Page 5 of 11 Figure 1 The molecular structures of four steroidal alkaloids from BFU. The steroidal alkaloids are imperialine (I), peimisine (II), verticinone (III), verticine (IV). Figure 1 The molecular structures of four steroidal alkaloids from BFU. The steroidal alkaloids are imperialine (I), peimis II), verticine (IV). Figure 1 The molecular structures of four steroidal alkaloids from BFU. The steroidal alkaloids are imperialine (I), peimisine (II), verticinone (III) verticine (IV) Figure 1 The molecular structures of four steroidal alkaloids from BFU. The steroidal alkaloids are imperialine (I), peimisine (II), verticinone (III), verticine (IV). Effect of TAFU on tumor growth and organ indices in tumor-bearing mice The results suggested that CE and TAFU showed the sig- nificantly higher cytotoxic effects than HE and WE, espe- cially TAFU showed the most effective inhibitory activity. In addition, two cancer cell lines (A2780 and LLC) were more sensitive to the CE and TAFU than the others. Moreover, we evaluated the cytotoxicity of verticine, veri- cinone, imperialine and peimisine in vitro, and their IC50 are shown in Table 1. The four alkaloids showed signifi- cant cytotoxic effects, and verticinone and peimisine showed markedly higher inhibitory effects than the others. To our knowledge, different fractions of BFU and the alka- loids were firstly studied regarding the cytotoxic activity against these tumor cell lines. To further evaluate the antitumor effects of TAFU, an in vivo antitumor study using S180 and LLC tumor models were performed. The inhibitory effects of TAFU at the high (80 mg/kg/day), medium (40 mg/kg/day) and low dose (20 mg/kg/day) on the transplanted S180 and LLC tumors are shown in Table 2. The higher dose of TAFU significantly inhibited the growth of transplant- able S180 sarcoma and LLC tumors in mice in dose- dependent manner. As a positive control drug, CTX showed higher inhibitory rate. As shown in Table 3, body weight of animals which re- ceived TAFU has no significant difference compared with that of Control group. In contrast, the body weight of mice with treatment of CTX significantly decreased com- pared to that of TAFU- and non-treatment. In addition, immune organs (the thymus and spleen) indices in the CTX-treated group were significantly lower than that of the TAFU and Control groups. The result indicated that TAF had significant antitumor activity in vivo, and lower toxicity compared with CTX on the basis of body weight and organ indices. The cytotoxic activities of TAFU against the four tumor cell lines at series concentrations with different treatment time were studied. As shown in Figure 2B, TAFU had cytotoxicity against the four kinds of tumor cells in a time-dependent manner. After incubation with IC50 concentrations of TAFU for 24, 72 h, its effects on cell morphology are shown in Figure 2C. Compared with the negative control, clas- sical apoptotic cells were observed in TAFU mediated cell death. Wang et al. Peimisine induced G0/G1 phase arrest and rising apoptosis rate clear, and the tumor cells showed obvious nucleolus cleavage and high extent of malignancy. To investigate the effect of peimisine on the apoptosis and the cell cycle distribution of A2780 cells, flow cy- tometry was used. As depicted in Figure 3A, the typical sub-G1 peak, which represented the apoptosis cell popu- lation, appeared in the DNA content after the treatment with peimisine. The percentage of apoptosis increased in a time-dependent manner and the changes in the cell cycle progression were notable (Figure 3A). Besides, the cell population of the S phase reduced concurrently with the increase of the G0/G1 phase population after the treatment with peimisine. The results suggested that pei- misine mainly induced G0/G1 phase arrest of A2780 cells in a time-dependent manner, which might be contribut- ing to the apoptosis. We investigated the anti-angiogenic effects of TAFU by immunostaining of tumor sections for the endothe- lial cell marker CD31, which is the most specific and sensitive endothelial marker. Figure 3C shows the typ- ical reduction of tumor blood vasculature number after TAFU and CTX treatment. As show in Figure 3C, the positive cells were counted from five different areas for each sample, and there were 34.85 ± 5.74, 38.45 ± 4.90 and 43.10 ± 5.89 for the higher dose of TAFU, CTX and Control groups in S180 tumor model respectively, and 6.10 ± 0.21, 7.50 ± 0.93 and 13.85 ± 1.57 for the higher dose of TAFU, CTX and Control groups in LLC tumor model, respectively. Immunohistochemical analysis of caspase-3 expres- sion in solid tumor is shown in Figure 3D. A profound increase in caspase-3 expression was observed in solid tumors from mice treated with TAFU and CTX. As show in Figure 3D, integrated optical density (IOD) of brown color of every observed field was quantified from eight representative fields for each sample, and there were 79.57 ± 9.37, 69.60 ± 8.88 and 36.77 ± 4.23 for the higher dose of TAFU, CTX and Control groups in S180 tumor model respectively, and 14.71 ± 0.83, 23.65 ± 6.33 and 7.01 ± 0.97 for the higher dose of TAFU, CTX and Control groups in S180 tumor model, respectively. Effect of TAFU on tumor growth and organ indices in tumor-bearing mice BMC Complementary and Alternative Medicine (2015) 15:29 Page 6 of 11 See legend on next page.) Figure 2 (See legend on next page.) Figure 2 (See legend on next page.) Figure 2 (See legend on next page.) Page 7 of 11 Wang et al. BMC Complementary and Alternative Medicine (2015) 15:29 (See figure on previous page.) Figure 2 The cytotoxic effects of the different fractions against tumor cells. (A), Effects of different concentrations of HE, WE, CE and TAFU on proliferation of LLC, A2780, HepG2 and A549 cells. (a), HE; (b), WE; (c), CE; (d), TAFU. Three cell lines were treated with four fractions for 72 hours. Significant differences compared with Control group were designated as P < 0.05, P < 0.01 and *P < 0.001. (B), Time and dose effects of different concentrations of TAFU on LLC, A2780, HepG2 and A549 cells growth. (a), LLC; (b), A2780; (c), HepG2; (d), A549. Significant differences of inhibitory effects between treatment of 24 h and 72 h were designated as P < 0.05, P < 0.01 and P < 0.001; Significant differences of inhibitory effects between treatment of 24 h and 48 h were designed as □P < 0.05 □□P < 0.01 and □□□P < 0.001; Significant differences of inhibitory effects between treatment of 48 h and 72 h were designed as #P < 0.05, ##P < 0.01 and ###P < 0.001. (C), Microscopic image of TAFU (IC50) treated LLC, A2780, HepG2 and A549 cells for 24 and 72 hours. Cells morphology was observed by a phase contrast inverted microscope (200×). (a), LLC; (b), A2780; (c), HepG2; (d), A549. All results were expressed as the mean ± S. E. M. (n = 3). Peimisine induced G0/G1 phase arrest and rising apoptosis rate The reported values are the means ± S.E.M. (n = 3). Discussion In recent years, increasing attention has been paid to genus of Fritillaria owing to its powerful potential thera- peutic benefits for various diseases. Previous studies have reported that the total alkaloids or monomers of alkaloids from the genus of Fritillaria (Fritillaria ebeiensis and Fritillaria cirrhosae) have antitumor activ- ities [11,13,14]. It is well known that some plants of the genus Fritillaria are on the edge of extinction which is regarded as major constraint for further development and utilization [32]. However, there are very mature artificial plantation technologies for BFU, the cultivated BFU become the mainstream of BFU in market [33], Apoptosis is an important biological mechanism that contributes to the maintenance of the integrity of multi- cellular organisms that is dependent on the expression of cell-intrinsic suicide machinery [14]. Apoptosis is characterised by key morphological features, such as cell shrinkage, membrane blebbing, chromatin condensation and generation of apoptotic bodies [35]. Effects of TAFU on histological changes, CD31 and caspase-3 expression of tumor tissues In order to further investigate the mechanisms of antitu- mor efficacy of TAFU, HE CD31 and caspase-3 immu- nohistochemical staining were performed on tumor tissues. The morphology of apoptosis cell in tumor tis- sues was observed by HE staining technique. The results suggested that apoptotic cells observed in the TAFU- or CTX-group were more significant in tumor tissues com- pared with that in the control group (Figure 3B). In Control group, the tumor architecture was intact and Table 1 IC50 values of the different fractions and alkaloids from BFU against tumor cell lines Fractions/compounds IC50 (μg/ml) LLC A2780 HepG2 A549 HE 660.11 ± 16.71 246.97 ± 18.78 288.95 ± 17.84 294.96 ± 24.46 WE 614.74 ± 19.55 208.38 ± 23.57 279.09 ± 13.06 461.26 ± 28.38 CE 97.55 ± 4.02 88.50 ± 5.26 128.26 ± 12.55 172.59 ± 6.59 TAFU 39.37 ± 2.36 27.65 ± 1.84 51.10 ± 2.84 58.60 ± 3.99 Verticine 16.53 ± 0.82 48.41 ± 2.58 53.38 ± 3.16 97.58 ± 3.72 Vericinone 3.03 ± 0.32 17.03 ± 0.89 14.64 ± 0.65 47.39 ± 1.90 Imperialine 66.26 ± 3.46 39.18 ± 2.21 84.26 ± 4.46 117.84 ± 6.29 Peimisine 20.75 ± 1.44 17.43 ± 0.80 92.07 ± 4.89 36.11 ± 1.70 The reported values are the means ± S.E.M. (n = 3). the different fractions and alkaloids from BFU against tumor cell lines Table 1 IC50 values of the different fractions and alkaloids from BFU against tumor cell lines Wang et al. BMC Complementary and Alternative Medicine (2015) 15:29 Page 8 of 11 Table 2 Inhibitory effects of the TAFU on the tumor growth of S180 and LLC tumor models Groups Dose (mg/kg) No. of animals Weight of tumor (g) Inhibition (%) S180 tumor model Control - 10 1.20 ± 0.30 - TAFU 20 10 1.22 ± 0.21 −1.46 40 10 0.99 ± 0.10 17.80 80 10 0.62 ± 0.06 48.56 CTX 20 10 0.53 ± 0.13* 55.92 LLC tumor model Control - 10 2.27 ± 0.16 - TAFU 20 10 2.38 ± 0.17 −4.68 40 10 2.11 ± 0.24 7.22 80 10 1.36 ± 0.20 39.96 CTX 20 10 0.94 ± 0.11* 58.37 The reported values are the means ± S.E.M. (n = 10). Significant differences compared to Control group are indicated by P < 0.05, P < 0.01 and *P < 0.001. Effects of TAFU on histological changes, CD31 and caspase-3 expression of tumor tissues Table 2 Inhibitory effects of the TAFU on the tumor growth of S180 and LLC tumor models which provides the adequate resources for further ex- ploitation and utilization of BFU. Moreover, antitumor effects of BFU have not yet been properly investigated. So, it was our primary goal to evaluate antitumor activ- ity of BFU in vitro and in vivo and explore its related mechanism for its action. Cytotoxic assay is used to search which fractions have potential antitumor properties. In our work, samples with higher total alkaloids (CE and TAFU) trended to have stronger cytotoxic activity against tumor cancer cells than the other two fractions (HE and WE), and they all inhibited cell proliferation in a dose- and time- dependent manner. The results suggested that the anti- tumor potential of the fractions might mainly be attrib- uted to the total alkaloids content. Moreover, we isolated four main alkaloids (imperialine, peimisine, verticine and verticinone) from TAFU, measured their cytotoxic effects, and found that they all showed significant cytotoxic activity in vitro, which proved that the steroidal alkaloids are truly involved in the antitumor activity. Besides, our previous study showed that the alkaloids from BFU had no signifi- cant cytotoxic effect on normal ARPE-19 cells compared to DMSO at concentrations ranging from 10 to 320 μg/ml, and they showed that IC50 values of these compounds is higher than 200 μg/ml [34]. The reported values are the means ± S.E.M. (n = 10). Significant differences compared to Control group are indicated by P < 0.05, P < 0.01 and P < 0.001. The reported values are the means ± S.E.M. (n = 10). Significant differences compared with Control group were designated as and those compared with CTX group as #P < 0.05, ##P < 0.01 and ###P < 0.001. Discussion The results of this study showed typical morphological characteristics of apoptosis, such as nuclear condensation and apoptotic Table 3 Organ weights index of the mice xenografted with S180 cells and LLC cells Groups Dose (mg/kg) Growth of weight (g) Organ indices (mg/g) Spleen Thymus Heart Liver Lung Kidney S180 tumor model Control - 4.08 ± 0.61 7.02 ± 0.70 2.24 ± 0.22 5.17 ± 0.40 63.71 ± 3.53 6.88 ± 0.52 13.01 ± 0.61 TAFU 20 2.36 ± 0.81### 6.86 ± 0.20## 2.43 ± 0.35## 4.34 ± 0.12 63.28 ± 2.10# 7.29 ± 0.37 13.00 ± 0.50 40 2.84 ± 0.74# 6.67 ± 0.17## 1.98 ± 0.12, # 4.67 ± 0.20 56.04 ± 1.11 6.62 ± 0.21 12.29 ± 0.37 80 2.45 ± 0.50## 5.43 ± 0.11, # 1.84 ± 0.17 4.55 ± 0.30 58.50 ± 0.82 6.39 ± 0.45 12.88 ± 0.63 CTX 20 −1.82 ± 0.64* 4.26 ± 0.16* 1.30 ± 0.15** 4.73 ± 0.18 52.72 ± 3.30* 6.75 ± 0.39 12.81 ± 0.59 LLC tumor model Control - 0.64 ± 0.48 6.07 ± 0.91 1.57 ± 0.32 5.93 ± 0.48 57.54 ± 3.99 6.90 ± 0.52 13.63 ± 0.60 TAFU 20 0.58 ± 0.68### 7.74 ± 0.57## 1.25 ± 0.13### 5.22 ± 0.32# 58.69 ± 1.32# 7.20 ± 0.33* 12.54 ± 0.55 40 0.37 ± 0.32, ## 7.53 ± 0.38## 1.18 ± 0.04## 5.25 ± 0.43# 53.44 ± 1.20# 7.33 ± 0.10 11.17 ± 0.22 80 0.29 ± 0.50, ## 5.68 ± 0.82# 1.09 ± 0.07, ## 4.86 ± 0.12 53.85 ± 4.96# 6.87 ± 0.60# 11.69 ± 0.40 CTX 20 −1.88 ± 0.71* 4.05 ± 0.42 0.41 ± 0.05*** 4.38 ± 0.17* 44.34 ± 3.59* 6.80 ± 0.54 11.96 ± 0.62 The reported values are the means ± S.E.M. (n = 10). Significant differences compared with Control group were designated as P < 0.05, P < 0.01 and *P < 0.001; and those compared with CTX group as #P < 0.05, ##P < 0.01 and ###P < 0.001. Table 3 Organ weights index of the mice xenografted with S180 cells and LLC cells Gro ps Dose Gro th of Organ indices (mg/g) Table 3 Organ weights index of the mice xenografted with S180 cells and LLC cells Wang et al. BMC Complementary and Alternative Medicine (2015) 15:29 Page 9 of 11 Figure 3 Mechanism of antitumor activity of alkaloids from BFU. Discussion (A), Effects of peimisine on A2780 cells cycle distribution and the apoptosis rate. The cells were treated with peimisine (15 μg/ml) for 0, 24, 48 and 72 h. (a), 0 h; (b), 24 h; (c), 48 h; (d), 72 h. (B), Histopathological examination of tumor tissue in S180 and LLC tumor models treated with TAFU, CTX and Tween 80 solution as Control by HE staining images (magnification: 40× and 100×), and there are larger areas of necrotic region in the high dose of TAFU and CTX groups than that of Control group. (a), tumor tissue in S180 tumor model; (b), tumor tissue in LLC tumor model. (C), Effects of TAFU on CD31 expression in mice treated with TAFU, CTX and Tween 80 solution as Control. Typical images of immunohistochemical staining of CD31 in tumor mice (magnification: 100×), and the positive cells were stained by brown. (a1), tumor tissue in S180 tumor model; (a2) microvessel density (MVD) of tumor tissue in S180 tumor model; (b1), tumor tissue in LLC tumor model; (b2) MVD of tumor tissue in LLC tumor model. (D) Effects of TAFU on caspase-3 expression in mice treated with TAFU, CTX and Tween 80 solution as Control. Typical images of immunohistochemical staining of caspase-3 in tumor mice (magnification: 400×), and the positive cells were stained by brown. (a1), tumor tissue in S180 tumor model; (a2) IOD of caspase-3 expression in tumor tissue of S180 tumor model; (b1), tumor tissue in LLC tumor model; (b2) IOD of caspase-3 expression in tumor tissue of LLC tumor model. All results were expressed as the mean ± S. E. M., significant differences compared with Control group were designated as P < 0.05 and *P < 0.01. Figure 3 Mechanism of antitumor activity of alkaloids from BFU. (A), Effects of peimisine on A2780 cells cycle distribution and the apoptosis rate. The cells were treated with peimisine (15 μg/ml) for 0, 24, 48 and 72 h. (a), 0 h; (b), 24 h; (c), 48 h; (d), 72 h. (B), Histopathological examination of tumor tissue in S180 and LLC tumor models treated with TAFU, CTX and Tween 80 solution as Control by HE staining images (magnification: 40× and 100×), and there are larger areas of necrotic region in the high dose of TAFU and CTX groups than that of Control group. (a), tumor tissue in S180 tumor model; (b), tumor tissue in LLC tumor model. Discussion (C), Effects of TAFU on CD31 expression in mice treated with TAFU, CTX and Tween 80 solution as Control. Typical images of immunohistochemical staining of CD31 in tumor mice (magnification: 100×), and the positive cells were stained by brown. (a1), tumor tissue in S180 tumor model; (a2) microvessel density (MVD) of tumor tissue in S180 tumor model; (b1), tumor tissue in LLC tumor model; (b2) MVD of tumor tissue in LLC tumor model. (D) Effects of TAFU on caspase-3 expression in mice treated with TAFU, CTX and Tween 80 solution as Control. Typical images of immunohistochemical staining of caspase-3 in tumor mice (magnification: 400×), and the positive cells were stained by brown. (a1), tumor tissue in S180 tumor model; (a2) IOD of caspase-3 expression in tumor tissue of S180 tumor model; (b1), tumor tissue in LLC tumor model; (b2) IOD of caspase-3 expression in tumor tissue of LLC tumor model. All results were expressed as the mean ± S. E. M., significant differences compared with Control group were designated as P < 0.05 and **P < 0.01. Page 10 of 11 Wang et al. BMC Complementary and Alternative Medicine (2015) 15:29 Page 10 of 11 Page 10 of 11 of the active alkaloids by bioassay guided isolation, deter- mination of cytotoxic activity of steroidal alkaloids in vitro, measurement of the inhibitory effects of TAFU on xenograft tumor and its relative mechanism. CE and TAFU with higher total alkaloids exhibited notable cytotox- icity against the four tumor cell lines, and TAFU signifi- cantly inhibited the growth of transplantable S180 sarcoma and LLC tumor in mice with lower toxicity. In addition, four main steroidal alkaloids were isolated from TAFU and identified as imperialine, peimisine, verticinone and verti- cine. Furthermore, all alkaloids showed significant cytotox- icity against four tumor cells and peimisine induced G0/G1 phase arrest and increased apoptosis rate. Moreover, the histological examination indicated that TAFU could change the morphological features of tumor cells, induce their apoptosis. The immunohistochemical examinations showed that TAFU remarkably increased caspase-3 expression and reduced MVD in tumor tissues of transplantable S180 and LLC tumor models. Notably, our finding is the first report on the antitumor activity of extracts and steroidal alkaloids from the cultivated BFU in vitro and in vivo and its mecha- nisms, however, further studies are needed to elucidate the precise mechanisms of its antitumor activity. body formation, in tumor cells treated with TAFU. Authors’ contributions DDW participated in its design and coordination and helped to draft the manuscript. KW participated in evaluation the antitumor activity of TAFU in vivo. SW conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Relevant studies have demonstrated that tumor growth and metastasis are dependent on angiogenesis. So, inhib- ition of tumor angiogenesis may affect the tumor growth and decrease metastatic potential of tumors [38]. MVD has been considered as a useful prognostic indicator reflecting tumor angiogenesis. The intratumoral MVD was examined by immunohistochemical analysis to as- sess whether TAFU induced inhibition of xenograft tumor growth was mediated through its anti-angiogenic effects. Microscopic examination of tumors showed IOD of CD31 stained cells in TAFU- and CTX-treated group were markedly less than that of control group. The re- sults suggested that the inhibit effects of TAFU on the number of microvessel may partially contribute to the inhibition of tumor growth in mice. Acknowledgements The authors are grateful to Science and Technology Support Program of Sichuan Province (2011SZ0274) from The Department of Science and Technology of Sichuan Province and the Chinese Government Scholarship from The China Scholarship Council for financial support. The funding agency did not have any role in collection, analysis, interpretation of data, writing of the report or the decision to submit the paper for publication. Author details 1 1Department of Pharmacognosy, West China College of Pharmacy, Sichuan University, No. 17, Duan 3, Renmin Nan Road, Chengdu 610041, Peoples’ Republic of China. 2State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Science, University of Macau, Macau 999078, Peoples’ Republic of China. Received: 19 July 2014 Accepted: 12 February 2015 Received: 19 July 2014 Accepted: 12 February 2015 Discussion Further- more, the apoptosis of A2780 cells induced by peimisine was also confirmed by the sub-G1 DNA accumulation. In addition, the results showed that peimisine induced an ac- cumulation of cells in G0/G1 phase with an increasing apoptotic rate, which indicated that alkaloids from BFU in- duced apoptosis likely through G0/G1 phase arrest. The further results of experiment in vivo showed that TAFU could significantly inhibit growth of transplant- able S180 sarcoma and LLC tumor in mice. It is well known that most of chemical antitumor drug could causes toxic side effects, while natural products often have very low clinical toxicity compared with them [36]. In this study, the toxicity of TAFU was evaluated based on changes of body weight and organ indices in ICR and C57BL/6 J mice. These results showed that CTX mark- edly decreased body weight, thymus and spleen indices in mice as compared with TAFU group and Control group. As the important immune organs, the spleen and thymus indices reflect the immune function of the organism [14]. By combining the tumor weight, body weight and organ indices measurement, it was indicated that TAFU had sig- nificant antitumor activities in vivo and fewer side effects compared with CTX. Competing interests p g The authors declare that they have no competing interests. Abbreviations As indicated in many other studies, apoptosis is a type of cell death process regulated in an orderly way by a series of signal cascades under certain situations [13]. Caspases family plays a crucial role in the mechanism of cell apoptosis. To further clarify the mechanism of its antitumor activity, we investigated whether caspase-3, the major downstream effector of apoptosis [37], was in- volved in TAFU-induced apoptosis in tumor tissues. The results showed that TAFU significantly inhibited trans- planted S180 and LLC tumors in a manner that causes caspase-dependent apoptosis by activating the caspase-3. BFP: Bulbus Fritillariae ussuriensis; CE: Chloroform extracts; CTX: Cyclophosphamide; DMEM: Dulbecco’s Modified Eagle’s medium; DMSO: Dimethyl sulphoxide; FBS: Fetal bovine serum; HE: n-hexane extracts; H & E: Hematoxylin and eosin; IC50: 50% inhibitory concentration; IOD: Integrated optical density; EE: EtOH extracts; MTT: 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenyl-formazan; MVD: Microvessel density; PI: Propidium iodine; TAFU: Total alkaloids of BFU; WE: Water extracts. . National Commission of Chinese Pharmacopoeia. Pharmacopoeia of Peoples Republic of China, Volume 1. Beijing: China Medical Science Press; 2010. p. 90–1. Conclusions l 1. National Commission of Chinese Pharmacopoeia. Pharmacopoeia of Peoples Republic of China, Volume 1. Beijing: China Medical Science Press; 2010. p. 90–1. In conclusion, this study showed investigation of cytotox- icity of different fractions from BFU in vitro, identification Page 11 of 11 Wang et al. 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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 21. Ma YS, Weng SW, Lin MW, Lu CC, Chiang JH, Yang JS, et al. Antitumor effects of emodin on LS1034 human colon cancer cells in vitro and in vivo: Roles of apoptotic cell death and LS1034 tumor xenografts model. 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https://openalex.org/W2167801352	https://europepmc.org/articles/pmc4109427?pdf=render	English	null	What is the role of visual skills in learning to read?	Frontiers in psychology	2,014	cc-by	3,356	Edited by: Korean kindergartners tend to learn to read Korean syllables holis- tically initially, similar to how Chinese characters are taught. A superior perfor- mance on visual skills was also found by Demetriou et al. (2005) for older Chinese as compared to Greek children. In addi- tion, Huang and Hanley (1994) found that both Taiwanese and Hong Kong children showed a clear advantage on the visual form discrimination task as compared to their British peers. , ) Indeed, the role of visual skills for early reading development may be stronger for reading Chinese than reading English. Pure visual skills are sometimes rela- tively strong correlates of Chinese chil- dren’s reading (e.g., Huang and Hanley, 1994, 1995; Ho and Bryant, 1997; Siok and Fletcher, 2001; Mcbride-Chang et al., 2005; Luo et al., 2013). Such visual tasks likely tap at least three visual skills that may be required for Chinese character read- ing. First, a focus on visual form constancy (e.g., is this square the same size as the one embedded in other designs on the previous page?) likely has some analogies with the fact that radicals within Chinese might appear as larger or smaller or even reversed in appearance across characters. Second, a focus on visual spatial skills, i.e., identifying the same form when it is in a different direction or placed dif- ferently, might be useful when Chinese character identiﬁcation requires children to reduce a compact character into compo- nent radicals. Third, visual memory may be useful in learning to read Chinese in at least two ways, namely, making asso- ciations between characters and sounds, many of which are arbitrary, sometimes referred to as visual verbal paired associate learning, and helping children to build a Interestingly, the Chinese written sys- tem has two versions, the simpliﬁed and the traditional. The simpliﬁed script, which has fewer visual features to distin- guish one character from another, may make more visual demands than does the traditional version. In one study of those learning to read traditional (in Hong Kong) as compared to simpliﬁed (in Mainland China) script, those learn- ing the simpliﬁed script outperformed those learning the traditional one on three visual tasks, namely visual discrimination, visual spatial relationships and visual clo- sure tasks (Mcbride-Chang et al., 2005), even across time. Peng et al. Edited by: Edited by: Tânia Fernandes, University of Porto, Portugal Reviewed by: Andrea Facoetti, Università di Padova, Italy Reviewed by: Andrea Facoetti, Università di Padova, Italy Reviewed by: Andrea Facoetti, Università di Padova, Italy Keywords: reading, visual skill, Chinese, orthography, visual processing mental memory of how different radical parts are located within a character. Inhoff and Liu (1998) found that Chinese readers used comparatively smaller visual perceptual spans than English readers. Szwed et al. (2014) found that readers of Chinese showed strong activations in intermediate visual areas of the occipital cortex; these were absent in French read- ers. Researchers have attributed these char- acteristics to perceptual learning resulting from learning to read Chinese characters (Rayner, 1998; Perfetti et al., 2010; Szwed et al., 2014). Although the issue of visual skills in relation to word reading has not been central to recent explorations of read- ing development, all visual word reading involves visual skill. Children constantly face tasks of differentiating visually sim- ilar letters or words. For example, dis- tinguishing “b” from “d,” “a” from “e,” or “book” from “boot” all require visual differentiation. Children’s orthographic knowledge and letter knowledge are causal factors in subsequent reading develop- ment in English (e.g., Badian, 1994; Lonigan et al., 2000). At a pure visual skill level, some researchers (e.g., Franceschini et al., 2012) suggest that core visual pro- cessing skills such as visual spatial atten- tion in preschoolers could be a causal factor in subsequent reading acquisition. In addition, some alphabetic readers with dyslexia may have visual processing deﬁcits (e.g., Valdois et al., 2004; Van der Leij et al., 2013). Following this hypothe- sis, Franceschini et al. (2013) showed that action video games that strengthened children’s visual attention also improved their reading speed in Italian without sacriﬁcing reading accuracy, similar to previous interventional research training facilitating visuospatial attention skills in Italian children with dyslexia (Facoetti et al., 2003). However, orthographic depth mediates the role of visual attention in reading (Bavelier et al., 2013; Richlan, 2014). English is a more opaque orthogra- phy than Italian, and Chinese is even more opaque than English. What is the evidence that learning to read Chinese trains one’s visual skills? A cross-cultural study (McBride-Chang et al., 2011b) found that Chinese and Korean kindergartners performed signif- icantly better than Israeli and Spanish children on a task of visual spatial relation- ships, the only visual task tested across all four cultures. Yanling Zhou 1, Catherine McBride-Chang 2 and Natalie Wong 2 1 Department of Early Childhood Education, The Hong Kong Institute of Education, Hong Kong 2 Developmental Centre, Department of Psychology, The Chinese University of Hong Kong, Hong Kong Correspondence: ylzhou@ied.edu.hk Edited by: (1975) found that the copying of Hebrew did not distinguish dyslexic from non- dyslexic children who were native English speakers unfamiliar with Hebrew. In con- trast, such copying skills did distinguish those with and without dyslexia in Chinese (McBride-Chang et al., 2011a). However, few, if any researchers, have gone fur- ther with this research, examining to what extent the ability to copy unfamiliar mate- rials is associated with the ability to write words in a native orthography. Given a proposed ﬁrst stage of word reading as primarily visual (Ehri, 2013), a ﬁrst stage of word writing might be, corre- spondingly, associated with visual-motor skills that can be measured using pure two-dimensional patterns. Perhaps such copying abilities are particularly linked to learning to write in Chinese or in ashkara. However, it is important to exam- ine these abilities independently within, as well as across, orthographies. While it may be the case that copying of unfa- miliar stimuli explains subsequent spelling skills for Hindi, Chinese, Korean, or even Dutch children within a group, for Importantly, most studies on the topic of visual skills and word reading were carried out at a single point in time, rather than in a longitudinal study, and there are few experimental studies on this topic. Thus, the issue of causality is not clear (e.g., McBride-Chang et al., 2011b; Yang et al., 2013). However, there is prob- ably a bidirectional association between pure visual skills and learning to read in the early years (e.g., McBride-Chang et al., 2011b). Future directions in this area should focus on at least three aspects of research in order to gain a better under- standing of the causal associations between literacy and visual skills. First, more research should explore how and which visual skills may promote word reading in the early grades. Second, researchers can consider more broadly how learning to read particular orthographies might facil- itate given visual skills. Third, an explo- ration of pure visual skills and word read- ing could be expanded further to visual- motor skills and writing. The second question focuses more on group differences across orthographies: Do orthographies facilitate visual ﬂexibil- ity and analysis in different ways? Perhaps not only are the visual characteristics of the orthography important, but the style of teaching and learning the orthogra- phy is additionally important for facili- tating visual skills. Edited by: For example, although Korean Hangul is ultimately a relatively simple alphasyllabary, it is taught initially to children more in the form of sylla- bles with different components, such that children have many different conﬁgura- tions to learn. Perhaps children’s visual memorization loads would be reduced if they were taught the basic phonological rules of Korean ﬁrst, but this is not the way in which they are instructed. Future research should consider both the dimen- sions of visual demands of the orthog- raphy and also teaching approaches. For example, young children are often taught to read Thai with spaces in between words indicated before they are gradu- ally coaxed to read Thai as it is writ- ten for adults—without spaces between words. Conceivably, those learning to read Thai with and without spaces might show different visual patterns of discrimination early on. Another area for research on this topic would be to compare children learn- ing to read orthographies that differ on the dimension of contained vs. expansive as deﬁned by Nag, at least in three aspects, i.e., alphabetic, ashkara, and Chinese, across the early years of literacy develop- ment (perhaps ages 4–7 years) to deter- mine whether those learning an alpha- bet are least sophisticated in visual skill, those learning an ashkara in the middle, and Chinese learners the best. Although McBride-Chang et al. (2011b) compared 5-year-olds learning Chinese, Korean, and alphabets, this study could be expanded to include ashkara learners and to examine the developmental trend with age across several dimensions of basic visual skill. The issue of how visual skills facilitate word reading is important to explore in a variety of orthographies. For example, Nag (2007, 2011) has presented a model of orthographically contained vs. exten- sive orthographies. At the most extensive level is Chinese, with thousands of pos- sible characters. At the most contained level are alphabetic orthographies with sets of around 24 to 30 letters each. In the middle, she placed Bengali, Hindi, and Kannada, each with 400+ possible sym- bols. Collectively, these are referred to as the ashkara languages. As with Chinese, small changes in visual symbols in ashkara scripts signal potentially large changes in phonological or meaning representations. For example, native readers of Arabic are slower to process it than a second lan- guage of Hebrew because of differences in visual complexity (Ibrahim et al., 2002; Abdelhadi et al., 2011). Edited by: (2010) found that electrophysiological response poten- tials (ERPs) in the brains of those expert readers who saw characters with one stroke either added or subtracted in a few mil- liseconds showed the same basic pattern: Both eye movement and neuroimag- ing studies have demonstrated that reading Chinese affects visual processing dif- ferently than does reading alphabetic orthographies (e.g., Inhoff and Liu, 1998; Perfetti et al., 2010; Szwed et al., 2014). July 2014 \| Volume 5 \| Article 776 \| 1 www.frontiersin.org www.frontiersin.org Visual skills in learning to read Zhou et al. The brains of simpliﬁed script readers appeared to register the alterations early in processing; those of traditional script readers did not. whether and which visual analysis skills might explain early literacy in diverse orthographies. Perhaps more varied visual skills would best explain performances in ashkara languages and Chinese given their visual complexities. For this research ques- tion, the focus is on individual differences within a group learning to read in a single orthography. whether and which visual analysis skills might explain early literacy in diverse orthographies. Perhaps more varied visual skills would best explain performances in ashkara languages and Chinese given their visual complexities. For this research ques- tion, the focus is on individual differences within a group learning to read in a single orthography. A third issue to consider makes an anal- ogy from word reading and visual recog- nition, considered above, to word writ- ing and visual copying. We have recently established that children who tend to write Chinese characters better tend also to show better abilities to copy 2-dimensional unfamiliar forms (e.g., Wang et al., 2013). These forms were foreign scripts that were coded by those unfamiliar with each (e.g., a Chinese research assistant evaluated chil- dren’s writing of Hebrew based purely on the visual representation of the writ- ing perceived). Unfamiliar scripts were selected to ensure that the 2-dimensional writing was equally unfamiliar to all chil- dren. (In contrast, copying of geometric shapes or one’s own script would be poten- tially problematic because those who are academically more skilled often tend to write all familiar stimuli better than those who are less so). Tan et al. (2005) have suggested that copying skills are impor- tant for learning to write/spell Chinese. However, there is very little research that has examined this question for orthogra- phies other than Chinese. Vellutino et al. Edited by: Even in simple alphabetic orthographies, young children might memorize words based on par- ticular visual features (e.g., M has two humps; the word “bed” in English actually looks like a bed). Therefore, it is impor- tant for researchers to continue to explore July 2014 \| Volume 5 \| Article 776 \| 2 Frontiers in Psychology \| Developmental Psychology Visual skills in learning to read Zhou et al. superior visuo-motor skills more gener- ally (as compared to those learning to read and write Dutch, for example). More research on the interface between literacy skills within and across orthographies and visual and visuo-motor skills, can poten- tially yield new directions that are theoret- ically interesting and possibly practically important. Franceschini, S., Gori, S., Rufﬁno, M., Viola, S., Molteni, M., and Facoetti, A. (2013). Report action video games make dyslexic children read better. Curr. Biol. 462–466. doi: 10.1016/j.cub.2013.01.044 Rayner, K. (1998). Eye movements in reading and information processing: 20 years of research. Psychol. Bull. 124, 372–422. Richlan, F. (2014). Functional neuroanatomy of developmental dyslexia: the role of ortho- graphic depth. Front. Hum. Neurosci. 8:347. doi: 10.3389/fnhum.2014.00347 Ho, C. S.-H., and Bryant, P. (1997). Learning to read chinese beyond the logographic phase. Read. Res. Q. 32, 276–289. doi: 10.1598/RRQ.32.3.3 Siok, W. T., and Fletcher, P. (2001). The role of phono- logical awareness and visual-orthographic skills in Chinese reading acquisition. Dev. Psychol. 37, 886–899. doi: 10.1037/0012-1649.37.6.886 Huang, H. S., and Hanley, J. R. (1994). Phonological awareness and visual skills in learning to read Chinese and English. Cognition 54, 73–98. To conclude, the role of visual skills in learning to read is apparently com- plex, and our understanding of this role depends upon the extent to which we look within as compared to between orthogra- phies. The types of visual skills, the types of orthographies, and the teaching meth- ods for literacy instruction all inﬂuence this association. Moreover, the associa- tion between visual skills and literacy development is likely bidirectional. This association can be expanded to focus on visuo-motor skills and writing. These issues cross-culturally represent some new avenues for future research. Szwed, M., Qiao, E., Jobert, A., Dehaene, S., and Cohen, L. (2014). Effects of literacy in early visual and occipitotemporal areas of chinese and French readers. J. Cogn. Neurosci. 26, 459–475. doi: 10.1162/jocn_a_00499 Huang, H. S., and Hanley, J. R. (1995). Phonological awareness and visual skills in learning to read Chinese and English. ACKNOWLEDGMENT Luo, Y. C., Chen, X., Deacon, S. H., Zhang, J., and Yin, L. (2013). The role of visual processing in learn- ing to read chinese characters. Sci. Stud. Read. 17, 22–40. doi: 10.1080/10888438.2012.689790 Vellutino, F. R., Steger, J. 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Dyslexia 19, 191–213. doi: 10.1002/ dys.1463 ACKNOWLEDGMENT REFERENCES doi: 10.1016/j.jneuroling.2010.03.004 This article was submitted to Developmental Psychology, a section of the journal Frontiers in Psychology. Copyright © 2014 Zhou, McBride-Chang and Wong. 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 licen- sor are credited and that the original publication in this journal is cited, in accordance with accepted aca- demic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Facoetti, A., Lorusso, M. L., Paganoni, P., Umilt, À. C., and Mascetti, G. G. (2003). The role of visuospatial attention in developmental dyslexia: evidence from a rehabilitation study. Brain Res. Cogn. Brain Res. 15, 154–164. doi: 10.1016/S0926-6410(02)00148-9 Perfetti, C. 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W1587536155.txt	https://www.ccsenet.org/journal/index.php/jas/article/download/50254/28357	en		Understanding Small-Scale Farmers’ Perception and Adaption Strategies to Climate Change Impacts: Evidence from Two Agro-Ecological Zones Bordering National Parks of Uganda	Journal of agricultural science	2,015	cc-by	9,215	Journal of Agricultural Science; Vol. 7, No. 10; 2015 ISSN 1916-9752 E-ISSN 1916-9760 Published by Canadian Center of Science and Education Understanding Small-Scale Farmers’ Perception and Adaption Strategies to Climate Change Impacts: Evidence from Two Agro-Ecological Zones Bordering National Parks of Uganda Christopher Ewaechabo Tiyo2, F. L. Orach-Meza1 & Eric L. Edroma1 1 School of Sciences, Nkumba University, Uganda 2 African Wildlife Foundation (AWF), Kampala, Uganda Correspondence: Christopher Ewaechabo Tiyo, African Wildlife Foundation (AWF), Kampala, Uganda. E-mail: assemvi@gmail.com Received: June 21, 2015 doi:10.5539/jas.v7n10p253 Accepted: August 5, 2015 Online Published: September 15, 2015 URL: http://dx.doi.org/10.5539/jas.v7n10p253 Abstract Agricultural production by small-scale farmers in Uganda is vulnerable to climate change because the agricultural regime is rainfed and subject to climatic changes and variability with significant impact on agricultural productivity, livelihoods and food security. This study analysed small-scale farmers perceptions and impacts of climate change on agricultural production and livelihoods and adaptation options to tackle the adverse effects of climatic changes in Karenga (lowland agroecology) and Kapchesombe (highland agroecology). Both study areas are adjacent to Kidepo Valley and Mount Elgon National Parks respectively. Analysed by using data obtained from 607 households, (41.5 percent males and 58.5 percent females) and were multistaged and purposively sampled. The study found out that that the small-scale farmers were aware of climate change events. Meteorological data analysed confirmed the warming. The largest proportion of the respondents was affected by climate change effects with more impacts felt in Kapchesombe (highland agroecology). The major coping strategies employed include: planting different crops, different planting dates, different crop varieties, soil conservation and crop diversification. Coping strategies employed to contain extreme weather events included terracing, tree-planting, digging drainage channels, planting cover crops, and food storage and meals regulations. Other challenges associated with climate change included: food insecurity due to crop failure, soil erosion, shift in spread of diseases and land degradation. Government should provide effective and productive agronomic farm inputs and production assets and working farm-to-farm extension programme so as to build the adaptive capacity of the vulnerable and improve agricultural production. This should be intertwined with relevant traditional methods. Keywords: climatic variability, small-scale farmer, adaptation, national parks 1. Introduction Climate change and variability is an impediment to development and affects agricultural production, food security and livelihoods in sub-Saharan Africa due to rain-dependent agricultural production system (Deressa et al., 2010; Nhemachena et al., 2008). Agricultural production in Uganda is vulnerable to climate change because the agricultural regime is rainfed and subject to climatic changes and variability which are now frequent affecting agricultural productivity, leaving rural communities livelihoods and food insecure (Okonya et al., 2013). Agriculture has for long been the cornerstone of Uganda’s economy in terms of contribution to the country’s domestic product (GDP). For instance, it comprises of about 23.7 percent of the total GDP, employs about 73.0 percent of the labour force, and accounts for 47.0 percent of the country’s total export (NDP, 2010). According to Okonya et al. (2013) and Hepworth (2010), the agricultural sector is dominated by small-scale farmers of mixed crop and livestock production with low productivity undermined by traditional farming practices such as lack of soil and water conservation practices, poor complimentary services such as farm-to-farm extension services and occurrence of extreme weather events such as prolonged drought, flash-floods and soil erosion. The current study postulates that these factors could undermine the adaptive capacity of the small-scale farmers and hence increase their vulnerability to the adverse effects of climate change. Global changes inform of global warming and climatic variability has threatened the development trajectory in 253 www.ccsenet.org/jas Journal of Agricultural Science Vol. 7, No. 10; 2015 the least developed countries with greatest impacts in the sub Saharan Africa and Uganda is not exclusive (Asiimwe, 2007; GoU, 2007; IPCC, 2014). Arguably climate change is impacting on a broad facets of life: health, agriculture, environment, water resources and fisheries, because of overreliance on the rainfall variable for most of the agro-based livelihoods (Cooper et al., 2008). Climatic models for Uganda have shown that the country experiences high variability and high temperatures and reduced rainfall and increased rainfall variability reduces crop yield and threaten food security and livelihoods (NDP, 2010; Hepworth, 2010). The effects of climatic shocks and extreme weather events take toll on the small-scale farmers and the fact that climate has changed in the past and will continue to change in the future underlines the need to understand how farmers perceive and adapt to climatic change impacts to guide future coping strategies to minimize the negative impacts (Hepworth and Goulden (2008). In Uganda for instance, studies have shown that much of the country’s agricultural production is rain-fed, meaning that changes in weather conditions have important implications for households’ total agricultural production and well-being (Asiimwe & Mpuga, 2007; Republic of Uganda, 2006). In line with structural transformations in the economy, the agricultural output as a share of the total GDP has declined over the years due to poor traditional practices that are dependent on natural weather patterns, so that variations in precipitation amounts results in large variations in total output and farm incomes for the small-scale farmers who are not well-endowed with resources and with low adaptive capacities. This volatility output due to climatic variability and extreme weather events could mean a large burden for low-income small-scale farmers unable to acquire adaptation technology and many times lacking farm-level extension services support, credit and insurance services and critical agronomic inputs (Okonya et al., 2013; Asiimwe & Mpuga, 2007). Parry et al. (1999) also noted that climatic changes and variability directly affects agricultural production given that the sector is sensitive to climatic changes and precipitation variability and therefore making small-scale farmers vulnerable. As Orindi et al. (2005) observes, Uganda is vulnerable to climatic changes and variability and this situation could amplify and worsen food security, households’ poverty, and poor health given the projections of warming temperature for the county. Climate change is unquestionably a natural process, however, significantly affected by human induced activities (Deressa et al., 2010). This study postulates that climate is changing, and given that it has in the past, and will continue in the future, therefore underpins the urge to understand how the small-scale farmers’ perception is and adapt to adverse effects of climate change in areas near protected areas (national parks) in Eastern and North Eastern Uganda. Studies have shown that African farmers have perceived and responded differently to tackle the adverse effects of climatic changes and rainfall variability in Tanzania, South Africa, Ethiopia, Ghana, and Uganda (Komba et al., 2012; Maponya et al., 2012; Tafesse et al., 2013; Fosu-Mensah et al., 2010; Okonya et al., 2013). However, none of these illustrates responses from small-scale farmers in areas adjacent to national parks and the conservation implications. The small-scale farmers in Karenga and Kapchesombe put a lot of emphasis on agricultural production, but climate change could adversely impact on their agricultural production and understanding their perception of and adaptation strategies is critical to help farmers in overcoming adaptation challenges. Further, a variety of coping strategies have been put forward by various studies which include: planting different crop varieties, changing land size, irrigation, crop diversification and changing from farming to non-farming strategies (Okonya et al., 2013; Maponya et al., 2012; Deressa et al., 2010; Fosu-Mensah et al., 2010; Okoye, 1998). Though informative, except for Okonya et al. (2013), these cases are not in Uganda and are at national or regional level in scope yet effects of adverse climatic changes are at local level and require area-specific adaptations based on ground factors. Literature shows that most of the studies on climate change impacts have been at continental, regional and national levels (Okonya et al., 2013; Maponya et al., 2013; Nhemachena et al., 2008; Kurukulasuriya et al., 2000a; Kandlinkar et al., 2000). This study postulates that climate change effects differs from one country to another and from one area to another and therefore adaptation will not be uniform rather area-specific. This study is different from other studies in that we examined the actual adaptation strategies undertaken by the small-scale farmers in Karenga (lowland) and Kapchesombe (highland) both areas being adjacent to Kidepo Valley and Mount Elgon National parks respectively. We contend that this had conservation implications. Some attempt has been made to analyse how farmers adapt to climate change (Maponya & Mpandeli, 2012; Okonya et al., 2013; Asiimwe & Mpuga, 2007; Deressa et al., 2010). For instance, Maponya and Mpandeli (2012) attempted to understand how farmers adapt to climate change in Limpopo, South Africa but did so among commercial large scale farming community that has access to essential adaptive technology and agronomic inputs. Okonya et al. (2013) analysed farmers’ perception of and coping strategies to climate change at national 254 www.ccsenet.org/jas Journal of Agricultural Science Vol. 7, No. 10; 2015 level. Though informative, the study was restricted to potato farmers throughout Uganda and failed to address adaptation and coping strategies challenges by small-scale farmers in areas adjacent to national parks. Asiimwe and Mpuga (2007) looked at rainfall shocks for households’ income and consumption in Uganda in general and failed to analyse perception of farmers to climate change in rural areas. Hepworth (2010) reviewed climate change vulnerability and adaptation preparedness in Uganda, though informative, it failed to address the perception of and adaptation to climatic changes and variability for it was a content review of the national adaptation strategy. Despite being highly variable in climatic changes, with extreme climatic shocks like floods, drought and extreme rainfall (NDP, 2010; Hepworth, 2010; Okonya et al., 2013), farm-level studies in areas adjacent to national parks on how the small-scale farmers perceive climate change and how they are responding to climate change impacts are limited. There is also limited knowledge on whether small-scale farmers perceive climate change and how they are coping with it in areas bordering national parks in Uganda. Perceptions of poverty stricken, non-equipped with adaptive technology and resource-constrained small-scale farmers in Karenga, and Kapchesombe lowland and highland agro-ecological zones near Kidepo Valley and Mount Elgon National Parks respectively are not documented. The conservation and policy implications of all this is still grey. Fussel (2007) and Deressa et al. (2009) argued that adaptation should be emphasised because human activities already have affected climate and it will take a while for mitigation measures to yield results. This study argues that understanding farmers’ perception is critical in directing future adaptation efforts in addressing adverse climate change impacts in the studied communities because adaptation should be area specific. This study postulates that, understanding farmers’ perception and adaptation strategies gives insight in guiding and supporting them in adopting relevant coping strategies that are site-specific in areas adjacent to the national parks. The study investigated how the small-scale farmers in the two agroecologies in areas bordering Kidepo Valley and Mount Elgon National Parks in Uganda perceived effects of changes in climatic variables on the agricultural production and how they have adjusted coping strategies to effects of climatic changes. It investigated how changes in climatic variables affected crop production in the two agro-ecological zones and how farmers are addressing climate change challenges. We hypothesised that perception of and adaptation to climatic changes is highly influenced by the socioeconomic and demographic factors, and the study is based on plausible methodological approach to studying perceptions of small-scale farmers based on comparing farm survey with data records from the meteorological stations (Thomas et al., 2007), adopted for it being informative in understanding levels of awareness and perception of the small-scale farmers. It also examined factors that influence perception and adaptation strategies. It also offered opportunity for the validation of farmers’ claims on climate change and the meteorological data is used to confirm that. Combining perception, meteorological climatic trends, agricultural (crop) production regressed against rainfall data and farmers coping strategies to climate change effects demonstrates the cause-effect relationship and set this study apart. 2. Materials and Methods 2.1 Survey Design and Study Area Both qualitative and quantitative methods of data collection were employed in households’ survey questionnaire whereby 607 questionnaires were administered to respondents. The questionnaire was structured and and thematic with sections on socio-economic and demographic characteristics, perceptions of and adaptation to climatic change impacts. Impacts on agricultural production, income and relationship between climatic variables (rainfall) and crop production was assessed. In-depth interviews were administered for focuss group after household surveys interviews with the small-scale farmers. The standard structured questionnaires were pretested on ten households in Karenga, Kaabong district and adjusted to capture data during the study. Data (rainfall and temperature) from the Department of the Meteorology was obtained and analysed and formed the basis for confirming the basis of small-scale farmers perception and claims on changes in climatic variables. Data on agricultural production was obtained from Ministry of Agriculture, Animal Industry and Fisheries and regressed against rainfall data and this formed the basis for comparing the rainfall variability effects on crop production and food security and comparison with claims by the small-scale farmers. Clearance was sought from relevant authorities and interviews were conducted in Karenga and Kapchesombe subcounties between January to June 2014. Representative sample of 622 households were interviewed in age range of 15-60+ years and 15 questionnaires were nullified due to error in data capture and only 607 questionnaires were valid and formed the unit of analysis. By gender, males were 41.5 percent and female 58.5 percent. The research assistants were first trained in data capture techniques before commencement of interviews administration. In this study, multi-stage and purposive sampling techniques were employed to select the respondents given the scope of the study involved Eastern and Northeastern Uganda. In the first stage, eastern Uganda was stratified into two agro-ecological zones that are lowland and highland for Karenga and 255 www.ccsenet.org/jas Journal of Agricultural Science Vol. 7, No. 10; 2015 Kapchesombe respectively. In the second stage, Karenga and Kapchesombe were purposely selected for bordering the national parks. In the third stage, the study areas were stratified using administrative parishes and in the fourth stage, the households were purposively sampled for being small-scale farmers. Table 1. Agroecological zones (AEZ) and characteristics of Karenga and Kapchesombe study areas Sn. Agroecological zones Characteristics Agricultural practices 1 Eastern highlands (Kapchorwa district) Covering the ranges of mt. Elgon, rainfall Rainfed mixed farming involving mostly stall-fed over 1400 mm; 1300-3600m asl; with rich volcanic soil cattle, small ruminants, vegetable production, Northeastern; mostly bordering Acholi, Teso, Limited Kapchorwa and Lango sub regions; reliable predominant; rainfed consisting of sorghum, rainfall; average rainfall of 1100mm; altitude at 970-1420m asl; moderately good soil millet, early maturing maize, sweet potato, some beans, groundnut and pigeon pea production 2 Karamoja wet zone cereals such as barley and wheat in Kapchorwa, and Arabica coffee livestock raring; crop cultivation Source: http://www.fao.org/agriculture/seed/cropcalendar/aezone.do?iscode=UGA 2.1 Statistical Analysis Data was coded and entered using MS Excel and analysed by the statistical package for social sciences (SPSS) version (13.0) and later exported to STATA (version 10.0) for multinomial logit regression analysis together with Chi-squared and t-test as the major statistical tools employed to analyse small-scale farmers perception and adaptation strategies to tackle adverse effects of climate change. Linear regression was used to analyse trend in climatic variables (temperature and rainfall) to establish change in climatic variables. 2.2 Analytical Methods The common approach to studying perception of farmers to climate change in developing countries in Africa is based on comparing farm survey or farm group discussion results with data records from meteorological stations (Deressa et al., 2010; Thomas et al., 2007); and is informative approach in terms of understanding the level of awareness of the small-scale farmers and the validation of farmers claims. Previous studies (Deressa et al., 2010; Nhemachena & Hassan, 2007) found out that perception and coping strategies to climate change are influenced by socioeconomic and environmental factors. We therefore hypothesised that factors which affect perception and the development of adaptation strategies include gender of the respondent, the agroecological location, age, education, years of stay in the community, family size asset value and main source of income. Seasonal variations in weather (precipitation, humidity and temperature) by standardization of seasonal and annual weather data helps in the determination of the climate change variability and trend to give picture of the behaviour of rainfall and temperature in comparison with the 30 years climatological period under review and is determined by: Y = MX + C (1) Where, M is the gradient and takes the value of negative or positive; When M is negative this shows that the trend is declining and when M is positive, the trend is increasing; R2 indicates the statistical significance of the trend process. Significance is great from 50% or more (R2 ≥ 0.5) and significance is less from below 50% (R2 ≤ 0.5); Y = the y axis and X is the x axis of the trend graph. 2.3 Dependent and Independent Variables In this study, the independent variables were climatic attributes (rainfall and temperature) and factors that influence perception and adaptation (gender, agroecological area, age, education, year in community, family size, assets, and source of income). The dependent variables were effects of climate change (whether a small-scale farmer has or not perceived climate change, whether a farmer has or not developed coping strategies to adverse effects of climate change). 3. Results 3.1 Small-Scale Farmers Perception on Long Term Changes in Climatic Variables In this study, climatic variables examined are the long-term temperature, rainfall and the occurrence of weather shocks and extreme events in the past ten to twenty years as experienced by small-scale farmers. In Table 2, 256 www.ccsenet.org/jas Journal of Agricultural Science Vol. 7, No. 10; 2015 albeit the largest proportion of farmers in the two sub counties perceived seasonal changes in rainfall amounts and timings, significant changes in rainfall were more reported by farmers in Kapchesombe (97.7%) than Karenga where 66 percent of the respondents observed this change. In relation to temperatures, over 50 and 90 percent of farmers in Karenga and Kapchesombe respectively perceived seasonal changes in temperature. This means that there were changes in temperature and this could have implications for agricultural production. The observed differences were statistically significant at 0.05 level of significance (Table 2). The general perception however, was, that more rainfall was received in the past ten years (Table 3) and this is consistent with NDP (2010) and Hepworth (2010) that stated that eastern Africa including Uganda will experience extreme weather events including high amounts of rainfall, in spite of the precipitation variability in the past years. Table 2. Seasonal rainfall and climate change variability for Karenga and Kapchesombe subcounties Variable Level of seasonal change in rainfall over the past 10 years Level of seasonal change in temperature over the past 10 years Karenga (n=303) Kapchesombe (n=304) Seasonal change 66.12% 97.7% No seasonal change 15.79% 1.65% Altered seasonal change 16.45% 0.66% Do not know 1.64% 0% Seasonal change 50.99% 90.1% No seasonal change 14.8% 4.62% Altered seasonal change 32.24% 5.28% Do not know 1.97% 0% Chi-square p-value 102.35 .000 113.8 .000 Table 3. Perception on seasonal rainfall variability in the area over the past 10 years Variable Frequency Percentage Rainfall change 566 93.7% Longer rainy season 298 49.3% Shorter rainy season 280 46.4% More rains 346 57.3% Longer dry spells 244 40.4% Shorter dry spells 364 60.3% More dry spells 166 27.5% 3.2 Relationship between Socio-Economic and Demographic Factors and Adaptation to Climate Change The empirical adoption literature variously point out relationships between socio-economic and demographic factors and adaptation to climate change (Tafesse et al., 2013; Nhemachena et al., 2008). The influence some of the factors have mixed results in literature. Factors considered influential in perception and adaptation were gender, age, education, years of stay in community, family size, assets and main sources of income and were analysed at bivariate level to determine the level of adaptation to climate change. Looking at gender, although the highest proportion of male (79.2%) and female (70.9%) farmers have been able to address the challenges of climate change, the result of chi-square (X2 = 4.999) show significant differences (p < 0.05) in the level of adaptation between male and female which suggests that male farmers are more likely to adapt to climate change than their female counterparts. This could be attributed to male dominance of the society in Karenga and Kapchesombe whereby the male are favoured in terms of land ownership, access to loans, credit and agricultural inputs such as agro-chemicals, fertilizers and extension services. This undermines the central role of women as the sole providers of bread to feed the family. With more climate change impacts and in disadvantageous position, they will take longer time to look for food to feed the family since they do not control production assets. The influence of age on perception and adaptation to climate change can have mixed influence on perception and 257 www.ccsenet.org/jas Journal of Agricultural Science Vol. 7, No. 10; 2015 adoption of adaptation strategies (Maponya & Mpandeli, 2013; Bekele et al., 2003). In this study, the findings in Table 4 revealed that age is a highly significant factor (p < .05) in explaining farmers’ adaptation to climate change. There was more likelihood of adaptation by small-scale farmers in the age bracket of 15-44 years. This could be attributed to the fact that whereas the old have the experience, the young with education and better information have better planning ability and horizon to take agricultural production step further than their ‘parents’ did in the olden days to make living and ends meet and address climate change challenges. An adventurous age bracket, they could also venture in long-term initiatives and measures that previously were seldom used such as agricultural practices aimed at water and soil conservation, livestock farming and off-farm enterprises as best adaptation and/or coping strategies. This finding is consistent with Maponya and Mpandeli (2013). Studies (Tizale, 2007; Anley et al., 2006) show that improving education and dissemination of knowledge is an important policy measure for stimulating local participation in various development and natural resources management initiative. This study hypothesized that educational level would positively influence adaptation to climate change. Analysis of influence of education revealed that, higher level of adaptation was observed among the small-scale farmers with higher levels of education and the effect of this relationship was statistically significant at 1 percent level. Consistent with Tizale (2007) and Anley et al. (2007), this result indicates that better education improves awareness and information on conservation agriculture and better coping strategies to climate change challenges, and adoption of better techniques, technologies and willingness to do better and embrace new ideas and changes necessary for adaptation. Education also encourages willingness to learn and adopt new ideas and need to excel in whatever one does and in this case in climate-smart agricultural production. Farming experience is accumulated over years and experienced farmers are expected to have rich knowledge in adaptation and would have participated in modernization effort in tackling climate change. It was hypothesized that those years of stay in community affected and influenced perception and adaptation positively. The result of this study revealed that the duration of stay in years in community was significant (X2 = 26.070; p = .000) factor associated with adaptation to climate change, whereby, those with a farming experience of less than 40 years adapted more than those aged forty years and above. This is disturbing and not consistent with Nhemachena et al. (2009) because, year of stay in community should have enabled and equipped farmers with better experience and farming skills and therefore better adaptation. However, one could not discount the fact that at old age, the effect of ‘diminishing’ returns sets in as effectiveness and efficiency decrease with advanced age. But also, beyond 30 years in farming, possibly farming is not passion any more in ones’ life and the zeal for new ventures retardation sets in. Studies in support (Yirga et al., 1996) of large family size labour pool diverted to off-farm activities as adaptation strategy is discounted by a different school of thought that does not believe that large family size is no adaptation strategy (Tafesse et al., 2013). In this study, it was postulated that family size had great bearing on perception and adaptation. However, the results of the chi-square test were contrary to the apriori expectation. According to the results of the statistical test, family size was found to have no significant (.086 > .05) influence on adaptation to climate change. This revelation is disturbing but it could mean that, modern farming geared to tackling climate change effect is not associated with family size but access to adaptation technology. The finding is sharp contrast with with Yirga et al. (1996) and Legas et al. (2006) who argued that family size was a proxy to labour availability and could influence the adaptation of new technology positively. Given the education modernization policy, in Uganda universal primary, secondary education and quota system offers opportunity for all school going children to acquire education up to tertiary level as requirement and therefore family size proxy for labour may not necessarily count in the study areas. In similar vein, analysis for relationship between asset value and adaptation revealed that family asset had no significant (X2 = 4.385; p value 0.112 > .05) influence on adaptation to climate change. This may be so because; the study looked at household assets as opposed to farm assets. Farm assets are part of production assets with more relevance in tackling climate change effects. Regarding main sources of income and adaptation to climate change, this variable was found to be statistically significant (p = .000). In particular, the level of adaptation for small-scale farmers who were dependent on farming only was 72.3%; whereas homesteads with off-farm income had 80.0%. Those that practiced mixed crop and livestock farming had 80.2%. The result suggests that being dependent on farming only does not reduce adaptation to climate change. Broadening income base by taking on strategies that include farming and off-farming ventures are effective climate change coping strategies. 258 www.ccsenet.org/jas Journal of Agricultural Science Vol. 7, No. 10; 2015 Table 4. Relationship between socio economic and demographic factors and adaptation to climate change Whether the respondent has been able to address challenges of climate change Variable Gender of respondent Response Row % Frequency Row % Male 50 20.8% 190 79.2% Female 98 29.1% 239 70.9% 148 25.6% 429 74.4% Karenga 122 41.6% 171 58.4% Kapchesombe 26 9.2% 258 90.8% 148 25.6% 429 74.4% 15-24 14 20.3% 55 79.7% 25-34 30 19.2% 126 80.8% 35-44 33 24.3% 103 75.7% 45-54 41 36.3% 72 63.7% > 55 30 29.1% 73 70.9% 148 25.6% 429 74.4% No education 104 31.1% 230 68.9% Primary 29 22.1% 102 77.9% Secondary 11 12.2% 79 87.8% Tertiary 4 18.2% 18 81.8% 148 25.6% 429 74.4% < 10 years 13 11.9% 96 88.1% 10-19 years 25 25.3% 74 74.7% 20-29 years 28 21.1% 105 78.9% 30-39 years 30 27.3% 80 72.7% 40 years 49 40.2% 73 59.8% 145 25.3% 428 74.7% 1-4 21 19.3% 88 80.7% 5-9 101 29.7% 239 70.3% 10-14 25 21.7% 90 78.3% >14 1 14.3% 6 85.7% 148 25.9% 423 74.1% < 50000/= 26 16.3% 134 83.8% 50000-100,000/= 21 24.4% 65 75.6% 100,001-150,000/= 10 34.5% 19 65.5% >150,000/= 22 25.0% 66 75.0% 79 21.8% 284 78.2% Total Age of small scale farmers Total Formal education level Total Years stayed in the community Total Family size Total Asset value (UGX shillings) Total Main source of income Total Yes Frequency Total Sub county No Farming 64 27.7% 167 72.3% Off-farm income 1 20.0% 4 80.0% Mixed crop & livestock 53 19.8% 215 80.2% 118 23.4% 386 76.6% 259 Chi-square p-value 4.999 .025 79.793 .000 11.899 .018 15.274 .002 26.070 .000 6.592 .086 4.383 .112 79.793 .000 www.ccsenet.org/jas Journal of Agricultural Science Vol. 7, No. 10; 2015 3.3 Analysis of Climatic Variables: Data from Department of Meteorology 3.3.1 Seasonal Precipitation Trend Analysis of data obtained from the department of meteorology aimed at understanding the evolution and changes in rainfall and temperature patterns and seasonal changes both within and between seasons across years, decades and the two agroecological zones of Karenga and Kapchesombe. Results indicate that mean rainfall for Kotido (Karenga) is 276.5 mm per annum mostly received between July to October and is unimodal and therefore not well balanced distribution and this has agricultural implications in seasons’ calendar for crop cultivation. Rainfall received for Kapchorwa (Kapchesombe) is 1052.9 mm per annum and well distributed throughout the year, with two seasons and favourable for crop production. The rainfall trend standardisation for Kotido shows very slight increase coming in August to October giving a limited period for crop production as the seasonal calendar is short. Figure 1. Kotido seasonal rainfall trend (1960-2011) Kotido variations in precipitation are due to low rainfall amounts received and high seasonal variations. In the period 1987-1990, rainfall decreased by 222.9 mm (30%); the years 1994-1998 saw a drop by 186.2 mm as 16.8% decline; in 2000-2003 precipitation decreased by 34.6 mm (3.9%) and in 2004-2009 rainfall decreased amount fell by 141 mm (9.8%). The trend in Figure 1 appreciates towards the last season of the year which has been found to be critical for agriculture whereby the seasonal trend witnesses slight increase in rainfall but this increase was (R2 < 0.5) found not to be significant. Generally, this unreliable rainfall could hurt agricultural production as less rain days become many with high temperatures and seasons characterized by late onset and early cessation of rainfall with high unreliability impacting negatively on crop production. Seasonal precipitation trend for Kapchorwa for the past thirty years showed slight increase. For instance 1966-1967 precipitation amounts increased by 74.1 mm (13.4%); 1968-1973 saw increase by 58.6 mm (3.5%). The period 1974-1977 had increase in precipitation by 47.7mm (4.7%). From 1979-1983 rainfall amount received increased by 37.5 mm (4.5%), and in the period 1989-1995, the area experienced increase in rainfall by 67.6 mm (4.8%). Although, the analysis indicates increase, the computed trend for precipitation (R2=0.0818) was not significant as R2 value was less than 0.5 (Figure 2). In-spite of this, rainfall amount in Kapchorwa has been increasing though slightly; but still better than for Kotido and could reliably support crop production. 260 www.ccsenet.org/jas Journal of Agricultural Science Vol. 7, No. 10; 2015 Figure 2. Kapchorwa seasonal rainfall trend 1960-1992 3.3.2 Seasonal Trend for Temperature Weather behaviour within the seasons in the past 30 years in seasonal changes in temperature indicates considerable changes. Results show (Figure 3) that temperatures in Kotido (Karenga) is increasing at significant rate (y = 0.1716 positive trend; R2 = 0.7021 high significance) (Figure 3). In other words, the local atmosphere in Kotido is warming up. With low amounts of rainfall, and unpredictable onset and cessation of rainfall, this causes shortage of water and with limited ground water to support irrigation; this could harm crop production significantly Figure 3. Kotido mean annual maximum temperature (oC) trend Figure 4 presents the seasonal temperature trend for Kapchorwa and it show that Kapchorwa is also warming up though not at the rate of Kotido (y = 0.0437; R2 = 0.1629) (Figure 4). This finding conflicts with small-scale farmers’ perception and view that temperatures were generally low over the past 10 years. What is clear though is that, being a highland agroecological area, altitude influences temperatures and amounts of precipitation modifying weather and eventually the overall climate, an advantage Kotido (Karenga) misses. 261 www.ccsenet.org/jas Journal of Agricultural Science Vol. 7, No. 10; 2015 Figure 4. Kapchorwa mean annual maximum temperature (oC) trend 3.4 Effects of Climate Change and Variability on Agricultural Production 3.4.1 Effects of Climate Change on Agricultural Production Results indicate that climate change and variability impact on small-scale farmers agricultural production is greatly felt already with 61.4% stated had bad effects; 19.1% very bad; 18.9% were slightly affected and only 0.5% not affected (Table 5). Those not affected took up broad off-farm adaptation strategies such as income generating enterprises, adoption of adaptation technologies such as water and soil conservation strategies necessary to cushion adverse climate change impacts. Table 5. Rainfall variability effects on agricultural production Variable Rating of rainfall variability and climate change impact on agricultural production in households Total Frequency Percentage Very bad 116 19.1% Bad 373 61.4% Slightly affected 115 18.9% Not affected 3 .5% 607 100.0% By cross tabulation, the results indicate that climate change and rainfall variability affected agricultural production and the effect was felt more in Kapchesombe (highland agroecology) (Figure 5). This could be attributed to the sensitivity of mountain ecosystems which is sensitive to any slight change in climatic variables. It could also be due to the recent drought in (year 2013) and still linked to various environmental and edaphic changes that cause reduction in water availability (Deressa et al., 2010). Challenges such as soil degradation, erosion and human population pressure affect and stress up environmental factors and exacerbate effects of climate change including on agricultural production. 262 www.ccsenet.org/jas Journal of Agricultural Science Vol. 7, No. 10; 2015 90.0% 80.9% 80.0% 70.0% 60.0% 50.0% 42.1% Percent 40.0% 30.0% 29.6% 27.6% 20.0% 10.6% 8.3% 10.0% 0.7% 0.3% 0.0% Very bad Bad Slightly affected Rainfall variability effects on households Karenga Not affected Kapchesombe Figure 5. Climate change and rainfall variability effects on agricultural production by agroecology 3.4.2 Effects of Climate Change on Crop Production Analysis of rainfall and crop production was done to show the relationship between rainfall influence on agricultural production in Kotido (Karenga) and Kapchorwa (Kapchesombe) (Figure 6). The results show crop production trends for Kotido and Kapchorwa from 1981 to 1995 and crops under consideration were cereals (maize) and legumes (beans). Trend for crop production for Kapchorwa appreciated higher than for Kotido (Figure 6). On average, Kotido recorded the highest precipitation than Kapchorwa with 222.86 and 207.95 mm respectively but these differences were not statistically significant as shown by the p-value of the t-test equal to 0.609 (Table 6). For crop production however, Kapchorwa had an average of 28,111.47 metric tonnes while the average for Kotido was 6,981 and the observed differences in the two means were statistically significant (p < .01). 50000 40000 30000 20000 10000 0 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 crop production (metric tonnes) 60000 kotido kapchorwa Figure 6. Crop production trend for Kotido and Kapchorwa 263 www.ccsenet.org/jas Journal of Agricultural Science Vol. 7, No. 10; 2015 Table 6. Results of the t-test on the average precipitation and crop production in the two districts Variable District Obs Mean Std. Dev. t p Precipitation Kotido 27 222.8593 84.50314 0.5156 0.609 Kapchorwa 15 207.9533 98.81145 Kotido 30 6981 4355.262 103505 0.000 Kapchorwa 15 28111.47 9551.284 Crop production (tons) Effects of rainfall changes and variability on agricultural production result of the regression analysis (Table 7), the coefficient for precipitation is negative which suggests that an increase in the rainfall by one mm is associated with a reduction in crop production by 8.93 metric tonnes. Nevertheless, it appears that the amount of rainfall received is not a significant factor associated with agricultural production as shown by the p-value of 0.458 > .05. In other words, other factors other than rainfall need to be addressed to improve crop production in the study area. This could include provision of water for agricultural irrigation, undertaking soil and water conservation measures, improving and providing complementary measures such as extension services, awareness and education and demonstrations and agronomic inputs such as access to credit, loans, marketing and production assets and infrastructure required to stimulate agricultural production. The results however show statistically significant mean differences in crop production in the two districts. Looking at the dummy coefficient for the period 1981-1995, Kapchorwa was able to produce 20,826 more metric tonnes of agricultural output than Kotido and this difference was statistically significant (p = .000) (Tables 7 and 8). Table 7. Effect of rainfall changes and variability on agricultural production-Kapchorwa Bean_maize Coef. Std. Err. T P>t Mm -8.93 11.93202 -0.75 0.458 Kapchorwa 20826.06 2188.845 9.51 0.000 _cons 9143.43 2961.575 3.09 0.004 Prob > F 0.0000 R-squared 0.7042 Table 8. Effect of rainfall changes and variability on agricultural production in the two study sites (by district) Bean_maize Kotido Kapchorwa Coef. Std. Err. t P>t Coef. Std. Err. t P>t Mm 6.747279 10.68563 0.63 0.533 -30.235 25.464 -1.19 0.256 _cons 5648.529 2540.902 2.22 0.035 34398.910 5826.585 5.9 .000 F 0.40 1.41 Prob > F 0.5335 0.2563 R-squared 0.0157 0.0978 3.4.3 How Changes in Climatic Variables and Extreme Weather Events Affected Crop Production Extreme weather events are already impacting on the small-scale farmers’ agricultural production, and 95.1% reported being affected and only 4.9% were not affected. By cross tabulation, 81.3% and 93.6% for Karenga and Kapchesombe reported experience of excessive and stormy rainfall respectively. Impact of such hazards were more on Karenga for instance, crop diseases 94.8%; pests 83.0%; drought 67.0%; floods 63.2% and soil erosion 55.6%. Except for incidences of excessive rainfall, small-scale farmers in Karenga take the brunt of the extreme weather events than their counterparts in Kapchesombe (Table 9). 264 www.ccsenet.org/jas Journal of Agricultural Science Vol. 7, No. 10; 2015 Table 9. Natural hazards experienced by small-scale farmers Agroecologies Hazards experienced Flood Karenga (lowland) Kapchesombe (highland) Frequency Percentage Frequency Percentage 182 63.2% 22 7.8% Storm/wind/excessive rainfall 234 81.3% 263 93.6% Drought 193 67.0% 65 23.1% Landslide 25 8.7% 24 8.5% Soil erosion 160 55.6% 104 37.0% Diseases 273 94.8% 70 24.9% Mudflow 67 23.3% 90 32.0% Lightening 80 27.8% 24 8.5% Pests 239 83.0% 83 29.5% Earthquake 107 37.2% 3 1.1% Climate change already is impacting negatively on the small-scale farmers and excessive rainfall was reportedly the highest. Excessive and stormy rainfall caused shredding of crop leaves, broke shoots and flowers; reduced leaf quality, soil erosion, cut off roads, caused root and stem tubers to rot thus causing food insecurity. Reportedly, there is high increase in fungal diseases, soil erosion and hence reduction in the crop yields, ultimately affecting income and livelihoods and quality of life. Affected crops were cassava, potato, tomato, and coffee. In some instances, it has caused premature harvests thus compromising the crop quality. Excessive rainfall caused floods, soil erosion thus leading to reduction of farmlands and declining yields. 3.5 Coping Strategies Undertaken to Adapt to the Effects of Climatic Change and Variability The small-scale farmers were asked to mention adaptation strategies and necessary changes they made to nutrilise the adverse effects of climatic change and variability in the past ten years and more. The study revealed that coping changes were made in crop agronomy, water and soil conservation. For instance, 84.1% households planted different crops, 71.3% used different planting dates, 64.3% planted different crop varieties, 61.5% made changes in soil and water conservation, 52.1% diversified crops and 45.4% used crops with shortened growing period (Table 10). The changes made were climate-smart strategies adopted to off-set adverse effects of climate change and variability. 265 www.ccsenet.org/jas Journal of Agricultural Science Vol. 7, No. 10; 2015 Table 10. Adaptation strategies used by farmers (n = 607) Variable Codes Frequency Percentage Plant different crops A 508 84.1% Use different planting dates B 428 71.3% Plant different crop varieties C 387 64.3% Soil conservation D 369 61.5% Crop diversification E 310 52.1% Shorten growing period F 272 45.4% Change use of fertilizers G 233 39.0% Better crop husbandry H 202 33.7% Move to different site I 161 26.9% Change landsite J 155 25.8% Irrigate the farms K 83 13.8% Improve storage & post-harvest storage/security L 64 10.8% Do water conservation M 36 6.0% Change crops to livestock N 25 4.2% Use subsidies O 12 2.0% Change from farming to non P 11 1.8% Use insurance Q 7 1.2% 4. Discussion In this study, the analysis of climatic data on rainfall and temperature revealed changes in precipitation where by very slight increase in precipitation has been noticed in the past ten years confirming that there is climate change. Most of the small-scale farmer admitted having observed seasonal changes and variations in rainfall and temperature. This is consistent with Christensen et al. (2007) and Hepworth et al. (2010) where IPCC also presumed increase in rainfall in the horn of and the eastern Africa. The increasing trend in temperature computed through this study confirms what government of Uganda indicated, that there are country-wide changes in climatic variables (Nsubuga et al., 2011) whereby temperatures are increasing and rainfall being erratic. Changes in temperature cause drought and affect crop physiology leading to poor yields. Drought further affects soil water and moisture, causing crop wilting and failure. The poor crop yields and harvests in Karamoja sub-region is largely related to erratic rainfall, unpredictable on set and cessation of rainfall. Strategies that aim at conservation agriculture should address water and soil conservation matters. Extension services to guide farmers, and provision of agronomic in puts is essential. This should be with dissemination of adaptation information using media outlet with widest possible coverage. This study found that rain days were few and there was increase in non-rain days. There were more warm days than cool days. This means that rainfall amounts to grow crops is generally lacking, a situation that can be further complicated by unpredictable onset and cessation. With poor ground water quantity and sources that are not favourable for irrigation, the future of agricultural production via irrigation in Karamoja is set to be in jeopardy. Unpredictable and increases in rainfall is said to be responsible for landslides on slopes of Mount Elgon in Bududa that destroyed homesteads, agricultural land and fields of crops and human lives (GOU, 2007). In Karenga, floods have destroyed farms reducing sizes of fields of crops, causing roots and tuber crops to rot hence worsening households’ livelihoods and food security. Increased temperatures are responsible for proliferation and extending the geographic range of insect vectors such as mosquitoes to higher fronts in Kapchorwa in Mount Elgon, where they were nonexistent in highland areas of Uganda. This affects the residents through malaria outbreaks once there is an encounter with the anopheles species, which is responsible for malaria parasites carrier (MoFPED, 2009). These findings are consistent with World Bank (2010) and Nhemachena et al. (2008) and Okonya et al. (2013) that stated that generally Africa is getting warmer but eastern Africa climate is changing and this will come along with many associated effects causing agricultural and households’ decline and food insecurity. Weather patterns in the country have been very unpredictable in terms of onset and cessation of seasons rainfall leading to low crop yields and instances of crop failure being high (Hepworth, 2010). Great variations in rainfall 266 www.ccsenet.org/jas Journal of Agricultural Science Vol. 7, No. 10; 2015 seasonality in terms of amount, onset and cessation are consistent with IPCC report of likely increase in climatic variables, especially increase in precipitation in eastern Africa (IPCC, 2014; Christensen et al., 2007). In the absence of irrigation technology, if such variations in climatic attributes persist, this could cause crop failure thus affecting agricultural production and livelihoods. In Karamoja sub-region, persistent drought has caused pests and vectors which caused crop failure and affected yields and household livelihoods, leaving the communities impoverished. Leading extreme weather events experienced were intensive rainfall, drought and floods affecting agriculture variously causing the rotting of pods, roots and tubers and poor seeds and crop decline presumably affecting yields and income as well as food security. On positive note, however, more rainfall enabled farmers to produce more crops with longer seasons’ rainfall to enhance household livelihoods especially in Kapchesombe. However, this study revealed that increase in rainfall caused drop in crop yield meaning that rainfall was not the only significant factor in agricultural production but others too; such as other production assets (agronomic input, information, demonstrations, and extension services) required should be provided so as to promote agricultural production and minimize vulnerability. In other words, small-scale farmers’ adaptive capacity needs to be enhanced to tackle adverse effects of climate change. The role of central government with development partners is central in this noble responsibility. Positive attributes and factors that promote agriculture need to be gender balanced, and deliberate effort need to be done to emancipate women with full empowerment for them to have full realisation of their potential. Given that they are basic producers, with knowledge and understanding of natural and environmental resources as they live closer to them, their role in climate change adaptation therefore would be positive once empowered with resources. 5. Conclusions, Recommendations and Policy Implications The small-scale farmers in Karenga and Kapchesombe are vulnerable communities to changes in changes in climatic variables and the high frequency of extreme weather events (drought, floods and destructive winds) coupled with stressors factors such as diseases, pests, floods and bushfires have resulted into the loss of gardens and low yields. Vulnerability has undermined small-scale farmers’ adaptive capacity and is almost in no position to acquire essential adaptation technology due to high household poverty levels because their agriculture is dependent on natural rainfall that is unpredictable. This has translated into poor income and livelihoods, food insecurity and hence low adaptive capacity. Government need a multi-approach to address the challenge of adverse climate change impacts that involves improvement of agricultural production assets, such as development of human and social capital through farmers capacity development to adopt climate-smart agricultural techniques to address climate changes through the acquisition of knowledge, agronomic inputs such as agrochemicals, fertilizers, tools and use of improved crop varieties that can cope up with high erratic seasonal changes. Agricultural modernization needs a holistic thinking and planning and working closely with small-scale farmers with climate change at the centre of agricultural modernization planning and implementation. The small-scale farmers in Karenga and Kapchesombe have limited capacity, resources, funds, extension service support and the essential adaptive technology to effectively respond to adverse effects of climatic variability and change. The government should expand farm-to-farm extension service to the farmers, who must be explained and should recognize the role of extension services and be able to access deployed resources and information at their disposal to tackle adverse effects of climatic variability and change. Accordingly, through extension services, farmers could then be provided to receive climate–smart agricultural skills and knowledge for crop production to engage in better profitable agriculture. Government should train and provide refresher training to extension workers in delivery of extension services that should be holistic including techniques of transferable skills in crop agronomy, resources delivery and use for effectiveness and designing demonstrational learning programmes to improve adaptive skills in climate-smart agricultural production and improved household livelihoods including off-farm adaptation enterprises establishments. The role of extension staff should be mainstreamed into the centre of development planning both at central and lower local government in the districts. Government needs to develop districts capacity to handle climate change effectively at lower grass-root levels by equipping production officers with knowledge and skills in explaining the roles of extension officers to small-scale farmers in Kaabong and Kapchorwa districts which must include use and adoption of appropriate adaptation technology, use of site-relevant adaptive technologies and crop husbandry. Delivery of information through demonstration plots, organized study tours and through media have great potential in inculcating and improving small-scale farmers adaptation capacities to climate change. Effort 267 www.ccsenet.org/jas Journal of Agricultural Science Vol. 7, No. 10; 2015 towards addressing cultural rigidity to adaptation can not be over-emphasised and deliberate awareness to address it be emphasized. The central government early warning systems and impending eco-disasters based on climatic forecasts should be relayed to the farmers through appropriate media channels with widest coverage at grass roots, including the local frequency Modulation (FM) radio stations and also print media. Disaster preparedness in prevention and saving of lives be improved. Farmers in Mount Elgon need to be given techniques in landslides prevention as well as shown areas prone to landslides be zoned and made known to the people. Lastly, climate change is there and to effectively adapt, farmers need to use available adaptation information proposed through this study. With reformed extension services, well packaged information and farmers demonstrations, tours, public talks and lectures, learnt techniques in agronomy integrated with indigenous coping strategies can help small-scale farmers to go a long way in adaptation to adverse climate change effects. 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This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). 270
https://openalex.org/W2943113867	https://discovery.ucl.ac.uk/id/eprint/10072659/1/s12014-019-9235-3.pdf	English	null	Discovery of non-invasive biomarkers for the diagnosis of endometriosis	Clinical proteomics	2,019	cc-by	12,778	Abstract Background: Endometriosis is a common gynaecological disorder affecting 5–10% of women of reproductive age who often experience chronic pelvic pain and infertility. Definitive diagnosis is through laparoscopy, exposing patients to potentially serious complications, and is often delayed. Non-invasive biomarkers are urgently required to accelerate diagnosis and for triaging potential patients for surgery. Methods: This retrospective case control biomarker discovery and validation study used quantitative 2D-difference gel electrophoresis and tandem mass tagging–liquid chromatography–tandem mass spectrometry for protein expression profiling of eutopic and ectopic endometrial tissue samples collected from 28 cases of endometriosis and 18 control patients undergoing surgery for investigation of chronic pelvic pain without endometriosis or prophylactic surgery. Samples were further sub-grouped by menstrual cycle phase. Selected differentially expressed candidate markers (LUM, CPM, TNC, TPM2 and PAEP) were verified by ELISA in a set of 87 serum samples collected from the same and additional women. Previously reported biomarkers (CA125, sICAM1, FST, VEGF, MCP1, MIF and IL1R2) were also validated and diagnostic performance of markers and combinations established. Methods: This retrospective case control biomarker discovery and validation study used quantitative 2D-difference gel electrophoresis and tandem mass tagging–liquid chromatography–tandem mass spectrometry for protein expression profiling of eutopic and ectopic endometrial tissue samples collected from 28 cases of endometriosis and 18 control patients undergoing surgery for investigation of chronic pelvic pain without endometriosis or prophylactic surgery. Samples were further sub-grouped by menstrual cycle phase. Selected differentially expressed candidate markers (LUM, CPM, TNC, TPM2 and PAEP) were verified by ELISA in a set of 87 serum samples collected from the same and additional women. Previously reported biomarkers (CA125, sICAM1, FST, VEGF, MCP1, MIF and IL1R2) were also validated and diagnostic performance of markers and combinations established. Results: Cycle phase and endometriosis-associated proteomic changes were identified in eutopic tissue from over 1400 identified gene products, yielding potential biomarker candidates. Bioinformatics analysis revealed enrichment of adhesion/extracellular matrix proteins and progesterone signalling. The best single marker for discriminating endo- metriosis from controls remained CA125 (AUC = 0.63), with the best cross-validated multimarker models improving the AUC to 0.71–0.81, depending upon menstrual cycle phase and control group. Results: Cycle phase and endometriosis-associated proteomic changes were identified in eutopic tissue from over 1400 identified gene products, yielding potential biomarker candidates. Bioinformatics analysis revealed enrichment of adhesion/extracellular matrix proteins and progesterone signalling. Irungu et al. Clin Proteom (2019) 16:14 https://doi.org/10.1186/s12014-019-9235-3 Irungu et al. Clin Proteom (2019) 16:14 https://doi.org/10.1186/s12014-019-9235-3 Clinical Proteomics Clinical Proteomics Abstract The best single marker for discriminating endo- metriosis from controls remained CA125 (AUC = 0.63), with the best cross-validated multimarker models improving the AUC to 0.71–0.81, depending upon menstrual cycle phase and control group. Conclusions: We have identified menstrual cycle- and endometriosis-associated protein changes linked to various cellular processes that are potential biomarkers and that provide insight into the biology of endometriosis. Our data indicate that the markers tested, whilst not useful alone, have improved diagnostic accuracy when used in combina- tion and demonstrate menstrual cycle specificity. Tissue heterogeneity and blood contamination is likely to have hindered biomarker discovery, whilst a small sample size precludes accurate determination of performance by cycle phase. Independent validation of these biomarker panels in a larger cohort is however warranted, and if successful, they may have clinical utility in triaging patients for surgery. Conclusions: We have identified menstrual cycle- and endometriosis-associated protein changes linked to various cellular processes that are potential biomarkers and that provide insight into the biology of endometriosis. Our data indicate that the markers tested, whilst not useful alone, have improved diagnostic accuracy when used in combina- tion and demonstrate menstrual cycle specificity. Tissue heterogeneity and blood contamination is likely to have hindered biomarker discovery, whilst a small sample size precludes accurate determination of performance by cycle phase. Independent validation of these biomarker panels in a larger cohort is however warranted, and if successful, they may have clinical utility in triaging patients for surgery. Keywords: Endometriosis, Biomarkers, Eutopic tissue, Ectopic tissue, Serum *Correspondence: john.timms@ucl.ac.uk 1 Department of Women’s Cancer, Institute for Women’s Health, University College London, Cruciform Building 1.1, Gower Street, London WC1E 6BT, UK Full list of author information is available at the end of the article © The Author(s) 2019. 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. Materials and methods Patient recruitment Patients and samples were sourced from the Univer- sity College London Hospital Gynaecology Depart- ment following ethical approval and informed consent. Patients were those referred by their general practition- ers or other clinicians for investigation of pelvic pain or for diagnosis and/or treatment of endometriosis. Those who agreed to undergo laparoscopic surgery for inves- tigation and treatment of endometriosis, pelvic pain or bilateral salpingoophorectomy for strong family his- tory of breast and ovarian cancer were approached and recruited. Inclusion criteria were as follows: ‘endome- triosis cases’ were defined as women diagnosed with endometriosis at laparoscopy and confirmed histologi- cally; ‘controls with pain’ were defined as symptomatic women with pelvic pain of unknown cause or chronic pelvic inflammatory disease without surgical evidence of endometriosis; ‘controls without pain’ were regularly cycling women with no known disease undergoing bilat- eral tubal ligation and/or prophylactic bilateral salpin- goophorectomy due to familial risk of breast and ovarian cancer and with no visual evidence of endometriosis at laparoscopy. The following women were excluded from the study; post-menopausal women, women with a posi- tive pregnancy test or unknown pregnancy status on day of surgery, those with other benign conditions or malig- nancies (particularly patients with fibroids and/or cancer were excluded as these conditions may compromise the integrity of the endometrium), women on any hormonal medication < 3 months prior to surgery and those whose surgical findings and pathological reports were inconsist- ent. Cycle phase was determined by a triple approach to ensure accuracy; chronologically, by histological dating and by sex steroid hormone determination. Women with unconfirmed menstrual cycle stage were excluded. Addi- tional patient data was collected including age, fertility history, treatment history (oral contraceptive and GnRH analogue use), menstrual cycle phase, pain history, histo- pathology findings and anatomic characteristics of dis- ease lesions. All patient records were handled per NHS confidentiality practices. Samples were anonymised and sequentially numbered. A lab coding system and data- base were developed for recording anonymised patient and sample information. While efforts have been made to develop blood- based diagnostic tests for endometriosis, few reported markers have been independently validated and none are in clinical use. As a result, definitive diagnosis of endometriosis remains surgical, requiring laparoscopy under general anaesthetic, exposing patients to poten- tially serious complications. Introduction additionally validate biomarkers reported in the litera- ture [4]. Endometriosis is estimated to affect 5–10% of women of reproductive age. Although some women with endo- metriosis may be asymptomatic, it can cause signifi- cant pain symptoms and infertility. The diagnosis of endometriosis can be difficult to achieve. Various stud- ies worldwide have documented a delay of 7–11 years from appearance of symptoms until diagnosis [1–3]. This delay is partly due to the fact that an invasive lap- aroscopy is needed to establish the diagnosis in some women. Imaging techniques are sensitive for diagnosis of ovarian endometriomas and deep endometriosis in experienced hands, but are less accurate for diagnosing other forms of endometriosis [4]. Materials and methods Patient recruitment Difficulty in establishing a diagnosis leads to patients spending years on visits to their doctors and unnecessary investigations, adding to the significant economic burden of the disease [5]. While early stage endometriosis is not without conse- quence, delay in diagnosis may allow disease progres- sion with reduced treatment efficiency and poorer outcomes. There is clearly an urgent and unmet need for a minimally invasive diagnostic test for endometrio- sis, though clinically useful blood-borne markers have yet to be found [6, 7]. The aim of a non-invasive test of endometriosis should be to expedite clinical diagno- sis by achieving a high positive predictive value (> 90%) or to triage patients for laparoscopic investigation for conclusive pathological diagnosis. A lower sensitiv- ity may be tolerated for the latter if specificity is high (> 90%). Ideally, a test would also need to function independently of menstrual cycle phase. Early diagno- sis of endometriosis may allow preventative measures that delay progression of the disease such as use of the progesterone only pill or Mirena coil. Women hop- ing to conceive or those who are refractory to medical management can be put forward for surgical treatment earlier. The first aim of our study was to identify potential biomarkers through proteomic profiling of eutopic and ectopic endometrial tissue specimens taken from women with a confirmed diagnosis of endometriosis and relevant controls undergoing exploratory surgery for chronic pelvic pain without endometriosis or pro- phylactic surgery due to strong familial risk of cancer. The second aim was to test these potential biomarkers in sera from a larger case control set of women, and Irungu et al. Clin Proteom (2019) 16:14 Page 2 of 16 Irungu et al. Clin Proteom (2019) 16:14 Irungu et al. Clin Proteom Sample collection There were no eutopic samples available for the pain group in proliferative phase or eligible ectopic sam- ples from endometriosis patients in proliferative phase. To improve proteomic coverage, immunodepletion of the 12 most abundant serum proteins was carried out on the pooled samples using Protein Purify 12 (PP12) Human Serum Protein Immunodepletion resin (R&D). Briefly, 500 μL of PP12 immunodepletion slurry was incubated with diluted endometrial tissue lysate (500 μg protein) on a rotary shaker for 30 min. Unbound material was recov- ered using Spin-X Filter units by centrifugation, the fil- trates were concentrated to 25 μL using 5 kDa molecular weight cut-off Vivaspin columns and protein concentra- tion determined using the Bradford method. A portion of each tissue specimen was fixed in 10% buffered formalin for histological examination, whilst remaining tissue was washed in sterile phosphate buff- ered saline to remove excess blood. Tissues were dried using lint-free paper, transferred into weighed Eppen- dorf tubes and snap-frozen in liquid nitrogen. Samples were transported to the lab and stored at − 80 °C. Cycle stage for each patient was determined by endometrial dating by an experienced histopathologist without prior knowledge of sample group according to [8]. Endometri- otic lesions were confirmed histologically. Samples were excluded where there was insufficient tissue (< 20 mg wet weight). 10 mL of blood was also collected from each patient before surgery by venepuncture into two vacu- tainer gel tubes. 5 mL of blood was used for determina- tion of oestradiol, progesterone, CA125 and CRP levels using standard assays. The remaining sample was allowed to clot at room temperature for 1 h, centrifuged at 3000g for 10 min at 4 °C, the serum collected, aliquoted and stored at − 80 °C. In total, 87 women met the inclusion criteria who had donated a serum sample; 45 with endo- metriosis, 21 controls with pain and no evidence of endo- metriosis and 21 controls with no pain and no disease (Table 1). Protein extraction, quality assessment, immunodepletion and pooling Each snap-frozen tissue sample was weighed and homog- enised by grinding in liquid nitrogen into a fine powder. Table 1 Characteristics of subjects included in the study and clinical grouping Table 1 Characteristics of subjects included in the study and clinical grouping Control Pain Endometriosis Number 21 21 45 Age (years) Mean (SD) 37.5 (8.5) 32.2 (6.5) 35.9 (5.8) Median (range) 36 (20–51) 33 (19–48) 36.0 (20–52) Symptoms Subfertility 4 8 16 Pain 0 22 38 Cycle phase Proliferative (1) 10 9 13 Secretory (2) 9 12 26 Inactive (0) 2 0 6 Sample collection Endometrial tissue biopsies (eutopic endometrium) were obtained by Pipelle cannula or curettage from women undergoing laparoscopy. Ectopic endometrial tissue Irungu et al. Clin Proteom (2019) 16:14 Page 3 of 16 Irungu et al. Clin Proteom (2019) 16:14 (endometriosis) samples were obtained from the same women at laparoscopy. Superficial lesions and deep infil- trating endometriosis samples only were used in this study, the rationale being that imaging techniques are sensitive enough for diagnosis of ovarian endometrio- mas, but are less accurate for diagnosing endometriosis elsewhere in the pelvis. Ground tissue was lysed in 2D lysis buffer (8 M urea, 4% w/v CHAPS and 10 mM Tris–HCl pH 8.3) using a ratio of 5 μL of lysis buffer per 1 mg of tissue at room tem- perature to extract protein. Samples were sonicated and centrifuged at 3000g for 10 min at 4 °C to pellet insolu- ble material. The supernatant was collected and protein concentration determined using a Bradford microtitre plate assay (Pierce) using a bovine serum albumin stand- ard curve. All samples were normalised to the same (lowest) protein concentration using 2D lysis buffer. To assess quality of the samples, 10 μg total protein from each sample were run on NuPAGE® Novex® 4–12% Bis–Tris 1.5 mm, pre-cast SDS-PAGE gels (Invitrogen) alongside 10 μg of total protein from human serum. Gels were stained with colloidal Coomassie Blue (Instant Blue gel stain; Expedion) and imaged on a GS-800 den- sitometer (BioRad). Samples were excluded if they com- prised largely of serum proteins (i.e. barely visible tissue protein bands) and/or had low protein staining above 20 kDa. Equal protein amounts from each sample were then pooled into 6 groups according to tissue type, case control status and menstrual cycle phase. These groups were: eutopic tissue from endometriosis cases in the secretory (ES; n = 19) or proliferative phase (EP; n = 9), eutopic tissue from patients with chronic pelvic pain with no evidence of endometriosis at laparoscopy in the secre- tory phase (PS; n = 7), eutopic tissue from asymptomatic controls scheduled for risk-reducing surgery with no evidence of disease at laparoscopy in the secretory (CS; n = 6) or proliferative phase (CP; n = 7) and ectopic tissue from endometriosis cases in the secretory phase (EcS; n = 11). There were no eutopic samples available for the pain group in proliferative phase or eligible ectopic sam- ples from endometriosis patients in proliferative phase. Sample collection To improve proteomic coverage, immunodepletion of the 12 most abundant serum proteins was carried out on the pooled samples using Protein Purify 12 (PP12) Human Serum Protein Immunodepletion resin (R&D). Briefly, 500 μL of PP12 immunodepletion slurry was incubated with diluted endometrial tissue lysate (500 μg protein) on a rotary shaker for 30 min. Unbound material was recov- ered using Spin-X Filter units by centrifugation, the fil- trates were concentrated to 25 μL using 5 kDa molecular weight cut-off Vivaspin columns and protein concentra- tion determined using the Bradford method. Ground tissue was lysed in 2D lysis buffer (8 M urea, 4% w/v CHAPS and 10 mM Tris–HCl pH 8.3) using a ratio of 5 μL of lysis buffer per 1 mg of tissue at room tem- perature to extract protein. Samples were sonicated and centrifuged at 3000g for 10 min at 4 °C to pellet insolu- ble material. The supernatant was collected and protein concentration determined using a Bradford microtitre plate assay (Pierce) using a bovine serum albumin stand- ard curve. All samples were normalised to the same (lowest) protein concentration using 2D lysis buffer. To assess quality of the samples, 10 μg total protein from each sample were run on NuPAGE® Novex® 4–12% Bis–Tris 1.5 mm, pre-cast SDS-PAGE gels (Invitrogen) alongside 10 μg of total protein from human serum. Gels were stained with colloidal Coomassie Blue (Instant Blue gel stain; Expedion) and imaged on a GS-800 den- sitometer (BioRad). Samples were excluded if they com- prised largely of serum proteins (i.e. barely visible tissue protein bands) and/or had low protein staining above 20 kDa. Equal protein amounts from each sample were then pooled into 6 groups according to tissue type, case control status and menstrual cycle phase. These groups were: eutopic tissue from endometriosis cases in the secretory (ES; n = 19) or proliferative phase (EP; n = 9), eutopic tissue from patients with chronic pelvic pain with no evidence of endometriosis at laparoscopy in the secre- tory phase (PS; n = 7), eutopic tissue from asymptomatic controls scheduled for risk-reducing surgery with no evidence of disease at laparoscopy in the secretory (CS; n = 6) or proliferative phase (CP; n = 7) and ectopic tissue from endometriosis cases in the secretory phase (EcS; n = 11). 2D‑DIGE profiling of endometrial tissue lysates Immunodepleted samples (80 μg total protein) were labelled differentially in triplicate with NHS-cyanine dyes (GE Healthcare) at a dye to protein ratio of 6 pmol/μg protein on ice for 30 min in the dark. Cy2 dye was used to label an internal standard pool prepared by mixing equal amounts of protein from all pools. Reactions were Irungu et al. Clin Proteom (2019) 16:14 Page 4 of 16 quenched by adding a 20-fold molar excess of l-lysine to dye and incubating on ice for 10 min in the dark. Pairs of differentially labelled samples (Cy3 and Cy5) and Cy2- labelled standard were mixed appropriately and reduced by addition of dithiothreitol (DTT) (65 mM final concen- tration) and carrier ampholines and pharmalytes added to a final concentration of 2% v/v with 1 μL of 2% bromo- phenol blue. Samples were then run on 9 2D-gels accord- ing to our standard procedures [9]. Briefly, Immobiline IPG strips (24 cm; pH 3–10 NL) (GE Healthcare) were rehydrated with labelled samples overnight and isoelec- tric focusing performed for 80 kVh at 16 °C. Strips were equilibrated with DTT reduction and iodoacetamide (IAM) alkylation steps, transferred onto 10% SDS-PAGE bonded gels cast between low fluorescence glass plates, overlaid with agarose and run for 16 h at 2.2 W per gel. Images were acquired by scanning gels on a Typhoon 9400 multi-wavelength fluorescence imager (GE Health- care) and analysed using DeCyder Software V6 (GE Healthcare), calculating standardised spot abundances (using the Cy2-labelled standard pool) for all matched spots across the 9 gel images. Differences in spot abun- dances between conditions were filtered by specifying a 1.5 threshold of average fold-change with P < 0.05 (Stu- dent’s t test). Pick lists of spots of interest were created and exported to an Ettan spot picking robot (GE Health- care) for excision from the same colloidal Coomassie Blue post-stained gels. an Acclaim PepMap 100 C18 pre-column (5 μm, 100 Å, 300 μm i.d × 5 mm) (Thermo Fisher) and washed for 3 min with 90% buffer A (H2O + 0.1% (v/v) FA) at a flow rate of 25 μL/min. Chromatographic separation was then performed on an Acclaim PepMap 100 C18 nano-LC col- umn (3 μm, 100 Å, 75 μm i.d × 25 cm) (Thermo Fisher) with a 60 min linear gradient of 10–50% buffer B (100% ACN + 0.1% (v/v) FA) at a flow rate of 300 nL/min. Protein identification by LC–MS/MS In-gel digestion was carried out by washing gel pieces three times in 200 µL of 50% acetonitrile (ACN) and then in 200 µL of 100% ACN. Gel pieces were dried in a SpeedVac for 20 min, reduced for 45 min with 20 µL of 10 mM DTT in 5 mM ammonium bicarbonate (AmBiC) pH 8.0 at 50 °C, alkylated with 20 µL of 50 mM IAM in 5 mM AmBiC, washed twice in 200 µL of 50% ACN and dried in a SpeedVac. Gel pieces were then overlaid with 50 ng modified trypsin (Promega) in 10 µL of 5 mM AmBiC and incubated for 16 h at 37 °C. The supernatant was collected and peptides extracted twice with 20 µL of 5% trifluoroacetic acid in 50% ACN and the supernatants pooled. Peptide extracts were vacuum dried, desalted using reversed phase C18 ZipTips™ (Merck Millipore) as per the manufacturer’s instructions, dried down in a SpeedVac and stored at − 20 °C prior to MS analysis. Digested samples were re-suspended in 6 μL of 0.1% (v/v) formic acid (FA) and analysed by LC–MS/MS using an Ultimate 3000 nano-flow reversed phase-liquid chro- matography (RPLC) system linked to an LTQ-Orbitrap XL mass spectrometer (Thermo-Scientific) equipped with a Picoview PV550 nano-electrospray ion source (New Objective Inc.). Samples were first injected onto 2D‑DIGE profiling of endometrial tissue lysates Tan- dem MS was performed in the data-dependent mode to automatically switch between MS (full ion scan) and MS/ MS (fragment ion scan) acquisition. Survey full scan MS spectra (m/z 390–1700) were acquired in the orbitrap with a resolution of 60,000 at m/z 400. The most intense (top 6 ions per survey scan) were sequentially isolated for fragmentation in the linear ion-trap by collision induced dissociation and dynamically excluded for 60 s. Acquired mass spectra were processed using Mascot Distiller ver- sion 2.5 (Matrix Science Ltd) and searched against the SwissProt database. The following parameters and search filters were used; MS tolerance was 10 ppm, MS/MS tol- erance was 0.5 Da, two missed cleavages were allowed, carbamidomethylation of cysteines was set as a fixed modification, methionine oxidation, acetylation (protein N-term) and deamidation (asparagine and glutamine) were set as variable modifications. Protein identifications were accepted where there were two unique peptides of score > 20 at a Mascot significant threshold of P < 0.05. Protein identifications were matched to specific spots in DeCyder with experimental molecular weights checked against theoretical values. Profiling of endometrial tissues using a TMT‑based 3D–LC– MS/MS strategy A 6-plex tandem mass tagging (TMT) approach with 3-dimensional peptide separation [strong anion exchange (SAX) chromatography, off-line high pH RPLC and low pH nano-RPLC coupled to MS/MS] was applied to the 6 tissue lysate pools described above to maximise quanti- tative proteomic coverage. Immunodepleted tissue lysate pools (100 μg each) were re-suspended in 100 mM tri- ethylammonium bicarbonate (TEAB), pH 8.5 and 0.1% SDS, reduced with 1 mM tris(2-carboxyethyl)phosphine for 1 h at 55 °C and alkylated with 7.5 mM IAM for 1 h at RT in the dark. Samples were digested overnight at 37 °C using 4 µg of modified porcine trypsin. Samples were then labelled with TMT reagents (Thermo Fisher). Briefly, each digested pool was labelled with 0.8 mg of one of six TMT reagents (re-suspended in 41 μL of ACN) for 1 h at RT as follows: CS—TMT126, CP—TMT127, PS—TMT128, EcS—TMT129, ES—TMT130 and EP— TMT131. Samples were then incubated with 0.25% hydroxylamine for 30 min at RT to quench the reaction. The six TMT-labelled samples were then combined. SDS Irungu et al. Clin Proteom (2019) 16:14 Page 5 of 16 was removed using detergent removal spin columns (Pierce) as per the manufacturer’s instructions, the sam- ples desalted using 1 cc Oasis HLB cartridges (Waters) as per the manufacturer’s instructions, dried in a SpeedVac and re-suspended in 300 μL of 100 mM TEAB pH 8.5. Gene ontology and pathway enrichment analysisf Gene ontology and pathway enrichment analysisf Differentially expressed proteins were imported into WebGestalt [10] and each clinical group analysed sepa- rately for enrichment of GO biological process, molecu- lar function and cellular component, GO Slim terms, protein interaction networks, KEGG pathways and disease association. Significantly enriched terms were identified using a hypergeometric test with a Benjamini– Hochberg (BH) correction at a significance of P < 0.05. The top 10 GO terms with the most significant P values were reported. In each comparison, the protein lists were analysed separately as up- or down-regulated proteins. Selection and verification of candidate biomarkersi For selection of proteins for verification, a biomarker scoring system was devised based on fold-change, TMT reporter ion ratio count, variability, number of unique peptide sequences, whether they were possible serum contaminants and their membership in 1 of 5 expres- sion clusters. Clustering was performed using Graphical Proteomics Data Explorer (GProX) based on reporter ion ratios for the different comparisons with a higher score given to candidates whose expressions differed between endometriosis and both control groups and in both cycle phases. Selection was also weighted based on prior knowledge of function, proteins known to be secreted and those for which commercial detection reagents were available. LUM, TPM2, CPM, PAEP and TNC were selected from the discovery profiling work. i Promising candidate markers reported in the literature (sICAM1, MCP1, MIF, IL1R2, VEGF and FST) were also tested using commercial ELISA kits. Assays were tested for reproducibility and technical sensitivity to ensure the protein of interest could be accurately detected in serum samples. Optimal dilutions and intra-assay CVs for the assays are shown in Additional file 1: Table S1. Assays were carried out according to the manufacturers’ instruc- tions using the complete study set. LC–MS/MS was carried out essentially as described above, using a 90 min linear gradient of 10–50% buffer B, with data-dependent acquisition of the top 3 ions for fragmentation by both CID and HCD (collision energy 40%) for optimal reporter ion measurement. For HCD, product ions were detected in the orbitrap at a resolu- tion of 7500. Proteome Discoverer version 2.4 was used for protein identification and quantification using Mascot for searching the SwissProt database. Search parameters were as described above, except that only one missed cleavage was allowed. A co-isolation threshold of 25% was set in the quantification method to limit the record- ing of reporter ion ratios from multiple peptides. Protein groups with a ratio above 1.5 or below 0.67 for each com- parison were considered to be differentially expressed. Profiling of endometrial tissues using a TMT‑based 3D–LC– MS/MS strategy p μ p For SAX chromatography, DEAE ceramic HyperD F slurry (300 μL; Pall Corporation) was added to a spin fil- ter unit and centrifuged at 3000 rpm for 2 min to remove storage buffer, then washed with 200 μL of 1 M NaCl in 100 mM TEAB pH 8.5, then three times with 200 µL of 200 mM TEAB pH 8.5 and then equilibrated by wash- ing with 100 mM TEAB pH 8.5, followed by centrifuga- tion at 3000 rpm for 2 min to remove the supernatant. The pooled sample was then incubated with the resin on a rotary shaker for 5 min. Un-bound peptides were removed by centrifugation followed by washing with 200 µL of 100 mM TEAB and the two fractions combined as the flow-through. Bound peptides were then sequen- tially eluted using 200 μL × 2 of increasing salt concentra- tion buffer; 100 mM TEAB plus 400 mM NaCl, 600 mM NaCl and 1 M NaCl. The two eluates at each salt con- centration were combined and desalted using 1 cc Oasis HLB cartridges, vacuum dried and stored at − 20 °C. For high pH RP-LC, peptide fractions were re-suspended in 45 μL of 20 mM ammonium formate pH 8.5 and frac- tionated on a Poroshell 300SB-C18 (5 μm, 2.1 × 75 mm) column using an Agilent 1100 series microflow pump. Briefly, 40 µL of re-suspended peptides were injected onto the column in 20 mM ammonium formate pH 8.5 and peptides fractionated using a 0–45% linear gradient of 20 mM ammonium formate pH 8.5 in 80% acetonitrile at a flow rate of 200 µL/min for 55 min. 30 fractions were collected for each of the 4 SAX fractions (total 120 frac- tions). Each fraction was dried, re-suspended in 200 μL of 0.1% FA and re-dried prior to MS analysis. Statistical analysis Data analysis was carried using Graphpad Prism software version 5.0.1 and the R statistical software environment. For samples where the protein of interest was determined to be below the limit of detection of the assay, the value of the lowest standard for that assay was used. The Sha- piro–Wilk test was applied to test data distribution with an unpaired t test used to compare groups for normally distributed data and the Mann–Whitney test used for non-normally distributed data. For each clinical group, the data was analysed independently of cycle phase and then by cycle phase to assess menstrual cycle depend- ency. A P value of < 0.05 was considered significant. To determine the diagnostic performance of each biomarker, Irungu et al. Clin Proteom (2019) 16:14 Irungu et al. Clin Proteom (2019) 16:14 Page 6 of 16 Page 6 of 16 Since multiple proteins were identified in many of the spots, it was not possible to assign which protein was differentially expressed with absolute certainty. There- fore, the most likely protein was assigned based on the number of matched peptides and concordance of theo- retical and experimental molecular weights. Differen- tially expressed proteins comprised cytoskeletal proteins, metabolic enzymes, extracellular matrix proteins, muscle proteins, those involved in protein folding and blood pro- teins, including haemoglobin. A simple biomarker score was used to prioritise candidates for further testing and was based on proteins displaying the same directional- ity of differential expression between endometriosis and both control groups and in ectopic versus eutopic tis- sue and that did not differ between control groups (PS/ CS). High-scoring proteins included lumican (LUM) and tropomyosin β chain (TPM2). Expression of LUM (identified in 3 spots) was higher in the secretory phase endometriosis group compared to both control groups (e.g. spot 708; ES/CS = 1.86, P = 0.005; ES/PS = 2.25, P = 0.004) and in ectopic compared to eutopic tissue (EcS/ES = 2.09, P = 0.008), but not in proliferative phase samples (Additional file 1: Table S2). Similarly, expression of two proteoforms of TPM2 was higher in ectopic versus eutopic tissue and in secretory phase endometriosis com- pared to control groups [e.g. spot 1548 (EcS/ES = 4.15, P = 0.0002; ES/CS = 3.15, P < 0.0001; ES/PS = 2.36, P = 0.0057)]. These differences suggest menstrual cycle dependency. LUM and TPM2 were selected for further testing as serum biomarkers. Discovery proteomic profiling by 2D‑DIGE In total, 122 pre-menopausal women were consented to the study with 87 women meeting the inclusion criteria. The set was divided into cases who were diagnosed with endometriosis (n = 45) and two control groups compris- ing women with pelvic pain (n = 21) or no known disease and no pain (n = 21). Eutopic and ectopic endometrial tissue specimens from these women were lysed and first quality-assessed by SDS-PAGE with colloidal Coomas- sie Blue staining. It was evident that some samples were heavily contaminated with blood proteins, impairing the ability to visualise tissue-derived proteins at that protein load, or had low protein staining overall (see Additional file 1: Figure S1). These tissue samples were subsequently excluded from further analysis. The remaining samples were pooled (based on equal protein amount) into six groups by clinical group and cycle phase with 6–20 tis- sue samples pooled per group (designated as CS, CP, PS, ES and EcS—see “Materials and methods” section). With blood protein contamination in mind, immunodepletion of the 12 most abundant serum proteins was undertaken to improve tissue protein coverage (Additional file 1: Fig- ure S1). Contamination was significant, as immunode- pletion resulted in protein yields of 20–25% of starting material, although the approach did reveal lower abun- dance (tissue-derived) proteins.i Statistical analysis ROC curve analysis was performed with the area under the curve (AUC) reported for each comparison [endome- triosis (E) vs. no-pain control (C) and endometriosis vs. pain control (P)]. Multi-variate analysis was carried out to assess the diagnostic performance of combined marker panels using logistic regression, reporting cross-validated (leave-one-out) AUC and sensitivity at fixed specificity. Discovery proteomic profiling by 3D–LC–MS/MS with TMT labelling To improve depth of coverage for candidate biomarker discovery, immunodepleted tissue pools were subjected to tryptic digestion, 6-plex TMT labelling and extensive peptide fractionation (120 fractions) using SAX, high pH RPLC and low pH nano RPLC linked on-line to tan- dem MS. Results are presented in Additional file 2. A total of 1581 proteins groups were identified of which 1433 (91%) had quantitative information across all clinical groups. To gain insight into the possible func- tional consequences of altered protein expression, a functional enrichment analysis was undertaken using GO biological process and KEGG pathway terms for the differentially expressed proteins (> 1.5-fold). The results were somewhat ambiguous. Analysis of pro- teins up-regulated in eutopic tissue from endometriosis patients, revealed enrichment of macromolecular com- plex subunit organization, mRNA metabolic process, protein complex disassembly, translational termination, aromatic/cyclic compound metabolic process, nitrogen compound metabolic process, catabolic process and p 2D-DIGE profiling was undertaken, analysing the 6 pools in triplicate for differential protein expression. Merged fluorescent gel images are shown in Additional file 1: Figure S2. Differential expression was assessed using Decyder image analysis software, where spot matching was performed and standardised spot abun- dances calculated with reference to a Cy2-labelled stand- ard pool (equal mix of all samples) run on all gels. Spot abundances were compared between clinical groups revealing 72 protein spots matching on all 9 gel images that displayed a > 1.5-fold change in standardised abun- dance (P < 0.05) between one or more clinical groups. Of these, 52 were detectable as well-defined colloidal Coomassie Blue-stained spots. These spots were excised, digested with trypsin and analysed by LC–MS/MS, resulting in 130 confident protein identifications (see Additional file 1: Table S2). Page 7 of 16 Irungu et al. Clin Proteom (2019) 16:14 Irungu et al. Discovery proteomic profiling by 3D–LC–MS/MS with TMT labelling Clin Proteom Table 2 GO enrichment analysis for differentially regu ES versus CS EP versus CP Upregulated Downregulated Upregulated Biological process Cellular macro- molecular complex subunit organiza- tion 5.34E−13 3.16E−05 mRNA metabolic process 5.34E−13 2.20E−05 0.0099 Cellular protein complex disassem- bly 3.46E−10 3.34E−05 Protein complex disassem- bly 4.90E−10 Translational termination 6.69E−09 0.0044 Cellular aromatic/ cyclic compound metabolic process 0.0092 Cellular nitrogen compound metabolic process 0.0139 Nucleic acid metabolic process Cellular catabolic process Response to wounding Actin filament- based process/ cytoskel- eton organisa- tion Acute inflam- matory response 0.0197 KEGG path- ways Metabolic pathways 7.26E−11 4.41E−07 Ribosome 4.72E−15 Focal adhe- sion 2.21E−05 Table 2 GO enrichment analysis for differentially regulated (≥ 1.5 fold-change) proteins ES versus CS EP versus CP ES versus PS EcS versus ES Upregulated Downregulated Upregulated Downregulated Upregulated Downregulated Upregulated Downregulate Biological process Cellular macro- molecular complex subunit organiza- tion 5.34E−13 3.16E−05 mRNA metabolic process 5.34E−13 2.20E−05 0.0099 4.85E−06 1.10E−25 Cellular protein complex disassem- bly 3.46E−10 3.34E−05 Protein complex disassem- bly 4.90E−10 Translational termination 6.69E−09 0.0044 Cellular aromatic/ cyclic compound metabolic process 0.0092 Cellular nitrogen compound metabolic process 0.0139 1.92E−10 Nucleic acid metabolic process 4.85E−06 Cellular catabolic process 3.42E−05 5.02E−10 Response to wounding 8.51E−14 Actin filament- based process/ cytoskel- eton organisa- tion 8.51E−14 Acute inflam- matory response 0.0197 2.58E−06 2.63E−09 1.57E−12 KEGG path- ways Metabolic 7 26E 11 4 41E 07 1 40E 05 2 75E 20 Table 2 GO enrichment analysis for differentially regulated (≥ 1.5 fold-change) proteins Table 2 GO enrichment analysis for differentially regulated (≥ 1.5 fold-change) proteins Table 2 GO enrichment analysis for differentially regulated (≥ 1.5 fold-change) proteins 1.10E−25 Page 8 of 16 Irungu et al. Clin Proteom (2019) 16:14 Irungu et al. Clin Proteom Table 2 (continued) ES versus CS EP versus CP ES versus PS EcS versus ES Upregulated Downregulated Upregulated Downregulated Upregulated Downregulated Upregulated Downregulated Regulation of actin cytoskel- eton 3.77E−06 4.82E−07 2.48E−11 ECM-receptor interaction 0.0001 1.40E−05 2.92E−09 Spliceosome 9.27E−06 9.31E−09 0.0005 Proteasome 3.55E−05 4.29E−05 1.36E−10 Oxidative phospho- rylation 5.93E−05 4.13E−07 Disease asso- ciation Carcinoma 9.60E−07 2.33E−07 8.97E−05 Neoplasm invasive- ness 7.87E−06 3.61E−08 5.76E−10 Cancer or viral infec- tions 6.40E−06 0.0007 Anemia, hemolytic 2.15E−13 3.69E−12 1.50E−09 Adhesion 5.10E−08 1.38E−06 1.39E−18 Hematologic diseases 1.30E−10 Protein defi- ciency 5.19E−08 2.13E−11 2.18E−10 Cardiovascu- lar diseases 5.04E−11 5.54E−10 Huntington’s disease 1.10E−06 3.87E−15 2.11E−12 Enrichment analysis was performed using WebGestalt. Discovery proteomic profiling by 3D–LC–MS/MS with TMT labelling Each clinical group was analysed separately for enrichment of biological process, KEGG pathways and disease association. Significantly enriched terms were identified using a hypergeometric test with a Benjamini-Hochberg (BH) correction with corrected P values shown for each comparison Table 2 (continued) Enrichment analysis was performed using WebGestalt. Each clinical group was analysed separately for enrichment of biological process, KEGG pathways and disease association. Significantly enriched terms were identified using a hypergeometric test with a Benjamini-Hochberg (BH) correction with corrected P values shown for each comparison acute inflammatory response, with only enrichment of mRNA metabolic process shared for all three com- parisons (Table 2). Down-regulated proteins were also enriched for macromolecular complex subunit organi- zation, mRNA metabolic process, protein complex dis- assembly and acute inflammatory response, with no enrichment common to the three comparisons. Com- paring ectopic and eutopic tissue, nitrogen compound metabolic process, response to wound healing and cytoskeleton organisation were enriched for up-reg- ulated proteins, whilst mRNA metabolic process and catabolic process were enriched for down-regulated proteins. KEGG pathway mapping revealed enrichment of genes involved in metabolic pathways, ribosome, proteasome, spliceosome and notably, regulation of the actin cytoskeleton, focal adhesions and extracellu- lar matrix (ECM)-receptor interactions, although there was no obvious pattern to the enrichment across the comparisons (Table 2). Disease association enrichment was also ambiguous, although there was a trend of down-regulated gene products linked with carcinoma and neoplasm invasiveness. This is perhaps not surpris- ing since endometriosis shares some features with can- cer, such as local and distant invasion, attachment and damage to affected tissues. Numerous muscle-related proteins were identified that were highly expressed in ectopic versus eutopic tissue, including TPM1, 2, 3 and 4, MYLK, MYL6 and 9, PDLIM7, CNN1, CALD1 and TAGLN, suggestive of significant amounts of mus- cle tissue in the ectopic samples (Additional file 2 and Table 3). ECM proteins including FN1, LUM, COL1A2, COL6A1, COL6A3, COL14A1, PRELP, OGN, DCN, BGN, FMOD and MFAP4, were also highly expressed in ectopic tissue. acute inflammatory response, with only enrichment of mRNA metabolic process shared for all three com- parisons (Table 2). Down-regulated proteins were also enriched for macromolecular complex subunit organi- zation, mRNA metabolic process, protein complex dis- assembly and acute inflammatory response, with no enrichment common to the three comparisons. Discovery proteomic profiling by 3D–LC–MS/MS with TMT labelling Com- paring ectopic and eutopic tissue, nitrogen compound metabolic process, response to wound healing and cytoskeleton organisation were enriched for up-reg- ulated proteins, whilst mRNA metabolic process and catabolic process were enriched for down-regulated proteins. KEGG pathway mapping revealed enrichment of genes involved in metabolic pathways, ribosome, proteasome, spliceosome and notably, regulation of the actin cytoskeleton, focal adhesions and extracellu- lar matrix (ECM)-receptor interactions, although there was no obvious pattern to the enrichment across the comparisons (Table 2). Disease association enrichment Protein groups were scored for biomarker potential with carboxypeptidase M (CPM) the highest scoring, displaying increased endometrial expression in endo- metriosis versus both control groups and in both cycle Irungu et al. Clin Proteom (2019) 16:14 Page 9 of 16 Irungu et al. Clin Proteom (2019) 16:14 Irungu et al. Clin Proteom Table 3 High-scoring proteins of interest identified by TMT 3D–LC–MS/MS profiling Acc. No. Discovery proteomic profiling by 3D–LC–MS/MS with TMT labelling Description Biomarker score Protein score Unique peptides PSMs Ratio ES/CS Ratio ES/PS Ratio EP/CP Ratio EcS/ES Ratio PS/CS P14384 Carboxypeptidase M CPM 26 154 2 6 1.619 2.525 2.451 0.319 0.633 P26599 Polypyrimidine tract-binding protein 1 PTBP1 24.6 264 7 11 2.646 2.771 0.953 0.377 0.787 Q14764 Major vault protein MVP 23 151 3 7 0.143 0.158 0.425 10.978 0.894 P29373 Cellular retinoic acid-binding protein 2 CRABP2 23 83 2 3 2.078 1.612 2.544 1.436 1.382 Q01995 Transgelin TAGLN 22.2 2119 17 95 1.105 1.324 2.171 18.832 1.240 Q01105 Protein SET 22.2 937 6 29 0.914 0.919 1.084 0.330 0.881 P00915 Carbonic anhydrase 1 CA1 22 5011 14 263 2.086 2.902 0.933 1.285 0.627 O94788 Retinal dehydrogenase 2 ALDH1A2 21.4 301 9 12 0.838 0.898 1.542 0.456 0.903 P16949 Stathmin STMN1 21.2 293 7 16 1.044 0.926 1.411 0.471 0.994 Q13308 Tyrosine-protein kinase 7 PTK7 21.2 362 3 13 0.510 0.677 1.497 0.293 0.807 P06703 Protein S100A6 21 762 5 47 1.379 1.653 0.795 3.364 0.774 P09466 Glycodelin PAEP 20.6 698 2 18 0.622 0.863 0.950 0.210 0.705 P02751 Fibronectin FN1 20.4 251 8 9 2.706 1.288 0.630 2.104 2.005 Q96KP4 Cytosolic non-specific dipeptidase CNDP2 20.2 1519 16 43 0.724 1.078 0.535 0.333 0.710 P00167 Cytochrome b5 CYB5A 20 692 7 29 0.835 0.880 0.205 0.979 0.948 P06401 Progesterone receptor PGR 20 37 1 1 1.415 1.762 2.420 0.321 0.792 P59665 Neutrophil defensin 1 DEFA1 19.6 137 3 11 0.325 2.476 1.315 4.989 0.133 P17661 Desmin DES 19.6 4279 24 216 0.904 0.930 1.381 3.056 1.063 Q05682 Caldesmon CALD1 19.4 677 6 25 1.162 0.950 1.260 7.685 1.223 Q7KZ85 Transcription elongation factor SPT6 SUPT6H 19.4 53 1 6 2.143 3.101 0.960 0.843 0.682 Q9HC84 Mucin-5B MUC5B 19.2 471 12 18 0.193 3.853 0.386 0.650 0.088 P12111 Collagen alpha-3(VI) chain COL6A3 19 687 12 27 1.933 1.254 1.209 18.478 1.597 P51884 Lumican LUM 19 1340 13 51 1.021 0.952 1.216 14.892 1.033 P60660 Myosin light polypeptide 6 MYL6 19 2726 12 92 1.378 0.878 1.148 5.003 1.382 P20774 Mimecan OGN 18.6 731 9 28 0.831 0.798 1.212 20.121 0.905 P09493 Tropomyosin alpha-1 chain TPM1 18.6 4361 8 186 1.004 1.007 1.245 10.119 0.964 P07951 Tropomyosin beta chain TPM2 18.6 4277 6 178 0.988 1.032 1.239 9.162 0.913 P06702 Protein S100A9 18.6 286 3 7 0.406 7.751 2.632 7.255 0.061 P67936 Tropomyosin alpha-4 chain TPM4 18.4 4288 12 201 1.014 1.023 1.230 7.301 0.918 P24821 Tenascin TNC 18.4 83 5 5 1.817 1.682 1.502 1.151 1.022 O00264 PGRMC1 18.4 191 4 8 1.013 1.035 1.561 0.609 1.228 P52907 F-actin-capping protein subunit a1 CAPZA1 18.2 145 4 7 3.031 1.078 0.885 1.459 1.280 P51888 Prolargin PRELP 18 84 3 5 0.504 0.471 1.466 33.466 1.145 Q05707 Collagen alpha-1(XIV) chain COL14A1 18 1210 18 49 1.294 0.834 0.898 10.120 1.426 P21333 Filamin-A FLNA 18 2103 41 77 1.227 0.967 1.509 2.216 1.357 Page 10 of 16 Irungu et al. Discovery proteomic profiling by 3D–LC–MS/MS with TMT labelling Notably, LUM and TPM2 were also identi- fied with increased expression in ectopic tissue, simi- lar to that observed by 2D-DIGE profiling, although were not altered appreciably in the eutopic tissue comparisons. phases (ES/CS = 1.62, ES/PS = 2.53, EP/CP = 2.45) (Table 3). Its expression was however lower in ectopic tissue (EcS/ES = 0.32). Similarly, progesterone receptor (PGR) was over-expressed in endometriosis eutopic tis- sue (ES/CS = 1.45, ES/PS = 1.76, EP/CP = 2.42), whilst decreased in ectopic tissue (EcS/ES = 0.32). Mem- brane-associated progesterone receptor component 1 (PGRMC1) was similarly over-expressed in prolifera- tive endometriosis eutopic tissue versus control (EP/ CP = 1.56), and under-expressed in ectopic tissue (EcS/ ES = 0.61). The progesterone-regulated gene glycode- lin (PAEP) was also under-expressed in ectopic tissue (EcS/ES = 0.21). Other proteins of interest were tenas- cin C (TNC), up-regulated in endometriosis compared to both control groups (ES/CS = 1.82, ES/PS = 1.68, EP/CP = 1.50), and transgelin (TAGLN), increased in proliferative phase endometriosis versus controls (EP/ CP = 2.17) and ectopic versus eutopic tissue (EcS/ ES = 18.83). Notably, LUM and TPM2 were also identi- fied with increased expression in ectopic tissue, simi- lar to that observed by 2D-DIGE profiling, although were not altered appreciably in the eutopic tissue comparisons. Multi-marker logistic regression models were gen- erated to assess if candidates would complement one another to improve classification. Generally, cross-val- idated models for discriminating the endometriosis and pain groups (all phases) performed similarly to those discriminating the endometriosis and non-pain groups with sensitivities of 62–67% at 80% specificity (Table 5). sICAM1 featured in the best models for both groups, whilst CA125 was only included in models for discrimi- nating endometriosis from the non-pain group. The best performing model [sICAM1, FST, TNC] for discriminat- ing endometriosis from pain controls (E vs. P; all phases), gave a sensitivity of 67% at 80% specificity. When con- sidering menstrual phase, for which both control groups were pooled, the best model [ICAM1, FST, oestradiol] gave 77% sensitivity at 80% specificity for detecting endo- metriosis in the proliferative phase, whilst the best model for secretory phase samples [CA125, MIF, PAEP], gave a sensitivity of 65% at 80% specificity. This again highlights cycle-dependency in the performance of the biomarker panels. Discovery proteomic profiling by 3D–LC–MS/MS with TMT labelling Testing candidates as serum biomarkers of endometriosis From the tissue profiling, TNC, CPM, TPM2, LUM and PAEP were selected for further testing as serological markers using samples from 87 women (control n = 21; pain control n = 21; endometriosis n = 45) (Table 1). Bio- marker candidates reported in the literature (sICAM1, MCP1, MIF, IL1R2, VEGF and FST) were also tested with CA125, progesterone, oestradiol and CRP. Commercial assays were first assessed using a test pool of all samples to determine optimal sample dilutions and reproducibil- ity (Additional file 1: Table S1) and then run on the full set. Serum measurements were correlated with clinical group and cycle phase. Only CA125 and sICAM1 were significantly elevated (P < 0.05) when comparing endo- metriosis to both control groups considering all cycle phases (Table 4A). Areas under the ROC curve for the endometriosis versus pain groups were 0.713 for CA125 and 0.722 for sICAM1. TNC was significantly elevated in the pain group versus the control and endometriosis groups. In the secretory phase, CA125 elevation main- tained significance when comparing endometriosis to both control groups, whereas sICAM1 was significantly raised only when comparing endometriosis and pain con- trols (Table 4B). Elevation of VEGF became significant between endometriosis and non-pain controls in secre- tory phase, whilst FST (lower in endometriosis) was the only candidate found to be significant when comparing groups in the proliferative phase. Together, these data Discovery proteomic profiling by 3D–LC–MS/MS with TMT labelling Clin Proteom (2019) 16:14 Irungu et al. Clin Proteom Table 3 (continued) Acc. No. Description Biomarker score Protein score Unique peptides PSMs Ratio ES/CS Ratio ES/PS Ratio EP/CP Ratio EcS/ES Ratio PS/CS P22105 Tenascin-X TNXB 18 85 3 3 1.548 1.570 1.155 2.143 0.973 P04792 Heat shock protein beta-1 HSPB1 17.8 1495 8 69 1.298 0.775 1.077 4.369 1.586 Q12805 Protein EFEMP1 17.6 62 2 3 2.222 1.596 0.921 1.974 1.373 Q32P28 Prolyl 3-hydroxylase 1 LEPRE1 17.6 139 3 4 1.399 1.023 1.415 0.547 1.260 P21291 Cysteine and glycine-rich protein 1 CSRP1 17.4 489 6 17 0.912 1.039 1.080 21.887 0.735 P02452 Collagen alpha-1(I) chain COL1A1 17.4 830 11 45 1.124 0.428 0.904 5.938 2.536 P18206 Vinculin VCL 17.4 1911 29 73 1.404 0.904 1.131 2.429 1.425 P35749 Myosin-11 MYH11 17.2 1059 14 40 0.986 1.006 1.228 3.765 1.372 P12004 Proliferating cell nuclear antigen PCNA 17.2 107 4 7 0.954 0.709 1.790 0.414 1.326 Selection of proteins with biomarker score, protein score, numbers of unique peptides and peptide spectrum matches (PSMs) and ratios of expression for the different tissue comparisons. Proteins in italics are of particular note with several selected for serum testing Irungu et al. Clin Proteom (2019) 16:14 Irungu et al. Clin Proteom Irungu et al. Clin Proteom Page 11 of 16 suggest cycle dependency in the serum levels of some of the candidate markers. phases (ES/CS = 1.62, ES/PS = 2.53, EP/CP = 2.45) (Table 3). Its expression was however lower in ectopic tissue (EcS/ES = 0.32). Similarly, progesterone receptor (PGR) was over-expressed in endometriosis eutopic tis- sue (ES/CS = 1.45, ES/PS = 1.76, EP/CP = 2.42), whilst decreased in ectopic tissue (EcS/ES = 0.32). Mem- brane-associated progesterone receptor component 1 (PGRMC1) was similarly over-expressed in prolifera- tive endometriosis eutopic tissue versus control (EP/ CP = 1.56), and under-expressed in ectopic tissue (EcS/ ES = 0.61). The progesterone-regulated gene glycode- lin (PAEP) was also under-expressed in ectopic tissue (EcS/ES = 0.21). Other proteins of interest were tenas- cin C (TNC), up-regulated in endometriosis compared to both control groups (ES/CS = 1.82, ES/PS = 1.68, EP/CP = 1.50), and transgelin (TAGLN), increased in proliferative phase endometriosis versus controls (EP/ CP = 2.17) and ectopic versus eutopic tissue (EcS/ ES = 18.83). Testing candidates as serum biomarkers of endometriosisi The aim of this study was a tissue-based discovery of potential new biomarkers of endometriosis with trans- lation to serum-based tests aimed at non-invasive diag- nosis. Promising biomarkers reported in the literature were also assessed. The main challenges experienced in the discovery was the heterogeneity of tissue samples and variable blood contamination. This was evidenced by the presence of variable levels of abundant fibro-muscular and serum proteins across the sample set that will have compromised the quality of the analysis. Whilst pooling would average out some of this heterogeneity, it is pos- sible that outlier samples may skew the data leading to a higher false discovery rate. Despite this, a number of dif- ferentially expressed proteins were identified as poten- tial biomarkers that were assayed in serum samples and which contributed to multivariate models that could discriminate endometriosis from either or both control groups with reasonable accuracy. Notably, we did not find CA125 or any of the previ- ously reported candidate biomarkers using the prot- eomic approaches described. CA125 exists at relatively low abundance and is an extremely large (up to 4 MDa), heterogeneously glycosylated protein. These proper- ties would make sampling of CA125 using these meth- ods unlikely—the protein is not likely to resolve well on 2D gels due to its large size and heterogeneity, whilst its heavy modification may hinder efficient tryptic diges- tion and decrease mass matching in database searches. Page 12 of 16 Irungu et al. Clin Proteom (2019) 16:14 Irungu et al. Clin Proteom Similarly, we postulate that the other candidates (sICAM1 MCP1 MIF IL1R2 VEGF and FST) exist at involved in disease progression. Although the enrichment analysis was somewhat ambiguous multiple proteins Table 4 (A) Median concentrations and ranges of individual biomarker candidates in serum and P values for comparison of clinical groups in all stages of the menstrual cycle. (B) Analysis by menstrual cycle phase showing significant differences in candidate biomarker levels. Testing candidates as serum biomarkers of endometriosisi P values < 0.05 are considered significant Candidate biomarker Units Control (C) Pain (P) Endometriosis (E) E versus C P value E versus P P value C versus P P value (A) CA125 U/mL 7.7 (1.3–28.0) 7.7 (0.6–273.0) 22.5 (0.70–386.6) 0.0007 0.0058 ns oestradiol pmol/L 245 (18–2512) 419 (79–1616) 345 (18–2110) ns ns ns progesterone nmol/L 1.9 (0.6–43.7) 6.1 (0.9–53.4) 2.3 (0.2–61.7) ns ns ns sICAM1 ng/mL 301.4 (81.9–502.3) 265.7 (85.83–624.7) 342.1 (92.3–730.8) 0.04 0.004 ns IL1R2 ng/mL 12.72 (7.65–19.23) 11.33 (6.41–23.29) 12.35 (0.57–23.2) ns ns ns MCP1 ng/mL 0.32 (0.10–1.55) 0.26 (0.07–1.39) 0.27 (0.03–2.25) ns ns ns MIF ng/mL 24.81 (2.20–106.3) 30.87 (2.22–282.8) 24.11 (2.25–314.6) ns ns ns VEGF ng/mL 0.33 (0.07–1.00) 0.32(0.09–0.95) 0.41 (0.15–1.41) ns ns ns FST ng/mL 0.82 (0.27–3.79) 0.73 (0.28–2.41) 0.67 (0.23–4.33) ns ns ns PAEP ng/mL 7.22 (1.03–51.82) 11.35 (4.92–38.84) 13.52 (0.70–79.68) ns ns ns LUM ng/mL 47.05 (0.60–101.1) 41.7 (0.18–111,3) 45.0 (6.39–133.0) ns ns ns TNC ng/mL 40.31 (11.27–152.2) 73.24 (13.47–154.7) 46.27 (7.09–182.4) ns 0.039 0.044 CPM ng/mL 3.83 (0.4–70.75) 4.32(0.02–38.03) 6.13 (0.4–94.62) ns ns ns CRP mg/L 0.7 (0.6–9.2) 0.6 (0.6–10.0) 0.75 (0.6–20.0) ns ns ns Candidate biomarker Secretory phase Proliferative phase E (n = 26) versus C (n = 9) E (n = 26) versus P (n = 12) E (n = 13) versus C (n = 10) E (n = 13) versus P (n = 9) Table 4 (A) Median concentrations and ranges of individual biomarker candidates in serum and P values for comparison of clinical groups in all stages of the menstrual cycle. (B) Analysis by menstrual cycle phase showing significant differences in candidate biomarker levels. P values < 0.05 are considered significant Table 4 (A) Median concentrations and ranges of individual biomarker candidates in serum and P values for comparison of clinical groups in all stages of the menstrual cycle. (B) Analysis by menstrual cycle phase showing significant differences in candidate biomarker levels. P values < 0.05 are considered significant involved in disease progression. Although the enrichment analysis was somewhat ambiguous, multiple proteins involved in cytoskeletal and ECM organisation and cell– matrix interactions were enriched. This supports findings from previous studies [11–14]. Testing candidates as serum biomarkers of endometriosisi P values < 0.05 are considered significant C = no pain control; P = pain control; E = endometriosis; ns = not significant Candidate biomarker Units Control (C) Pain (P) Endometriosis (E) E versus C P value E versus P P value C versus P P value (A) CA125 U/mL 7.7 (1.3–28.0) 7.7 (0.6–273.0) 22.5 (0.70–386.6) 0.0007 0.0058 ns oestradiol pmol/L 245 (18–2512) 419 (79–1616) 345 (18–2110) ns ns ns progesterone nmol/L 1.9 (0.6–43.7) 6.1 (0.9–53.4) 2.3 (0.2–61.7) ns ns ns sICAM1 ng/mL 301.4 (81.9–502.3) 265.7 (85.83–624.7) 342.1 (92.3–730.8) 0.04 0.004 ns IL1R2 ng/mL 12.72 (7.65–19.23) 11.33 (6.41–23.29) 12.35 (0.57–23.2) ns ns ns MCP1 ng/mL 0.32 (0.10–1.55) 0.26 (0.07–1.39) 0.27 (0.03–2.25) ns ns ns MIF ng/mL 24.81 (2.20–106.3) 30.87 (2.22–282.8) 24.11 (2.25–314.6) ns ns ns VEGF ng/mL 0.33 (0.07–1.00) 0.32(0.09–0.95) 0.41 (0.15–1.41) ns ns ns FST ng/mL 0.82 (0.27–3.79) 0.73 (0.28–2.41) 0.67 (0.23–4.33) ns ns ns PAEP ng/mL 7.22 (1.03–51.82) 11.35 (4.92–38.84) 13.52 (0.70–79.68) ns ns ns LUM ng/mL 47.05 (0.60–101.1) 41.7 (0.18–111,3) 45.0 (6.39–133.0) ns ns ns TNC ng/mL 40.31 (11.27–152.2) 73.24 (13.47–154.7) 46.27 (7.09–182.4) ns 0.039 0.044 CPM ng/mL 3.83 (0.4–70.75) 4.32(0.02–38.03) 6.13 (0.4–94.62) ns ns ns CRP mg/L 0.7 (0.6–9.2) 0.6 (0.6–10.0) 0.75 (0.6–20.0) ns ns ns Candidate biomarker Secretory phase Proliferative phase E (n = 26) versus C (n = 9) E (n = 26) versus P (n = 12) E (n = 13) versus C (n = 10) E (n = 13) versus P (n = 9) (B) CA125 0.006 0.003 ns ns oestrogen ns ns ns ns progesterone ns ns ns ns sICAM1 ns 0.026 ns ns IL1R2 ns ns ns ns MCP1 ns ns ns ns MIF ns ns ns ns VEGF 0.033 ns ns ns FST ns ns 0.011 ns PAEP ns ns ns ns LUM ns ns ns ns TNC ns 0.037 ns ns CPM ns ns ns ns CRP ns ns ns ns Table 4 (A) Median concentrations and ranges of individual biomarker candidates in serum and P values for comparison of clinical groups in all stages of the menstrual cycle. (B) Analysis by menstrual cycle phase showing significant differences in candidate biomarker levels. Testing candidates as serum biomarkers of endometriosisi Control groups were pooled (C + P) for some of the analyses Models AUC Sensitivity Specificity E versus C (all phases) CA125, sICAM1, CPM 0.768 0.667 0.8 CA125, sICAM1, VEGF 0.777 0.644 0.8 CA125, sICAM1, FST 0.77 0.644 0.8 CA125, sICAM1 0.778 0.6 0.9 CA125, sICAM1, IL1R2 0.758 0.6 0.9 CA125, sICAM1, MCP1 0.757 0.6 0.9 E versus P (all phases) sICAM1, FST, TNC 0.679 0.667 0.8 sICAM1, TNC 0.708 0.622 0.8 sICAM1, TNC, Oestradiol 0.68 0.622 0.8 sICAM1, PAEP, TNC 0.695 0.622 0.8 sICAM1, MIF, PAEP 0.697 0.622 0.8 sICAM1, LUM 0.665 0.444 0.9 E versus C + P (all phases) CA125, sICAM1, FST, CPM 0.706 0.578 0.8 CA125, sICAM1, VEGF, PAEP 0.71 0.578 0.8 CA125, sICAM1, PAEP 0.719 0.578 0.8 CA125, sICAM1, MIF, PAEP 0.704 0.578 0.8 CA125, MIF 0.621 0.467 0.9 E versus C + P (Proliferative) sICAM1, FST, Oestradiol 0.769 0.769 0.8 sICAM1, MIF, FST 0.781 0.692 0.8 sICAM1, FST 0.802 0.692 0.8 CRP, sICAM1, FST 0.802 0.692 0.8 CA125, sICAM1, FST 0.814 0.692 0.8 sICAM1, MIF, FST 0.781 0.615 0.9 E versus C + P (Secretory) CA125, MIF, PAEP 0.705 0.654 0.8 CA125, sICAM1, MIF 0.725 0.615 0.8 CA125, MIF, TNC 0.683 0.615 0.8 CA125, MIF, PAEP 0.705 0.538 0.9 CA125, MIF, TNC 0.683 0.577 0.9 Table 5 Performance of cross-validated multi-marker models for discriminating endometriosis from control groups particularly in ectopic versus eutopic tissue. One such protein, tenascin C (TNC), was reported to be regulated across the menstrual cycle and aberrantly in endometrio- sis [18–20]. Our data suggest that its cyclic expression is lost in the endometrium of endometriosis patients, and whilst we did not observe TNC overexpression in ectopic lesions, its increased expression may promote adhesion and invasion of endometrial cells at ectopic sites. Tropomyosins TPM1 and TPM2 play a role in mus- cle contraction, motility, maintenance of cell shape and cell–matrix interactions through stabilisation of actin filaments. Expression of TPM2 was higher in ectopic versus eutopic tissue and increased in secretory phase eutopic tissue from endometriosis patients compared to both control groups. This may point to its involvement in mediating cellular structural changes that allow move- ment and invasion of endometrial cells. The similarly expressed smooth muscle actin-binding protein TAGLN, may also be involved in this process and supports previ- ous findings [21]. Testing candidates as serum biomarkers of endometriosisi Adhesion to the perito- neal ECM and invasion of retrograde-shed endometrial cells is one of the vital stages in implantation of ectopic endometrial cells and it is tempting to speculate that the adhesion/ECM proteins identified herein play important Similarly, we postulate that the other candidates (sICAM1, MCP1, MIF, IL1R2, VEGF and FST) exist at relatively low abundance in tissue, reducing the chance of their identification. This highlights that coverage of the proteome is still somewhat limited despite the multi- dimensional approach used in our methodology.iif Tissue profiling identified numerous differentially expressed proteins implicated in the implantation of endometriotic tissue beyond the endometrium and/or Irungu et al. Clin Proteom (2019) 16:14 Page 13 of 16 Page 13 of 16 Irungu et al. Clin Proteom roles in this process. Lumican (LUM) has a role in cell Table 5 Performance of cross-validated multi-marker models for discriminating endometriosis from control groups Models were generated by logistic regression using up to 4 candidates with cross-validation by leave-one-out. The best performing models (by sensitivity) and area under the ROC curve (AUC) are reported for each comparison at fixed specificities of 0.90 or 0.80. E = endometriosis; C = no pain controls; P pain controls. Testing candidates as serum biomarkers of endometriosisi It is noteworthy that CA125 is currently the best single serum marker for ovarian cancer, although its median level in the endometriosis samples fell below the clinical threshold of 35 U/mL used for ovarian cancer detection. CA125 has been investigated extensively as a circulating marker of endometriosis, although lacks diagnostic accu- racy when used alone. Our data supports this notion, with serum CA125 giving 40% sensitivity at 90% specific- ity in our cohort. Its performance was also cycle-depend- ent, being better at discriminating secretory phase samples. Cyclic differences in CA125 levels in endome- triosis have been reported previously [29–32], although the diagnostic benefit of taking cycle stage into account is unclear. Soluble ICAM1 has also been investigated as a circulating marker with conflicting reports on its use- fulness as a biomarker [33–37]. Our data do not support its use alone as a diagnostic marker, however its inclusion in our best classification models, suggests its value when combined with other markers, particularly CA125, FST and TNC. underpowered the study. Thus, validation of these mod- els in a larger independent cohort is necessary and should include samples from patients with other gynaecological conditions presenting with similar symptoms, particu- larly gynaecological malignancies. This would allow bet- ter assessment of the specificity of the biomarker panels. Secondly, our best biomarker model for discriminating endometriosis from the more relevant pain control group [sICAM1, FST, TNC] provided a sensitivity of 67% at 80% specificity, and so its usefulness as a triage test for guiding surgery is debatable. However, taking cycle phase into account, one model provided 61.5% sensitivity at 90% specificity, and so might be acceptable for triaging. Thirdly, whilst we show improved model performances by taking cycle-phase into account (particularly for pro- liferative phase), this type of testing may be difficult to implement in clinical practice should the models be validated. Testing candidates as serum biomarkers of endometriosisi Increased expression of TPM1, TPM2 and TAGLN may be the result of a higher smooth muscle content of deep endometriotic lesions. Another selected candidate was CPM; a GPI-anchored extracellular pepti- dase that functions in processing of peptide hormones, chemokines and growth factors. It has a reported role in inflammation and stem cell mobilisation and has been shown to be up-regulated in endometrial epithelial cells during the proliferative phase [22]. The increased expres- sion in endometriosis observed herein, may support the inflammatory response, although its lower expression in ectopic lesions is somewhat at odds with this. Of particular note was the differential expression of the progesterone receptor (PGR). Several genes found to be dysregulated in the endometrium of endometriosis patients are known progesterone targets (Foxo1a, Mig6 and Cyp26a1) and their overall pattern of expression suggested prolongation of the proliferative phase [23]. Indeed, incomplete transition of the endometrium from the proliferative to the secretory phase is a hallmark of endometriosis that has been attributed to progesterone resistance [24]. Our finding of reduced PGR expression in ectopic tissue has been previously reported as a possi- ble mechanism of progesterone resistance [25]. However, whether the observed higher expression of PGR observed in eutopic endometrium from endometriosis patients is involved in progesterone resistance, or a response to it, is unclear. In part agreement with the literature [26–28], we also showed reduced expression of PGRMC1 and the progesterone target gene PAEP in endometriotic lesions; changes that may also contribute to progesterone resist- ance in ectopic lesions.i roles in this process. Lumican (LUM) has a role in cell migration and proliferation during embryonic develop- ment, tissue repair and tumour growth through regula- tion of matrix metalloprotease activity [15] and collagen fibrillogenesis [16]. In the endometrium, LUM expression was reported to increase in the secretory phase [17], in agreement with our findings. We also found multiple col- lagens and other ECM glycoproteins and integrin ligands to be overexpressed in endometriotic samples, and CA125 was identified as the best single marker for dis- criminating endometriosis from controls and featured Irungu et al. Clin Proteom (2019) 16:14 Page 14 of 16 Page 14 of 16 Irungu et al. Clin Proteom prominently in the best-performing multimarker models. Competing interests The authors declare that they have no competing interests. Conclusions In conclusion, we have identified molecular changes associated with endometriosis in eutopic and ectopic tissue and have derived non-invasive, cycle phase-spe- cific diagnostic models for endometriosis with respect- able performance characteristics that are similar, if not better, than those reported previously. Our study has some weaknesses. Firstly, models were only tested by cross-validation on the same dataset with some evi- dence of overfitting and subgrouping by cycle phase has Authors’ contributions DM, ES and JFT conceived and designed the study. DM and ES provided clini- cal samples. SI and JW performed sample processing, proteomic profiling and ELISA assays. SI and OB carried out data analysis and model testing. SI, DM and JFT drafted and edited the manuscript. All authors read and approved the final manuscript. Author details 1 1 Department of Women’s Cancer, Institute for Women’s Health, University College London, Cruciform Building 1.1, Gower Street, London WC1E 6BT, UK. 2 Reproductive Medicine Unit, University College London Hospital, Elizabeth Garrett Anderson Wing, Lower Ground Floor, 235 Euston Road, London NW1 2BU, UK. Additional files Previous studies have tested panels of serum or plasma biomarkers, including those investigated here. Kocbek et al. reported a model using the ratio of leptin to gly- codelin/PAEP and age, with 83.6% sensitivity and 83.8% specificity for distinguishing ovarian endometriosis from controls independently of cycle phase [38]. Although also tested in this study, ICAM1, VEGF, CRP and MCP1 were not included in the reported best models, whilst CA125 was not assessed. In another study, 28 plasma proteins were assessed in a large patient cohort, with an indepen- dently validated model comprising CA125, annexin V, VEGF and sICAM1/or PAEP giving 81–90% sensitivity and 63–81% specificity for detecting endometriosis in the menstrual phase [39]. Other reported models including CA125, MCP1 and/or MIF showed reasonable diagnos- tic accuracies [40, 41]. Whilst MCP1 added little to our models, MIF was in the best models for discriminating endometriosis from both control groups, particularly for the secretory phase. Our data thus corroborate CA125, sICAM1, PAEP, MIF and FST as potentially useful diag- nostic markers when combined in multivariate models. Additional file 1: Table S1. ELISA assays used with optimal dilutions and intra-assay CVs. Table S2. List of differentially expressed protein identifica- tions from 2D-DIGE profiling. Figure S1. Quality control of tissue lysates and immunodepletion test. Figure S2. Overlaid fluorescent 2D gel images from main 2D-DIGE profiling experiment. Additional file 1: Table S1. ELISA assays used with optimal dilutions and intra-assay CVs. Table S2. List of differentially expressed protein identifica- tions from 2D-DIGE profiling. Figure S1. Quality control of tissue lysates and immunodepletion test. Figure S2. Overlaid fluorescent 2D gel images from main 2D-DIGE profiling experiment. Additional file 2. Results of TMT-based LC-MS/MS profiling with candi- date scoring. Additional file 2. Results of TMT-based LC-MS/MS profiling with candi- date scoring. Additional file 2. Results of TMT-based LC-MS/MS profiling with candi- date scoring. Abbreviationsf 2D-DIGE: 2D-difference gel electrophoresis; TMT–LC–MS/MS: tandem mass tagging–liquid chromatography–tandem mass spectrometry; AUC: area under the receiver operating characteristic curve; DTT: dithiothreitol; IAM: iodoaceta- mide; LC–MS/MS: liquid chromatography–tandem mass spectrometry; ACN: acetonitrile; AmBiC: ammonium bicarbonate; FA: formic acid; RPLC: reversed phase-liquid chromatography; SAX: strong anion exchange; TEAB: triethylam- monium bicarbonate. References 1. Ballard K, Lowton K, Wright J. What’s the delay? A qualitative study of women’s experiences of reaching a diagnosis of endometriosis. Fertil Steril. 2006;86(5):1296–301. 23. Burney RO, Talbi S, Hamilton AE, Vo KC, Nyegaard M, Nezhat CR, Lessey BA, Giudice LC. Gene expression analysis of endometrium reveals progesterone resistance and candidate susceptibility genes in women with endometriosis. 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Localization of laminin, fibronectin, E-cadherin, and integrins in endometrium and endome- triosis. Fertil Steril. 1997;67(2):266–72. 34. Barrier BF, Sharpe-Timms KL. Funding The study was funded by a Commonwealth Scholarship Commission Award to SI (KECS-2012-221) with additional financial support from the University College London Hospitals (UCLH) Charity Trustees. The work was supported by the National Institute for Health Research UCLH Biomedical Research Centre. 18. Sasano H, Nagura H, Watanabe K, Ito K, Tsuiki A, Sato S, Yajima A, Kusakabe M, Sakakura T. Tenascin expression in normal and abnormal human endometrium. Mod Pathol. 1993;6(3):323–6. 19. Harrington DJ, Lessey BA, Rai V, Bergqvist A, Kennedy S, Manek S, Barlow DH, Mardon HJ. Tenascin is differentially expressed in endome trium and endometriosis. J Pathol. 1999;187(2):242–8. Consent for publication 15. Brezillon S, Pietraszek K, Maquart FX, Wegrowski Y. Lumican effects in the control of tumour progression and their links with metalloprotein- ases and integrins. FEBS J. 2013;280(10):2369–81. Received: 15 November 2018 Accepted: 1 April 2019 21. Dos Santos Hidalgo G, Meola J, Rosa ESJC, Paro de Paz CC, Ferriani RA. TAGLN expression is deregulated in endometriosis and may be involved in cell invasion, migration, and differentiation. Fertil Steril. 2011;96(3):700–3. 22. Hood BL, Liu B, Alkhas A, Shoji Y, Challa R, Wang G, et al. Proteomics of the human endometrial glandular epithelium and stroma from the proliferative and secretory phases of the menstrual cycle. Biol Reprod. 2015;92(4):106. Ethics approval and consent to participate 16. Ezura Y, Chakravarti S, Oldberg A, Chervoneva I, Birk DE. Differential expression of lumican and fibromodulin regulate collagen fibrillogen- esis in developing mouse tendons. J Cell Biol. 2000;151(4):779–88. Patients were recruited at the University College London Hospital Gynaecol- ogy Department following informed consent. Ethical approval for the study was granted by the UCLH Research Ethics Committee. 17. Lucariello A, Trabucco E, Boccia O, Perna A, Sellitto C, Castaldi MA, De Falco M, De Luca A, Cobellis L. Small leucine rich proteoglycans are differently distributed in normal and pathological endometrium. Vivo. 2015;29(2):217–22. Availability of data and materials The raw datasets used during the current study are available from the cor- responding author upon request. The raw datasets used during the current study are available from the cor- responding author upon request. Page 15 of 16 Page 15 of 16 Irungu et al. Clin Proteom (2019) 16:14 Irungu et al. Clin Proteom Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 20. Tan O, Ornek T, Seval Y, Sati L, Arici A. Tenascin is highly expressed in endometriosis and its expression is upregulated by estrogen. Fertil Steril. 2008;89(5):1082–9. Received: 15 November 2018 Accepted: 1 April 2019 Received: 15 November 2018 Accepted: 1 April 2019 35. Somigliana E, Vigano P, Candiani M, Felicetta I, Di Blasio AM, Vignali M. Use of serum-soluble intercellular adhesion molecule-1 as a new marker of endometriosis. Fertil Steril. 2002;77(5):1028–31. 39. Vodolazkaia A, El-Aalamat Y, Popovic D, Mihalyi A, Bossuyt X, Kyama CM, et al. Evaluation of a panel of 28 biomarkers for the non-invasive diagnosis of endometriosis. Hum Reprod. 2012;27(9):2698–711. 38. Kocbek V, Vouk K, Bersinger NA, Mueller MD, Lanisnik Rizner T. Panels of cytokines and other secretory proteins as potential biomarkers of ovarian endometriosis. J Mol Diagn. 2015;17(3):325–34. 40. Seeber B, Sammel MD, Fan X, Gerton GL, Shaunik A, Chittams J, Barnhart KT. Panel of markers can accurately predict endometriosis in a subset of patients. Fertil Steril. 2008;89(5):1073–81. 37. Mosbah A, Nabiel Y, Khashaba E. Interleukin-6, intracellular adhe- sion molecule-1, and glycodelin A levels in serum and peritoneal fluid as biomarkers for endometriosis. Int J Gynaecol Obstet. 2016;134(3):247–51. 39. Vodolazkaia A, El-Aalamat Y, Popovic D, Mihalyi A, Bossuyt X, Kyama CM, et al. Evaluation of a panel of 28 biomarkers for the non-invasive diagnosis of endometriosis. Hum Reprod. 2012;27(9):2698–711. 40. Seeber B, Sammel MD, Fan X, Gerton GL, Shaunik A, Chittams J, Barnhart KT. Panel of markers can accurately predict endometriosis in a subset of patients. Fertil Steril. 2008;89(5):1073–81. 41. Agic A, Djalali S, Wolfler MM, Halis G, Diedrich K, Hornung D. Combination of CCR1 mRNA, MCP1, and CA125 measurements in peripheral blood as a diagnostic test for endometriosis. Reprod Sci. 2008;15(9):906–11. 41. Agic A, Djalali S, Wolfler MM, Halis G, Diedrich K, Hornung D. Combination of CCR1 mRNA, MCP1, and CA125 measurements in peripheral blood as a diagnostic test for endometriosis. Reprod Sci. 2008;15(9):906–11. 36. Steff AM, Gagne D, Page M, Hugo P, Gosselin D. Concentration of solu- ble intercellular adhesion molecule-1 in serum samples from patients with endometriosis collected during the luteal phase of the menstrual cycle. Hum Reprod. 2004;19(1):172–8. 35. Somigliana E, Vigano P, Candiani M, Felicetta I, Di Blasio AM, Vignali M. Use of serum-soluble intercellular adhesion molecule-1 as a new marker of endometriosis. Fertil Steril. 2002;77(5):1028–31. 36. Steff AM, Gagne D, Page M, Hugo P, Gosselin D. Concentration of solu- ble intercellular adhesion molecule-1 in serum samples from patients with endometriosis collected during the luteal phase of the menstrual cycle. Hum Reprod. 2004;19(1):172–8. 37. Mosbah A, Nabiel Y, Khashaba E. Interleukin-6, intracellular adhe- sion molecule-1, and glycodelin A levels in serum and peritoneal fluid as biomarkers for endometriosis. Int J Gynaecol Obstet. 2016;134(3):247–51. 38. Kocbek V, Vouk K, Bersinger NA, Mueller MD, Lanisnik Rizner T. Panels of cytokines and other secretory proteins as potential biomarkers of ovarian endometriosis. J Mol Diagn. 2015;17(3):325–34. References Expression of soluble adhesion molecules in sera of women with stage III and IV endometriosis. J Soc Gynecol Investig. 2002;9(2):98–101. Irungu et al. Clin Proteom (2019) 16:14 Page 16 of 16 Page 16 of 16 35. Somigliana E, Vigano P, Candiani M, Felicetta I, Di Blasio AM, Vignali M. Use of serum-soluble intercellular adhesion molecule-1 as a new marker of endometriosis. Fertil Steril. 2002;77(5):1028–31. 36. Steff AM, Gagne D, Page M, Hugo P, Gosselin D. Concentration of solu- ble intercellular adhesion molecule-1 in serum samples from patients with endometriosis collected during the luteal phase of the menstrual cycle. Hum Reprod. 2004;19(1):172–8. 37. Mosbah A, Nabiel Y, Khashaba E. Interleukin-6, intracellular adhe- sion molecule-1, and glycodelin A levels in serum and peritoneal fluid as biomarkers for endometriosis. Int J Gynaecol Obstet. 2016;134(3):247–51. 38. Kocbek V, Vouk K, Bersinger NA, Mueller MD, Lanisnik Rizner T. Panels of cytokines and other secretory proteins as potential biomarkers of ovarian endometriosis. 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https://openalex.org/W4220686494	https://air.unimi.it/bitstream/2434/923767/2/Cohort%20of%20Preterm%20Infants%20at%20School%20Age.pdf	English	null	Cognitive, Behavioral and Socioemotional Development in a Cohort of Preterm Infants at School Age: A Cross-Sectional Study	Pediatric Reports	2,022	cc-by	7,116	Citation: Ionio, C.; Lista, G.; Veggiotti, P.; Colombo, C.; Ciuffo, G.; Daniele, I.; Landoni, M.; Scelsa, B.; Alfei, E.; Bova, S. Cognitive, Behavioral and Socioemotional Development in a Cohort of Preterm Infants at School Age: A Cross- Sectional Study. Pediatr. Rep. 2022, 14, 115–126. https://doi.org/10.3390/ pediatric14010017 Keywords: preterm birth; school-age; executive function; IQ; social competence Article Cognitive, Behavioral and Socioemotional Development in a Cohort of Preterm Infants at School Age: A Cross-Sectional Study Chiara Ionio 1,* , Gianluca Lista 2 , Pierangelo Veggiotti 3, Caterina Colombo 2, Giulia Ciuffo 1, Irene Daniele 2, Marta Landoni 1 , Barbara Scelsa 3 , Enrico Alfei 3 and Stefania Bova 3 1 CRIdee, Unità di Ricerca sul Trauma, Dipartimento di Psicologia, Università Cattolica, 20123 Milano, Italy; giulia.ciuffo@unicatt.it (G.C.); marta.landoni@unicatt.it (M.L.) 1 CRIdee, Unità di Ricerca sul Trauma, Dipartimento di Psicologia, Università Cattolica, 20123 Milano, Italy; giulia.ciuffo@unicatt.it (G.C.); marta.landoni@unicatt.it (M.L.) 2 Neonatologia, Patologia e Terapia Intensiva Neonatale, Ospedale dei Bambini “Vittore Buzzi”, ASST Fatebenefratelli Sacco, 20154 Milano, Italy; gianluca.lista@asst-fbf-sacco.it (G.L.); caterina.colombo@asst-fbf-sacco.it (C.C.); irene.daniele@asst-fbf-sacco.it (I.D.) 3 Neurologia Pediatrica, Ospedale dei Bambini “Vittore Buzzi”, ASST Fatebenefratelli Sacco, 20154 Milano, Italy; pierangelo.veggiotti@asst-fbf-sacco.it (P.V.); barbara.scelsa@asst-fbf-sacco.it (B.S.); enrico.alfei@asst-fbf-sacco.it (E.A.); stefania.bova@asst-fbf-sacco.it (S.B.) * Correspondence: chiara.ionio@unicatt.it; Tel.: +39-027-234-3642 or +39-338-442-5218; Fax: +39-027-234-2280 2 Neonatologia, Patologia e Terapia Intensiva Neonatale, Ospedale dei Bambini “Vittore Buzzi”, ASST Fatebenefratelli Sacco, 20154 Milano, Italy; gianluca.lista@asst-fbf-sacco.it (G.L.); caterina.colombo@asst-fbf-sacco.it (C.C.); irene.daniele@asst-fbf-sacco.it (I.D.) 3 Neurologia Pediatrica, Ospedale dei Bambini “Vittore Buzzi”, ASST Fatebenefratelli Sacco, 20154 Milano, Italy; pierangelo.veggiotti@asst-fbf-sacco.it (P.V.); barbara.scelsa@asst-fbf-sacco.it (B.S.); enrico.alfei@asst-fbf-sacco.it (E.A.); stefania.bova@asst-fbf-sacco.it (S.B.) ( ) ( ) * Correspondence: chiara.ionio@unicatt.it; Tel.: +39-027-234-3642 or +39-338-442-5218; Fax: +39-027-234-2 Abstract: More than 50% of children who survive prematurity have an atypical course of development at school age, as environmental demands become more demanding. This study examines the effects of preterm birth on the cognitive, behavioral and socioemotional development of 185 children at ages ﬁve and seven years. Weaknesses were found in attention, working memory, processing speed and the ability to correctly interpret emotions at both ages ﬁve and seven. Signiﬁcant correlations were found in regression and moderation models. These ﬁndings suggest that school-age children who were preterm infants are at increased risk of exhibiting impairments in several developmental domains that may affect their overall quality of life. 1. Introduction It is during this time, when the demands of the environment are becoming more pressing and difﬁcult to manage, that these deﬁcits begin to manifest themselves more clearly and prominently [8]. throughout their development [5]. Impairments in the child’s IQ, executive functions and general well-being appear to progress into adulthood [6]. Deﬁcits in executive functions such as attention, inhibition and processing speed have been frequently observed in this population, especially during school age [7]. It is during this time, when the demands of the environment are becoming more pressing and difﬁcult to manage, that these deﬁcits begin to manifest themselves more clearly and prominently [8]. However, to our knowledge, the literature focuses less on the behavioral and socio- emotional development of this population, although there is some evidence of the so-called behavioral phenotype of preterm infants, which is characterized by a high risk for the development of symptoms and disorders related to social problems [9], in combination with internalizing and externalizing problems [10]. These problems appear to be long- term and have a signiﬁcant impact on the quality of life of this population [11]. In this context, Moreira, Magalhaes and Alves point out that the percentage of children showing behavioral problems at school age is even higher than at pre-school age [12]. This can be explained by the fact that most of the instruments used to study these difﬁculties are based on the assessments of the parents, whose perceptions become more accurate as the children grow older. At school age, parents seem to be increasingly concerned about their children’s possible deﬁcits and are more aware of developmental expectations for this age group, so they more often compare their children’s development with that of their peers [13]. Several authors suggest that social perception may play an important role in determining social–emotional problems in preterm infants [14]. This skill involves the ability to selectively infer and extract the most important non-verbal social cues in the current situation. Wocadlo and Rieger found that children born very prematurely have problems recognizing facial expressions and that these difﬁculties seem to be related to their poor social skills [15]. 1. Introduction Modern neonatology is faced with an ever-increasing percentage of preterm infants worldwide and, in particular, higher rates of very low birth weight (VLBW) or extremely low birth weight (ELBW) infants surviving preterm birth. In the 1960s, the World Health Organization referred to preterm infants as newborns born before 37 weeks of gestation or before 259 days have passed since the last menstrual cycle [1]. Studies have shown that preterm birth can be a signiﬁcant risk factor for developmental impairment [2]. A preterm infant begins his or her extrauterine life in the Neonatal Intensive Care Unit (NICU), where he or she is hypostimulated due to the lack of uterine control and rhythmic stimulation, but also hyperstimulated by the light, the noise of the unit and the medical interventions, which can sometimes be particularly invasive and painful [3]. This situation is stressful for the child, also because he has no or limited physical contact with his parents [3], and for his parents, who may not be physically, emotionally and psychologically ready to welcome the birth. In addition, because of the long hospital stays in the NICU, preterm infants may also be exposed to risks of long-term psychological effects that may manifest later. In fact, longitudinal studies have provided evidence that preterm infants experience more long-term neurodevelopmental problems compared to term infants. Therefore, it is of great importance for researchers to study the impact of these potentially atypical developmental trajectories and the quality of life of these children [4]. 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: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Regarding cognitive development, research clearly shows that between 30% and 60% of very preterm infants experience various cognitive challenges and learning difﬁculties https://www.mdpi.com/journal/pediatrrep Pediatr. Rep. 2022, 14, 115–126. https://doi.org/10.3390/pediatric14010017 Pediatr. Rep. 2022, 14 116 throughout their development [5]. Impairments in the child’s IQ, executive functions and general well-being appear to progress into adulthood [6]. Deﬁcits in executive functions such as attention, inhibition and processing speed have been frequently observed in this population, especially during school age [7]. 1. Introduction Consistent with these ﬁndings, the results of a recent paper by Williamson and Jakobson show that this population has difﬁculty correctly interpreting other people’s emotions because of an inability to decode non-verbal cues (body movements, facial expressions and situational cues) that could be used for this type of inferencing during social interactions [16]. These problems, which play an important role in these children’s social difﬁculties, also contribute to the exacerbation of emotional and behavioral problems [17]. Indeed, their difﬁculty in recognizing relevant social information may lead them to misinterpret different social situations and, consequently, respond with inappropriate behavior [18]. Following the literature and emphasizing the potential risk of long-term maladjust- ment in this population, the present study examined school-age children’s vulnerability to cognitive, behavioral and socioemotional deﬁcits following preterm birth. Speciﬁcally, in the cognitive domain, we hypothesized that preterm infants might have cognitive impair- ments in IQ and executive functions that affect their academic performance and learning abilities. Following previous literature [19], we hypothesized that cognitive functioning may be poorly explained and predicted by atypical developmental trajectories and physical factors [20]. In addition, we hypothesized that age may have an impact on behavior and that there is a relationship between behavior and deﬁcits in social cognition. 2. Materials and Methods 2.1. Data Collection This study is part of a broader longitudinal research project conducted in 2013 in col- laboration with CRIdee (Department of Psychology, Catholic University of the Sacred Heart, Milan, Italy) and Buzzi Children’s Hospital in Milan (Italy), following preterm infants from birth to 7 years of age. It was approved by the Ethics Committee of Azienda Ospedaliera, Istituti Clinici di Perfezionamento, Milano (protocol number: 1171, 11 December 2012). The aim of this broad study was to investigate, from a longitudinal perspective, the impact of preterm birth on child development, paying particular attention to the parental couple and, consequently, to the effects and consequences that affect the whole triad. For this reason, families were followed in two phases from the birth of the child until the age of 7 years. Pediatr. Rep. 2022, 14 117 The ﬁrst phase began at the birth of the child, followed by a further assessment at 3 months of age and ended at the completion of the ﬁrst year of life [21]. The second time point began at age 4 and continued through annual observations until the premature child was 7 years old. The ﬁrst phase began at the birth of the child, followed by a further assessment at 3 months of age and ended at the completion of the ﬁrst year of life [21]. The second time point began at age 4 and continued through annual observations until the premature child was 7 years old. y In the present paper, the second phase of the longitudinal study was analyzed, which included a neuropsychological assessment of the children at 4–7 years of age. Before the tests were administered to the children, the parents signed an informed consent form and a privacy statement. 2.3. Measures 2.3.1. Cognitive Proﬁle 2.2. Participants Participants were 185 preterm infants aged 5 and 7 years (86 males, 99 females; gesta- tional age (GA) = 28.46 ± 2.08; birth weight (BW) = 1044.89 ± 267.37). Exclusion criteria were the presence of congenital anomalies, severe sensory impairments, severe brain in- juries and other neurological complications and parents’ lack of Italian language skills. In addition, 42.1% of the babies were twins; 62.8% were delivered spontaneous vaginally, 14.2% vacuum-assisted vaginally and 23% were delivered by caesarean section. In the ﬁrst minute after birth, the mean Apgar score was 5.68 (SD = 1.620) and in the ﬁfth minute it was 7.78 (SD = 1.102). The number of days spent in the neonatal intensive care unit ranged from 29 to 144 (M = 70.39; SD = 29.44). Given the importance of parents’ role in infant development, we also considered their age and degree. The mean age of the mothers was 36.52 years (SD = 6.165), and that of the fathers was 41.35 years (SD = 6.509). Most mothers had a high school degree (32%) or a bachelor’s degree (32%), 24% had a postgraduate degree and 12% attended high school without having a degree. As for the fathers, most of them had a bachelor’s degree (47.4%), 15.8% had attended secondary school, 15.8% had a high school diploma, 10.5% had a junior high school diploma and ﬁnally, the remaining 10.6% had a primary school diploma (5.3%) or attended university (5.3%). These data suggest that they all had a reasonable level of education. Moreover, only 3.8% of the mothers were unemployed, while all the fathers were employed. 2.3.2. Behavioral and Socio-Emotional Proﬁle 2.3.2. Behavioral and Socio-Emotional Proﬁle NEPSY-II (Italian Edition Cured by Urgesi, Campanella and Fabbro, 2011) We administered the Social Perception subtests of the NEPSY-II to the seven-year-old children to assess their ability to recognize facial expressions and decode the intentions of others: Theory of Mind (SO1) and Emotion Recognition (SO2). Theory of Mind (SO1) assesses constructs such as beliefs, intentions, deceptions, emotions, fantasy and imagina- tion and the ability to understand that others have thoughts, ideas and feelings differently from one’s own. Emotion Recognition (SO2) assesses the ability to recognize emotional expressions in photographs of children’s faces. p p g p Child Behavior Checklist (CBCL; Achenbach, 1991; Achenbach and Rescorla, 2000) Parents were asked to complete the Child Behavior Checklist for Ages 1.5–5 (CBCL 1.5–5; Achenbach & Rescorla, 2000) and the Child Behavior Checklist for Ages 4–18 (CBCL 4–18; Achenbach, 1991). These questionnaires provide a multi-axial assessment of subjects’ social, emotional and behavioral skills. We used them primarily to assess internalizing and externalizing problems in ﬁve- and seven-year-old children. Scores were divided into three categories: normal range (<93rd percentile), subclinical or borderline range (93rd to 97th percentile) and clinical or elevated range (>97th percentile). The Cronbach’s alpha for this assessment was 0.85. Neuropsychological Evaluation Battery for Developmental Age (BVN 5–11, Tres- soldi et al., 2005) The BVN is a test battery that examines the main cognitive functions (language, visual perception, memory, practices, attention, higher executive functions, reading, writing and arithmetic) in children aged ﬁve to eleven. It analyses the development of cognitive functions under normal conditions and identiﬁes certain developmental and/or acquired pathologies. We chose the Tower of London subtest (TOL), which assesses the ability to design and plan the necessary strategies to solve a problem correctly. It was administered to 7-year-old children. The Cronbach’s alpha for this test battery was 0.454. NEPSY-II (Italian Edition Cured by Urgesi, Campanella and Fabbro, 2011) The NEPSY-II is a neuropsychological battery of cognitive skills tests for preschoolers and school-aged children between three and sixteen years old. The Italian version consists of 33 subtests related to six different cognitive domains. We decided to use the subtests from the Executive Function & Attention domain, which consists of 3 subtests: Auditory Attention and Response Set (A3), Inhibition (A4) and Grouping Animals (A6). They were administered to seven-year-old children. The Cronbach’s alpha for this test battery was 0.856. Wechsler Intelligence Scale for Children-IV (WISC-IV; Italian Edition Curated by Orsini and Pezzuti, 2012) Wechsler Intelligence Scale for Children-IV (WISC-IV; Italian Edition Curated by Orsini and Pezzuti, 2012) Wechsler Intelligence Scale for Children-IV (WISC-IV; Italian Edition Curated by Orsini and Pezzuti, 2012) The Wechsler Intelligence Scale for Children-IV (WISC-IV) is a clinical instrument designed to assess the cognitive abilities of children between the ages of six and sixteen. The test includes ﬁfteen subtests, ten of which are core tests and ﬁve of which are additional optional subtests. The instrument measures the child’s global IQ and thus describes his or her general cognitive ability. In addition to the IQ, the assessment of the child’s proﬁle is complemented and enriched by four indexes: Verbal Comprehension Index, Perceptual Reasoning Index, Working Memory Index and Processing Speed Index. In order to obtain a comprehensive proﬁle of the cognitive development of our seven-year-old children, we decided to administer all the subtests. The scores ranged from 40 to 160. The Cronbach’s alpha for this study was 0.852. p y Wechsler Preschool and Primary Scale of Intelligence, Third Edition (WPPSI-III, Wechsler, 2008) Wechsler Preschool and Primary Scale of Intelligence, Third Edition (WPPSI-III, Wechsler, 2008) The Wechsler Preschool and Primary Scale of Intelligence, Third Edition (WPPSI-III) is a clinical instrument that focuses on the cognitive abilities of children between the ages of two years, six months and seven years, three months. It consists of 14 subtests divided into three groups: the main tests, the additional tests and the optional ones. We administered all the core subtests to our ﬁve-year-old children. The results were added to the Verbal Intelligence Quotient (VIQ), the Performance Intelligence Quotient (PIQ), the Processing Speed Quotient (PSQ) and the Full-Scale IQ (FSIQ). The Cronbach’s alpha for these results was 0.76. Pediatr. Rep. 2022, 14 118 Neuropsychological Evaluation Battery for Developmental Age (BVN 5–11, Tres- soldi et al., 2005) 3.1. Cognitive Proﬁle As for the mean, the results of the scales of WPPSI-III, listed in Table 1, show that the values of all indices are in the average range, except for the quotient of processing speed (M = 94.54), which is in the medium to low range. The results of the scales of the WISC-IV show how the index of working memory (M = 93.94) and the index of processing speed (M = 88.38) differ from the others, positioning themselves in the medium to low range. Finally, we looked at the children’s performance on the Executive Function & Attention subtest of the NEPSY-II. The results were below average for Inhibition (M = 7.9), Auditory Attention (M = 4.1) and Response Set (M = 4.8). Table 1. Means and standard deviations. Variables Mean SD Range WPPSI-III Indices FSIQ 104.68 14.55 64–128 VIQ 108.68 14.73 38–133 PIQ 100.29 14.52 29–138 PSQ 94.54 16.22 14–132 WISC-IV Indices FSIQ 101.72 14.78 64–128 VCI 99.66 20.96 41–140 PRI 99.22 21.93 34–124 WMI 93.94 23.05 19–121 PSI 88.38 23.80 11–129 BVN5-TOL 97.85 22.52 11.5–118.2 CBCL 1.5–5 Total Problems 1.65 2.93 1–3 Internalizing Problems 1.32 0.66 1–3 Externalizing Problems 1.43 1.51 1–3 CBCL 4–18 Scores Total Problems 1.15 0.51 1–3 Internalizing Problems 1.45 0.75 1–3 Externalizing Problems 1.15 0.51 1–3 Social problems 1.10 0.40 1–3 Note: Mean and standard deviation obtained from ﬁve- and seven-year-old children. WPPSI-III: Wechsler Preschool and Primary Scale of Intelligence, Third Edition: Verbal Intelligence Quotient (VIQ), Performance Intelligence Quotient (PIQ), Processing Speed Quotient (PSQ) and Full-Scale IQ (FSIQ). Wechsler Intelligence Scale for Children-IV (WISC-IV): Verbal Comprehension Index, Perceptual Reasoning Index, Working Memory Index, Processing Speed Index. Neuropsychological Evaluation Battery for Developmental Age (BVN 5–11). Child Behavior Checklist (CBCL). Table 1. Means and standard deviations. Note: Mean and standard deviation obtained from ﬁve- and seven-year-old children. WPPSI-III: Wechsler Preschool and Primary Scale of Intelligence, Third Edition: Verbal Intelligence Quotient (VIQ), Performance Intelligence Quotient (PIQ), Processing Speed Quotient (PSQ) and Full-Scale IQ (FSIQ). Wechsler Intelligence Scale for Children-IV (WISC-IV): Verbal Comprehension Index, Perceptual Reasoning Index, Working Memory Index, Processing Speed Index. Neuropsychological Evaluation Battery for Developmental Age (BVN 5–11). Child Behavior Checklist (CBCL). Correlation analysis showed a positive correlation between weight and total intellec- tual quotient (TQ) of the WPPSI-III (ﬁve-year-old children; r = 0.221, p < 0.05) and between weight and the WPPSI-III QPV (r = 0.287, p < 0.01) (Table 2. 2.4. Statistical Analysis For the cognitive proﬁle, we ﬁrst compared the mean scores with the clinical cut-off to assess the children’s executive functions and IQ. We also examined correlations between cognitive outcomes and some physical indicators such as birth weight, Apgar scores in the ﬁrst and ﬁfth minute and hospitalization days. To better investigate the inﬂuence of hospitalization days on cognitive performance, we decided to perform a linear regression for the subscales of the WPPSI-III and WISC-IV. To avoid collinearity problems, linear regressions were conducted separately for WPPSI-III and WISC-IV values. g p y For the behavioral proﬁle, paired-sample t-tests were performed to analyze signiﬁcant differences in behavior when the variable age was included. Finally, following previous studies, we decided to investigate whether there was a moderation effect of behaviors on cognitive outcomes. We ran a moderation model using JAMOVI in which we included internalizing and externalizing symptoms, social problems and the total measured with CBCL after ﬁve years as a moderator, hospitalization days as a predictor and IQ overall, assessed with the WPPSI-III, as a dependent variable. Pediatr. Rep. 2022, 14 119 * p < 0.05, p < 0.001. 3.1. Cognitive Proﬁle Furthermore, signiﬁcant correlations were found between hospitalization days and total intelligence quotient (TQ) (r = 0.945, p < 0.05), verbal intelligence quotient (VIQ) (r = 0.881, p < 0.05) and performance intelligence quotient (PIQ) (r = 0.925, p < 0.05). No signiﬁcant correlations were found between the Apgar scores and the WPPSII-III scales (p > 0.05). Pediatr. Rep. 2022, 14 120 Table 2. Correlation analysis. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1. WPPSI-III QI TOT — — 2. WPPSI-III VIQ 0.780 — — 3. WPPSI-III PIQ 0.765 ** 0.273 * — — 4. WPPSI-III QVP 0.459 0.098 0.380 — — 5. Hospital days 0.945 * 0.881 * 0.925 * 0.597 — — 6. Weight 0.221 * 0.184 0.079 0.287 _0.523 — — 7. Apgar 1 0.055 0.197 0.038 0.115 _0.627 −0.1 — — 8. Apgar 5 0.109 0.194 0.029 0.090 −0.318 −0.1 1.000 — — 9. WISC-IV QI 0.485 ** 0.249 0.388 * 0.353 NaN 0.038 0.142 −0.062 — — 10. WISC-IV ICV 0.384 * 0.506 ** 0.063 0.408 * NaN 0.048 −0.011 0.042 0.311 — — 11. WISC-IV IRP 0.326 0.186 0.248 0.485 NaN 0.153 0.054 0.077 0.285 0.724 — — 12. WISC-IV IML QI Total 0.025 0.133 0.184 0.304 NaN 0.104 0.020 0.038 0.103 0.785 0.688 — — 13. WISC-IV IVE 0.258 0.071 0.212 0.333 NaN 0.109 0.173 0.016 0.245 0.583 0.680 0.717 — — 14. CBCL Social QI Total 0.261 0.160 0.155 0.227 NaN −0.14 −0.115 0.097 −0.445 −0.006 −0.006 −0.008 −0.267 — — 15. NEPSY-II Total 0.574 0.337 0.484 0.406 * NaN 0.174 0.107 −0.071 0.764 ** 0.363 * 0.445 * 0.149 0.239 0.407 — — 16. BVN Total 0.349 0.014 0.417 * 0.331 NaN 0.08 0.134 0.039 0.215 −0.005 −0.014 −0.132 −0.031 0.134 0.387 * — — * p < 0.05, ** p < 0.001. Pediatr. Rep. 2022, 14 121 Finally, signiﬁcant regression equations were found. The ﬁrst regression results (F (1, 3) = 17.9, p = 0.024) showed that hospitalization days explained 85% of the vari- ance for the performance quotient (TQ) of the WPPSI-III scale (R2 = 0.85, F (2, 55) = 5.56, p < 0.01). Hospitalization days signiﬁcantly predicted the performance quotient IQ after ﬁve years (β = 0.387, p < 0.05). 3.1. Cognitive Proﬁle Similarly, we found that hospitalization days predicted the Verbal Intelligence Quotient (VIQ) (β = 0.223, p < 0.05) with a model (F = (1, 3) = 10.4, p = 0.048) which explained 77% of the variance (R2 = 0.77). Hospitalization days also predicted IQ overall (β = 0.326, p < 0.05; F (1, 3) = 25.1, p = 0.015), explaining 89% of the variance (R2 = 0.89) (Table 3). No signiﬁcant regression models were found between the WPPSI-III scales and child indicators (p > 0.05). Table 3. Signiﬁcant linear regression between hospitalization days and WPPSI-III subscales. Dependent Variable Predictors B SE p R2 95% Conﬁdence Interval Hospital stay Quotient (TQ) of the WPPSI-III scale 0.326 0.0652 0.015 0.893 0.344 1.55 Hospital stay Verbal Intelligence Quotient (VIQ) of the WPPSI-III scale 0.223 0.0691 0.048 0.776 0.0125 1.75 Hospital stay Overall score of the WPPSI-III scale 0.387 0.0915 0.024 0.856 0.229 1.62 Table 3. Signiﬁcant linear regression between hospitalization days and WPPSI-III subscales. 4. Discussion The aim of this study was to examine children’s cognitive, behavioral and socioemo- tional development. In particular, regarding the cognitive proﬁle, the analysis showed that the scores obtained by the ﬁve-year-olds on the WPPSI-III were all in the average range (90–109). However, the mean value of the processing speed quotient (M = 94.54) was slightly different from the others and was in the middle to low range. These results suggest that preterm infants may develop learning difﬁculties and have difﬁculty in performing cognitive and perceptual tasks. g p p Based the correlational analyses performed, it appears that the children’s IQ increased with increasing birth weight and hospitalization days, and that their processing speed seems to be better with increasing birth weight. These initial results seem to be consistent with what has been reported in the international literature, in which it is highlighted that this population tends to have deﬁcits in certain cognitive domains from preschool age and that this is generally associated with low birth weight. Contrary to expectations, no signiﬁcant correlations were found between index scores and gestational age [2,20]. In addition, scores of seven-year-olds on the WISC-IV appeared to be consistent with those of ﬁve-year-olds on the WPSSI-III. Although all scores were in the average range (85–115), the indices for working memory and processing speed appeared to be more deﬁcient, conﬁrming the possibility that these children may have difﬁculty learning, retaining or processing information at school age. As for the IQ scores, the average scores at ages ﬁve and seven might be related to the schooling of the parents [19]. As we know, the parents of the children in our sample had a relatively high level of education, as most of them had a college degree. According to Mento and Bisiacchi [20], the hypothesis could be that their level of education provides their children with a developmentally stimulating environment that can counteract the effects of biological deﬁcits. Finally, days of hospitalization have been shown to predict cognitive outcomes. It is well known that a prolonged stay of a preterm infant in the stressful environment of the NICU increases the risk of lifelong disability [22]. Thus, a growing number of studies associate repeated neonatal stress with poor neurological outcomes [23]. However, in our study, regression models showed a positive relationship between hospitalization days and cognitive outcomes, i.e., the longer the hospitalization, the better the outcomes. 3.2. Behavioral and Socio-Emotional Proﬁles To assess the behavior of the children, we ﬁrst considered their scores on the CBCL 1.5–5 and CBCL 4–18 questionnaires, which were in the average range. The results are presented in Table 4. The difference between the mean scores for internalizing and exter- nalizing was not signiﬁcant (p < 0.05), while a signiﬁcant difference occurred for the Total Problems scale. In addition, signiﬁcant correlations were found between IQ and higher scores for social problems (r = −0.445, p < 0.01), IQ and higher scores for ToM (r = 0.764, p < 0.001) and ToM and executive function (r = 0.387, p < 0.05) (Table 3). Table 4. Paired samples t-test analyses. Table 4. Paired samples t-test analyses. Sample Mean SD t df Internalizing Problems 5-year-olds 5.38 5485 1552 0.125 7-year-olds 7.69 5844 Externalizing Problems 5-year-olds 7.27 5182 0.553 0.583 7-year-olds 8.00 4787 Total Problems 5-year-olds 12.52 7683 2.755 0.0007 7-year-olds 20.21 12.451 Note. Comparison between ﬁve- and seven-year-old children with respect to Internalizing, Externalizing and Total Problems of the CBCL 1.5–5 and CBCL 4–18. No signiﬁcance was found in moderation models for externalizing symptoms, social problems and total CBCL score (p > 0.005). Signiﬁcant predictive support was found for both hospitalization days (p < 0.01) and internalizing symptoms (p < 0.01) on the CBCL. The interaction between the two variables was also signiﬁcant (p < 0.01). Both low (b = 0.292, s.e. = 0.058, p < 0.01) and high (b = 0.503, s.e. = 0.060, p < 0.01) levels of internalizing symptoms were signiﬁcant in predicting IQ overall. As Figure 1 shows, children with mild internalizing symptoms had a better total IQ score with low hospitalization days, which increased over the course of hospitalization days. Children with severe internalizing symptoms had a lower IQ score at baseline that increased over time. 122 Pediatr. Rep. 2022, 14 Figure 1. Simple Slope Plot. Figure 1. Simple Slope Plot. 4. Discussion Similarly, recent evidence suggests that developmental care in the NICU signiﬁcantly affects the mental development of preterm infants by improving their cognitive development index (MDI) [24]. In Kleberg’s work [25], the MDI of infants receiving care according to the Newborn Individualized Developmental Care and Assessment Program (NIDCAP) was higher than the corresponding score in control children, suggesting that Pediatr. Rep. 2022, 14 123 NIDCAP-based care may have a positive impact on the cognitive development of very preterm infants. One of the aims of this program is to enhance parents’ abilities to identify and respond to their infants’ cues. By becoming more sensitive to the baby’s needs, they are thus able to provide adequate stimulation [24]. It could be hypothesized that this improved ability may strengthen parent–infant interaction even after discharge from hospital and enhance joint parent–infant attention activities considered beneﬁcial to long-term cognitive development [26]. Although the preterm infants in this study did not receive a structured NIDCAP program, it is conceivable that some of the individual interventions they underwent (e.g., Kangaroo mother care) may explain our results. However, further research is needed to better explain these ﬁndings. p p g Regarding the behavioral proﬁle, our results showed that, contrary to our expectations, the scores of the preterm infants were in the average range at both ﬁve and seven years of age. However, it should be noted that our data were based on indirect measurements using questionnaires completed by parents (CBCL 1.5–5 and CBCL 4–18). For this reason, they may be less reliable and further investigation may be required. For this purpose, the SOLE VLBWI questionnaire, a recently validated self-report instrument developed to assess the quality of life of school-age children born preterm with a VLBWI, can be used [27]. A signiﬁcant difference was discovered in the overall scale of perceived problems for the age variable. Parents appeared to perceive more problems in their children at age seven than at age ﬁve. In fact, parents’ perceptions appeared to become more accurate as children aged [12]. Parents are more concerned about potential deﬁcits and become more aware of developmental expectations during the school years [13]. In addition, our results also revealed a moderating effect that internal behavioral symptoms have in altering the relationship between hospital stay length and IQ. Indeed, poor cognitive performance in preterm infants has been found to be strongly associated with behavioral problems, conﬁrming previous ﬁndings. 4. Discussion understand the social context, may engage in inappropriate behavior [34]. Further research should be conducted to clarify the causal relationship between these variables. 5. Strengths and Limitations This study has some limitations. First, as mentioned earlier, the behavioral proﬁle data were collected by indirect measures using questionnaires completed by parents (CBCL 1.5–5 and CBCL 4–18). For this reason, they may be less reliable and further research is needed. Second, there was a lack of a control group of term infants due to a high dropout rate during follow-up. Third, we did not evaluate emotional and cognitive capabilities of parents that may have played an important protective or risk role in children’s capabilities/difﬁculties. Overall, this study highlights the importance of being alert to potential deﬁcits associ- ated with preterm birth over the long-term and the importance of assessing the cognitive and behavioral domains of preterm infants. These ﬁndings provide an opportunity to implement interventions to improve out- comes for these infants during the period of maximal plasticity. These ﬁndings could also be a starting point for future research. They underscore the importance of structuring follow-up interventions for these infants to address the socioemotional and behavioral domains that are often examined. 6. Conclusions Early identiﬁcation of behavioral and cognitive problems in very preterm children should be encouraged to address them appropriately and promptly, especially since very preterm or very low birth weight infants are also at increased risk for attention- deﬁcit/hyperactivity disorder, depression and anxiety in adolescence. Weaknesses were found in attention, working memory, processing speed and the ability to correctly interpret emotions at both ages ﬁve and seven. These ﬁndings support early screening for these complications in extremely preterm infants, including those who are less immature and do not have cognitive impairments. Author Contributions: Conceptualization, C.I., C.C., G.L. and S.B.; methodology, C.I., S.B. and G.C.; validation, C.I. and G.C.; formal analysis, C.I. and G.C.; investigation, C.I., C.C. and G.L.; writing— original draft preparation, C.I., G.C. and M.L.; writing—review and editing, C.I., G.C., P.V., B.S., S.B., E.A., I.D. and M.L. 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: The study was approved by the Ethics Committee of Azienda Ospedaliera, Istituti Clinici di Perfezionamento, Milano. Protocol number: 1171, 11 December 2012. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The dataset is not publicly available due the hospital privacy policy. Data Availability Statement: The dataset is not publicly available due the hospital privacy policy. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 4. Discussion Few studies have examined whether behavior can explain differences in the frequency of cognitive problems between preterm and term infants [28]. In a cohort of 194 preterm infants studied at age ﬁve years, lower IQ was associated with more hyperactive behavior and inadequate social skills [29]. Literature shows that hospitalization from birth represents a signiﬁcant risk factor in children born preterm [30,31], as in the general population (e.g., childhood illness and chronic physical conditions) [32]. Our ﬁndings seem to move in this direction, however, more detailed research and studies comparing these results with those of a control sample are needed to explain this moderation effect. p With regard to the socioemotional proﬁle, we have paid particular attention to the area of social cognition, in which this population seems to be more vulnerable. Several authors suggest that social cognition may play a crucial role in determining socio-emotional problems in preterm infants [14]. Our analysis revealed that the scores obtained from the Theory of Mind subtest (NEPSY-II) were in the average range (8–12), while performance in emotion recognition was below average. This result conﬁrms previous ﬁndings showing that preterm infants may have greater difﬁculty interpreting the emotions of others [16]. This misinterpretation could also exacerbate emotional and behavioral problems [2]. Finally, we examined the relationship between cognitive impairment and socioemotional outcomes. We found a positive correlation between IQ and Theory of Mind; high IQ scores were associated with higher ToM scores. We also found a negative correlation between IQ and social problems (CBCL 4–18). Regarding executive functions, we found a positive correlation between TOL (BVN 5–11) and Theory of Mind; higher TOL subtest scores were associated with higher ToM scores. There was also a positive correlation between the Inhibition subtest (NEPSY-II) and Theory of Mind; higher scores on the inhibition subtest were associated with higher scores on ToM. Our ﬁndings support the hypothesis that adaptive social and emotional functioning is closely related to the functioning of higher-level cognitive processes, such as executive functions, which are used to decode social and emotional information and develop knowledge about the social world [33]. As we have said, these difﬁculties may have a cascading effect. A child who does not properly Pediatr. Rep. 2022, 14 124 understand the social context, may engage in inappropriate behavior [34]. Further research should be conducted to clarify the causal relationship between these variables. , , g , y, 4. 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https://openalex.org/W4226349989	http://jurnal.fkip.unila.ac.id/index.php/jpp/article/download/24053/pdf	English	null	Bibliometric Mapping on the Research “Merdeka Belajar” Using Vosviewer	JPP (Jurnal Pendidikan Progresif)	2,022	cc-by-sa	4,481	To cite this article: Batubara, I, H., Sari, I, P., Andini, P., Jannah, M., Saragih,S., Sinaga, B., Syahputra, E., & Lubis, B, S. (2022). Bibliometric Mapping on the Research “Merdeka Belajar” Using VosViewer. Jurnal Pendidikan Progresif, 12(2), 477-486. doi: 10.23960/jpp.v12.i2.202207. Jurnal Pendidikan Progresif e-ISSN: 2550-1313 \| p-ISSN: 2087-9849 http://jurnal.fkip.unila.ac.id/index.php/jpp/ Bibliometric Mapping on the Research “Merdeka Belajar” using VosViewer Vol. 12, No. 2, pp. 477-486, 2022 Vol. 12, No. 2, pp. 477-486, 2022 Vol. 12, No. 2, pp. 477-486, 2022 DOI: 10.23960/jpp.v12.i2.202207 DOI: 10.23960/jpp.v12.i2.202207 V Bibliometric Mapping on the Research “Merdeka Belajar” using VosViewer Ismail Hanif Batubara1,2,, Indah Purnama Sari3, Putri Andini1, Miftahul Jannah1, Sahat Saragih Bornok Sinaga2 Edi Syahputra2, Baihaqi Siddik Lubis4 1Department of Mathematics Education, Universitas Muhammadiyah Sumatera Utara, Indonesia 2Departmen of Mathematics Education, Universitas Negeri Medan, Indonesia 3Departmen of Technology Information, Universitas Muhammadiyah Sumatera Utara, Indonesia 4Departmen of Primary School Teacher Education, Universitas Muhammadiyah Sumatera Utara, Indonesia Corresponding email: ismailhanif@umsu.ac.id  INTRODUCTION scientometrics, are part of the research evaluation methodology (Tibaná-Herrera, Fernández-Bajón, & De Moya-Anegón, 2018), and from various literature that has been widely produced (Azad & Parvin, 2022), it is possible to carry out bibliometric analysis using its own methods (Ellegaard & Wallin, 2015), (Lima & Partidario, 2020). One of the purposes of this bibliometric analysis is to map a research topic with certain keywords, abstracts, or titles (Miguel, Hidalgo, Stubbs, Posadas, & Ortiz Jaureguizar, 2013) that have been carried out by various researchers from all over the world (Md Khudzari, Kurian, Tartakovsky, & Raghavan, 2018), (Tibaná- Herrera et al., 2018). Bibliometric is also a tool to measure information recorded in scientific publications (Donthu, Kumar, & Pattnaik, 2020) with mathematics and statistical methods that later from these results are analyzed using VosViewer software (Khalil & Gotway Crawford, 2015), (Nalau & Verrall, 2021). This software will produce visualization results and then be interpreted by readers (Anand & Gupta, 2020a) with the benefit of finding research gaps as well as article data that has been published on certain topics. The rapid development of science and technology in the 21st century today has brought very rapid changes to the world of education (Irvan & Muslihudin, 2020), (Dachi & Batubara, 2020). The independent learning movement is a movement to change education in Indonesia to become an international standard. Merdeka Belajar is a new policy program of the Ministry of Education and Culture of the Republic of Indonesia launched by Nadiem Anwar Makarim (Yudhawasthi & Christiani, 2022)(Verawati, 2020). The Programme for International Student Assessment (PISA) in 2019 showed that the assessment results in Indonesian students only occupied the sixth position from the bottom for mathematics and literacy, Indonesia occupied the 74th position from 79 countries. Responding to this, Nadiem also made the Merdeka Belajar Kampus Merdeka program one of the solutions to improve the literacy skills of students. Through this program, there are wide opportunities for students to enrich and improve their insights and competencies in the real world in accordance with their passions and ideals (Yudhawasthi & Christiani, 2022). We believe that learning can happen anywhere; the learning universe is limitless, not only in classrooms, libraries and laboratories, but also in villages, industries, workplaces, places of service, research centres, and in the community. Received: 09 March 2020 Accepted: 10 April 2020 Published: 11 April 2020 Abstract: Bibliometric Mapping on the Research “Merdeka Belajar” Using Vosviewer Objectives: To find out the development of research on the topic of Merdeka Belajar. Methods: The method is done by tracing articles in national journals with Publish or Peris then stored in the form of a Research Information System (RIS) and analyzed with VosViewer. Findings: The topic of Merdeka Belajar started in 2019 and increased rapidly every year. The authors who published the most on this topic were Reyna Virgina Nona, Fauzan Putraga Albahri, and Abdus Salam as of February 15, 2022. The most total link authors are Ummu Aiman, Nanda Alfan Kurniawan., Roimil Latifa, Ainur Rofieq, Randi Saputra, and Eko Susetyarini. The most dominant topics appeared were “Merdeka Belajar Kampus Merdeka”, “MBKM”, “Covid” and “Kebijakan Merdeka Belajar” Conclusion: Through bibliometric studies, researchers are able to determine topics related to the researched and open gaps for other researchers while looking at maps between topics, and authors with each other. Keywords: mapping map, merdeka belajar, bibliometric, VosViewer, publish or perish. Abstrak: Studi Bibliometric Terhadap Merdeka Belajar Dengan VosViewer. Tujuan: Untuk mengetahui perkembangan penelitian terhadap topik Merdeka Belajar. Metode: Metode yang dilakukan adalah dengan menelusuri artikel di jurnal nasional dengan publish or peris kemudian disimpan dalam bentuk Research Information System (RIS) dan dianalisis dengan VosViewer. Temuan: Topik Merdeka Belajar dimulai sejak tahun 2019 dan meningkat pesat setiap tahun. Nama author yang paling banyak mempublikasikan topik ini adalah Reyna Virgina Nona, Fauzan Putraga Albahri, dan Abdus Salam per tanggal 15 Februari 2022. Total link author yang paling banyak adalah Ummu Aiman, Nanda Alfan Kurniawan, Roimil Latifa, Ainur Rofieq, Randi Saputra, and Eko Susetyarini. Topik yang paling dominan muncul adalah “Merdeka Belajar Kampus Merdeka”, “MBKM”, “Covid” and “Kebijakan Merdeka Belajar”. Kesimpulan: Melalui studi bibliometric peneliti mampu menentukan topik yang berkaitan dengan yang diteliti dan membuka gap bagi peneliti lainnya sekaligus melihat map antar topik, dan author yang satu dengan yang lainnya. Kata kunci: mapping map, merdeka belajar, bibliometrik, VosViewer, publish or perish 477 Jurnal Pendidikan Progresif, Vol. 12, No. 2, pp. 477-486, August 2022 478  INTRODUCTION And this research object is 232 documents downloaded from Publish or Perish by doing a search from Google Scholar and the keyword “Merdeka Belajar”. All documents that appear either in the title, abstract, or keywords totaling 232 documents are then stored in the form of RIS. The reason for taking the Google Scholar index is because the topic taken related to Indonesian, namely Merdeka Belajar, which is very minimally found in Scopus indexed journals. This is one of the gaps in this research. In mapping, the map will appear topics from science. The input is bibliographic data, keywords, citations, and so on will be raised with the help of this software. This is the state of the art of this research, namely mapping a map on the topic of independent learning with the aim of knowing the bibliographic data of authors who have published the word Merdeka Belajar in national journals indexed by Google Scholar, data that once put the word Merdeka Belajar as a keyword in the journal, citation of writing in a journal that once contained the title Merdeka Belajar, the relationship between topics in the research title, as well as the relationship between authors with co-authors and so on as a novelty carried out in this study. The selection of the topic of Merdeka Belajar is one of the analysis gaps set by researchers considering that the Merdeka Belajar-Kampus Merdeka Program is one of the programs rolled out by the Minister of Education and is still ongoing today. This study began from mid-January 2022 to February 15, 2022. This research design consists of three stages, namely the pre-field stage, the field activity stage and the post-field stage. In the first stage the researcher conducts a selection of research topics and themes. In this case, the theme carried was the theme of “Merdeka Belajar” which has been published in accredited or non-accredited national journals. Furthermore, researchers select data analysis applications using the Publish or Perish application, and the last step in this first stage is to choose indexing journals such as web of Science, scopus, and Google sholar. However, because the title carried was “Merdeka Belajar” then researchers take google scholar indexing. In the second stage, the Research field activity stage conducted a search of all journals that had published about Merdeka Belajar through the Publish or Perish application.  INTRODUCTION Through the close interaction between universities and the world of work, universities will be present as a spring for the progress and development of the nation, helping to colour the nation’s culture and civilization directly, which will later be relearned with several studies such as bibliometric. Basically, bibliometric studies have a positive impact in various matters related to the scientific literature, including literature identification (Sarirete, 2021), the direction of research symptoms and knowledge growth in various different disciplines, guessing the breadth (comprehensiveness) of secondary literature, recognizing authorship and direction of symptoms on documents of various subjects, compiling documents on the shelf appropriately and regularly, studying the obsolescence and dissemination of scientific literature, to (Tibaná- Herrera et al., 2018) know the gap of research and enrich the amount of research for the field of information science, and can develop library collections more purposefully (Irianti, 2016). All bibliometric studies are conducted to find out the mapping of a study. A mapping map is a process Bibliometric studies are one of the important areas of study for library and information science because it has practical applications in measuring the scope and quality of books, journals, and articles (Guler, Waaijer, Mohammed, & Palmblad, 2016), (Marsh, 2020). Bibliometric analysis or methods, sometimes referred to as Batubara et al., Bibliometric Mapping on the Research “Merdeka Belajar”... 479 that allows a person to recognize elements of knowledge as well as their configuration, dynamics, mutual dependencies, and interactions (Burki, Burki, & Najam, 2021). Mapping is used for technology management purposes, including the definition of research programs, decisions regarding activities related to technology, the design of knowledge base structures, as well as the creation of educational and training programs. In relation to bibliometry, science mapping is a method of visualization of a field of science. This visualization can be done with the help of VosViewer software and done by creating a map of the relationship between one topic and another topic (Md Khudzari et al., 2018), one keyword with another keyword, and so on (Sweileh, Al- Jabi, Zyoud, & Sawalha, 2018). development map on the topic of Merdeka Belajar conducted with the help of the VosViewer application (Kasilingam, Keepers, & Wuest, 2021), (Tanudjaja & Kow, 2018). Subject of this research taken in this study are all journals indexed by google scholars, both accredited and non-accredited, that have been published and indexed by Google Sholar.  INTRODUCTION Then the obtained document is stored in the form of RIS for further analysis. The last stage is to analyze the data of 232 selected documents to see the relationship between one topic and another using RESULT AND DISCUSSIONS Year of Publication of Research Development “Merdeka Belajar” Search results through Google Scholar show that the development of research on “Merdeka Belajar” towards Open access has existed since 2019. In that year, there was 1 document that could be accessed via the internet easily, then from year to year increased rapidly to the position of 62 documents in 2020, as many as 137 documents in 2021, and as many as 32 documents in 2022 as of February 15, 2022. For more details, the development map of Merdeka Belajar research from year to year can be seen in the table below: The data analysis in this study was entirely using the VosViewer application program. Where Vosviewer will analyze document files that have been changed in the form of RIS to bring up mapping data based on authors and topics. A RIS file is a bibliographic citation file stored in a format developed by Research Information Systems. This file contains a series of lines bounded by a two-character code and corresponding values (Lima & Partidario, 2020), (Batubara, Nur, Lubis, & Arianto, 2021). RIS files provide information such as title, author, publication date, keyword, publisher, issue number, and start and end pages (Nalau & Verrall, 2021). For this research, the RIS file taken and which will be analyzed is a RIS file related to “Merdeka Belajar” either in the research title, Abstract or Keywords. Table 1. Year of publication “merdeka belajar” Table 1. Year of publication “merdeka belajar” No. Year Document 1 2 3 4 2019 2020 2021 2022 1 62 137 32 Furthermore, the RIS data will be analyzed using VosViewer (Anand & Gupta, 2020b), (Dolhey, 2019). VosViewer will analyze the data and categorize RIS data taken from hundreds of these journals to be categorized into several research clusters (Kasilingam et al., 2021), (Khalil & Gotway Crawford, 2015) about Merdeka Belajar. The results that will be raised are the form of visualization of hundreds of articles based on clusters (Kasilingam et al., 2021), (Dolhey, 2019) relationships or the relationship of Merdeka Belajar keywords with others that appear in research that has been done. Terms or research topics that are rarely studied are still From the table, it can be seen that the map of research development every year continues to increase.  METHODS This research is qualitative research. The method carried out in this research is the study of bibliometric literature on the research Jurnal Pendidikan Progresif, Vol. 12, No. 2, pp. 477-486, August 2022 480 VOsViewer. The resulting data is then used as a result of research and reports. related to the topic of “Merdeka Belajar”, either directly or indirectly (Ghobakhloo, Fathi, Iranmanesh, Maroufkhani, & Morales, 2021). As well as being the next relevant research gap (Redeker, Kessler, & Kipper, 2019). This research is a bibliometric study so that the instruments used in this study are documents obtained through observations from data tracing about articles that have published about Merdeka Belajar. Applications used such as Publish or Perish and VosViewer are the main applications operated to find out and see the mapping map of this research. RESULT AND DISCUSSIONS The selection of the name of the journal in 2020 and 2021 is based on the top order of the number of link authors. Based on the table above, it can also be seen that almost 90 per cent of journal names that appear are journals that intersect with education. This is because the word Merdeka Belajar is part of the education minister’s program. journal names in 2021. The selection of the name of the journal in 2020 and 2021 is based on the top order of the number of link authors. Based on the table above, it can also be seen that almost 90 per cent of journal names that appear are journals that intersect with education. This is because the word Merdeka Belajar is part of the education minister’s program. Map of Co-Author “Merdeka Belajar” RESULT AND DISCUSSIONS This indicates that the topic of Merdeka Belajar is very important to study and has many benefits and relationships with other disciplines. Name of Journal Containing “Merdeka Belajar” On Mapping Map of Research The name of the journal that has published about “Merdeka Belajar” both as a title, abstract, keyword, and content can be seen in the table below: Batubara et al., Bibliometric Mapping on the Research “Merdeka Belajar”... 481 Table 2. Name of journal containing “merdeka belajar” No. Year Names of Journal 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 2019 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2021 2021 2021 2021 2021 2021 2021 2021 2021 2021 2021 2021 2021 2021 2021 2021 2021 2021 2022 2022 2022 2022 2022 Jurnal Manajemen Pendidikan Islam Tarbawi Jurnal Ilmu Pendidikan Jurnal Bahasa, Sastra dan Pengajaran Jurnal Tawadhu Jurnal Pendidikan dan Pembelajaran Jurnal Kependidikan Jurnal Hospitality Jurnal Riset Pedagogik Jurnal Ilmiah Wahana Pendidikan Jurnal Basic Edu Jurnal Eksakta Pendidikan Jurnal Optimisme Jurnal Sibermas Jurnal Pustaka Ilmiah Jurnal Sosial Ekonomi dan Politik Jurnal Pendidikan Matematika Uniba Jurnal Syntax Transformation Sasambo Jurnal Abdimas Jurnal Pedagogy Jurnal Kreatif Online Jurnal Inovasi Riset Akademik Jurnal Kumparan Fisika Jurnal Biogenerasi Jurnal Gentala Pendidikan Dasar Jurnal Ilmiah Sekolah Dasar Jurnal Pemberdayaan Masyarakat Madani Jurnal Ilmiah Fenomena Jurnal Dedikasi Pendidikan Jurnal Edumatic Jurnal Geocivic Jurnal Pendidikan Kimia Jurnal Lensa Pendas Pionir: Jurnal Pendidikan Jurnal Review Pendidikan dan Pengajaran Jurnal ESTUPRO Jurnal Pendidikan Guru Jurnal Pengabdian Masyarakat IPTEK Jurnal Education Development Jurnal Teknologi Informasi dan Komunikasi Jurnal Abdi Masyarakat Indonesia JAMSI The name of the journal above is just a few of the names of journals that the authors wrote from 232 journals that have published the word Merdeka Belajar. Due to time and space constraints, the author only wrote down all journal names that appeared in 2019 and 2022 as of the February 15, 2022 access date. While in 2020, the author only wrote 16 journal names and 18 Jurnal Pendidikan Progresif, Vol. 12, No. 2, pp. 477-486, August 2022 482 journal names in 2021. Map of Co-Author “Merdeka Belajar” From figure 2 above, you can see that several authors have links between each other. While not connected to each other means never writing a journal together in one document. From figure 2 above, you can see that several authors have links between each other. Figure 3. Topics occurance Figure 3. Topics occurance have been analyzed. Topic reduction is made if the topic only appears under seven times. Topics that only appear under seven times will automatically be eliminated. The dominant topic appeared, namely “Merdeka Belajar Kampus Merdeka” 58 times followed by “MBKM” 49 times and “Covid” 29 times. The results of the visualization of the topic can be seen in figure 4: Map of Co-Author “Merdeka Belajar” After the analysis, there were 29 authors who wrote a lot about Merdeka Belajar from hundreds of journals with criteria of at least two journals documents that had been written and published. The name of the authors and the total link is shown in Figure 1 below: Figure 1. Name of authors and total links From the figure above, it can be concluded that the author, on behalf of Nona, RV., Albahri, FP., and Salam, A. are the author who discusses the most about Merdeka Belajar, which has been searched from Publish or Perish data through Google Scholar search documents. While those with the most total links are Aiman, U., Kurniawan, Na., Latifa, R., Rofieq, A., Saputra, R., and Susetyarini, E. The visualization view of the author and co-author of the document that has been analyzed using VosViewer is shown in figure 2 below: Figure 1. Name of authors and total links Figure 1. Name of authors and total links From the figure above, it can be concluded that the author, on behalf of Nona, RV., Albahri, FP., and Salam, A. are the author who discusses the most about Merdeka Belajar, which has been searched from Publish or Perish data through Google Scholar search documents. While those From the figure above, it can be concluded that the author, on behalf of Nona, RV., Albahri, FP., and Salam, A. are the author who discusses the most about Merdeka Belajar, which has been searched from Publish or Perish data through Google Scholar search documents. While those with the most total links are Aiman, U., Kurniawan, Na., Latifa, R., Rofieq, A., Saputra, R., and Susetyarini, E. The visualization view of the author and co-author of the document that has been analyzed using VosViewer is shown in figure 2 below: with the most total links are Aiman, U., Kurniawan, Na., Latifa, R., Rofieq, A., Saputra, R., and Susetyarini, E. The visualization view of the author and co-author of the document that has been analyzed using VosViewer is shown in figure 2 below: Figure 2. Visualization author and co-author Figure 2. Visualization author and co-author Batubara et al., Bibliometric Mapping on the Research “Merdeka Belajar”... 483 While not connected to each other means never writing a journal together in one document. While not connected to each other means never writing a journal together in one document. Research Development Topic and Visualization The research topics that most emerged from the 232 journals that have been analyzed are shown in Figure 3 below: From figure 3 above, it can be seen that there are 15 topics that most often arise from hundreds of topics. There are 232 journals that Figure 4. Network visualization between topics Figure 4. Network visualization between topics Jurnal Pendidikan Progresif, Vol. 12, No. 2, pp. 477-486, August 2022 Jurnal Pendidikan Progresif, Vol. 12, No. 2, pp. 477-486, August 2022 484 topics is getting yellower, then it will be tighter and greener then the topic is less and less researched. From figure 4 above, there are 6 clusters of topics that appear in the form of six colours. Cluster 1 was in red consists of 9 topics, including Era Merdeka Belajar, Guru, Covid Pandemic, and so on. Cluster 2 in green consists of 7 topics, including Pandemic, Article, and activity. Cluster 3 blue consists of 7 topics, including medium, students, term. REFERENCES Anand, S., & Gupta, S. (2020b). Provisioning ecosystem services: Multitier bibliometric analysis and visualisation. Environmental and Sustainability Indicators, 8(August), 1–12. Cluster 4 was in yellow consists of 7 topics, including Application, Technology, and New Normal. Cluster 5 was in purple consists of 4 topics including MBKM, Merdeka Belajar Kampus Merdeka and the last cluster was cluster 6 with dark blue colour consisting of 2 topics; one of them is hard skills. Azad, A. K., & Parvin, S. (2022). Bibliometric analysis of photovoltaic thermal (PV/T) system: From citation mapping to research agenda. Energy Reports, 8, 2699–2711. Batubara, I. H., Nur, K., Lubis, A. T., & Arianto, N. (2021). The Effectiveness of Learning Using Social Media during the Covid 19 Pandemic in Higher Education. Budapest International Research and Critics Institute (BIRCI-Journal): Humanities and Social Sciences, 4(2), 2177–2183. https://doi.org/10.33258/birci.v4i2.1908  CONCLUSIONS Based on the results and discussions, it can be concluded that the development of research on Merdeka Belajar has existed since 2019. Since then, this research topic has grown rapidly to 232 documents in 2022 as of February 15 2022. The most discussed article on the topic is an article with a type of Research article by using a search “Google Scholar” with the keyword “Merdeka Belajar”. Reyna Virgina Nona, Fauzan Putraga Albahri, and Abdus Salam are the authors who wrote the most articles about “Merdeka Belajar” as of February 15, 2022, which were searched through Publish or Perish with the Google Scholar search menu. While those who have the total links are Aiman, U., Kurniawan, Na., Latifa, R., Rofieq, A., Saputra, R., and Susetyarini, E. All authors have links between each other, and overall it is known that the total clusters are 210 with 319 links and 325 link strengths. Network visualization shows that the relationship between research topics is shown with lines between descriptors in each field. The more lines between descriptors, the closer the relationship between documents. While density visualization shows that the relationship between Burki, M. A. K., Burki, U., & Najam, U. (2021). 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https://openalex.org/W2872999080	https://escholarship.org/content/qt4gk9c5cv/qt4gk9c5cv.pdf?t=pls59n	English	null	Structural Definition of a Unique Neutralization Epitope on the Receptor-Binding Domain of MERS-CoV Spike Glycoprotein	Cell reports	2,018	cc-by	11,732	Permalink https://escholarship.org/uc/item/4gk9c5cv Journal Cell Reports, 24(2) ISSN 2639-1856 Authors Zhang, Senyan Zhou, Panpan Wang, Pengfei et al. Publication Date 2018-07-01 DOI 10.1016/j.celrep.2018.06.041 Peer reviewed Title Structural Definition of a Unique Neutralization Epitope on the Receptor-Binding Domain of MERS-CoV Spike Glycoprotein Powered by the California Digital Library University of California eScholarship.org Article Structural Deﬁnition of a Unique Neutralization Epitope on the Receptor-Binding Domain of MERS- CoV Spike Glycoprotein Structural Deﬁnition of a Unique Neutralization Epitope on the Receptor-Binding Domain of MERS- CoV Spike Glycoprotein Structural Deﬁnition of a Unique Neutralization Epitope on the Receptor-Binding Domain of MERS- CoV Spike Glycoprotein Authors Senyan Zhang, Panpan Zhou, Pengfei Wang, ..., Shuying Wang, Xinquan Wang, Linqi Zhang Authors Senyan Zhang, Panpan Zhou, Pengfei Wang, ..., Shuying Wang, Xinquan Wang, Linqi Zhang In Brief Zhang et al. report the structural and functional analysis of the potent MERS- CoV neutralizing antibody MERS-4. MERS-4 recognizes a unique epitope and indirectly disrupts interaction between the receptor binding domain and the receptor DPP4. This mechanism provides a valuable addition for the combined use of antibodies against MERS-CoV infection. Data and Software Availability 5ZXV 5YY5 SUMMARY exact mechanism of its introduction into, and disappearance from, the human population still remains a mystery (Graham et al., 2013). By contrast, the MERS-CoV epidemic has persisted for more than 5 years with no signs of abating (http://www.who. int/csr/don/10-november-2017-mers-oman/en/). Apart from the sudden initial outbreak in Saudi Arabia, MERS-CoV has spread to other countries outside the Arabian Peninsula carried by in- fected travelers (Bermingham et al., 2012), leading to an outbreak in South Korea in 2015 (Choi, 2015). These are continuous reports of human MERS-CoV infection in affected regions, largely due to contact with dromedary camels, which are believed to be a major reservoir host for MERS-CoV and the immediate source of human infection (Haagmans et al., 2014; Hemida et al., 2014). Compared with SARS-CoV with a fatality rate of approximately 10% (Peiris etal.,2004),MERS-CoVappearstobemoredeadly,withitsfatality rate reaching as high as 35% (de Wit et al., 2016). All of these facts indicate that MERS-CoV will remain as a severe and long-time threat to global health and highlight the urgent need for effective prophylactic and therapeutic measures. The major mechanism of antibody-mediated neutral- ization of the Middle East respiratory syndrome coro- navirus (MERS-CoV) involves competition with the cellular receptor dipeptidyl peptidase 4 (DPP4) for binding to the receptor-binding domain (RBD) of the spike (S) glycoprotein. Here, we report a unique epitope and unusual neutralizing mechanism of the isolated human antibody MERS-4. Structurally, MERS-4 approached the RBD from the outside of the RBD-DPP4 binding interface. Such binding re- sulted in the folding of the b5-b6 loop toward a shallow groove on the RBD interface critical for ac- commodating DPP4. The key residues for binding are identiﬁed through site-directed mutagenesis. Structural modeling revealed that MERS-4 binds to RBD only in the ‘‘up’’ position in the S trimer. Further- more, MERS-4 demonstrated synergy with several reported antibodies. These results indicate that MERS-4 neutralizes MERS-CoV by indirect rather than direct competition with DPP4. This mechanism provides a valuable addition for the combined use of antibodies against MERS-CoV infection. Scientiﬁc progress achieved since the SARS-CoV epidemic has greatly increased our capacity to respond to MERS-CoV. Like that of SARS-CoV, the spike (S) glycoprotein of MERS- CoV plays a critical role in mediating viral entry and in inducing a protective antibody response in infected individuals (Raj et al., 2013; Zumla et al., 2016). Highlights Highlights d MERS-4 binds RBD from the outside of the RBD–DPP4 binding interface d MERS-4 favors binding to the RBD in the ‘‘up’’ position in the S trimer d MERS-4 neutralizes MERS-CoV by indirect rather than direct competition with DPP4 d MERS-4 is a valuable addition to the combined use of MERS- CoV antibodies Cell Reports Article Structural Deﬁnition of a Unique Neutralization Epitope on the Receptor-Binding Domain of MERS-CoV Spike Glycoprotein Senyan Zhang,1,6 Panpan Zhou,2,6 Pengfei Wang,1,6 Yangyang Li,2 Liwei Jiang,2 Wenxu Jia,2 Han Wang,2 Angela Fan,2 Dongli Wang,1 Xuanling Shi,2 Xianyang Fang,1 Michal Hammel,3 Shuying Wang,4 Xinquan Wang,1,5,7,* and Linqi Zhang2,* 1The Ministry of Education Key Laboratory of Protein Science, Beijing Advanced Innovation Center for Structural Biology, Collaborative Innovation Center for Biotherapy, School of Life Sciences, Tsinghua University, Beijing 100084, China 2Comprehensive AIDS Research Center, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing 100084, China 3Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA 4Department of Microbiology and Immunology, National Cheng Kung University Medical College, Tainan 701, Taiwan 5Collaborative Innovation Center for Biotherapy, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu, China 6These authors contributed equally 7Lead Contact *Correspondence: xinquanwang@mail.tsinghua.edu.cn (X.W.), zhanglinqi@mail.tsinghua.edu.cn (L.Z.) https://doi.org/10.1016/j.celrep.2018.06.041 SUMMARY The isolation of potent neutral- izing monoclonal antibodies has been reported shortly after the identiﬁcation of MERS-CoV. This was achieved by using various technological platforms such as phage or yeast display of anti- body library (Jiang et al., 2014; Tang et al., 2014; Ying et al., 2014), immunization of experimental animals (Chen et al., 2017a; Du et al., 2014; Li et al., 2015; Pascal et al., 2015; Wang et al., 2015a), and direct isolation from human survivors of MERS-CoV infection (Chen et al., 2017b; Corti et al., 2015; Wang et al., 2018). Currently, close to 20 different neutralizing monoclonal antibodies have been characterized in cell culture and experimental animal models. A large majority target the re- ceptor-binding domain (RBD) of the MERS-CoV S glycoprotein Cell Reports 24, 441–452, July 10, 2018 ª 2018 The Authors. 441 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Cell Reports 24, 441–452, July 10, 2018 ª 2018 The Authors. 441 der the CC BY license (http://creativecommons.org/licenses/by/4.0/). INTRODUCTION We and others have demonstrated that MERS-CoV RBD possesses core and recep- tor-binding subdomains (Chen et al., 2013; Lu et al., 2013; Wang et al., 2013, 2015a). A large majority of neutralizing antibodies against MERS-CoV, including MERS-27, 4C2, D12, JC57-14, m336, MCA1, and CDC-C2, have been found to target the recep- tor-binding subdomain and overlap with the DPP4 binding sur- face (Chen et al., 2017b; Li et al., 2015; Wang et al., 2015a, 2018; Ying et al., 2015; Yu et al., 2015). Therefore, these anti- bodies share at least one similar aspect of neutralization by directly competing with the cellular receptor DPP4 for binding to RBD. Here, we report the structural and functional analysis of our previously isolated potent neutralizing antibody MERS-4 and its variant MERS-4V2, which reveals their unique epitope speci- ﬁcity and unusual mechanism of action. In contrast to all the re- ported RBD-targeting neutralizing antibodies that compete with DPP4 for binding to the RBD, MERS-4 and MERS-4V2 ap- proached the RBD from the outside of the RBD-DPP4 binding interface. Site-directed mutagenesis identiﬁed several key resi- dues critical for binding and the neutralizing activity of MERS-4 and MERS-4V2. Structural comparisons of the RBD in unbound, DPP4-bound, and antibody-bound states revealed signiﬁcant conformational changes in the RBD when bound to MERS-4 or MERS-4V2. To the best of our knowledge, this is the ﬁrst report demonstrating that a MERS-CoV neutralizing antibody can indi- rectly interfere with DPP4 binding through conformational changes. Its unique epitope speciﬁcity and unusual mechanism of action enable MERS-4 to synergize with other antibodies, providing a valuable addition for the combined use of antibodies against MERS-CoV infection. As shown in Figure 1A, MERS-4 Fab and MERS-4V2 scFv shared the same mode of binding to the RBD. A superimposition of MERS-4 Fab with MERS-4V2 scFv revealed that their respec- tive recognition was largely mediated through interactions with the b5-b6, b6-b7, and b7-b8 loops in the receptor-binding sub- domain of the RBD (Figure 1B). Negligible structural differences were found between the two, with an overall root-mean-square deviation (RMSD) of 0.6 A˚ for 204 aligned Ca atoms. At the bind- ing interface, the b7-b8 loop of RBD inserted into the cavity be- tween the antibody heavy and light chains, forming interactions with all CDR loops of the antibody except for HCDR3 (Figures 1C and 1D). INTRODUCTION The 2012 emergence of the Middle East respiratory syndrome co- ronavirus(MERS-CoV)inSaudiArabiawasthesecondmajorintro- duction of a highly pathogenic coronavirus into the human popula- tion since the outbreak of SARS-CoV in China in 2003 (Assiri et al., 2013; Zakietal.,2012).The globalspreadofSARS-CoVresultedin more than 8,000 infections and nearly 800 deaths worldwide (Pei- ris et al., 2004). Fortunately, the SARS-CoV epidemic rapidly died off because of conventional public health measures, even if the and interfere with the binding of the cellular receptor dipeptidyl peptidase 4 (DPP4). We previously reported the isolation of the two neutralizing antibodies MERS-4 and MERS-27 via screening of a yeast-displayed library of human scFv (single-chain variable domain fragment) using the MERS-CoV RBD as bait (Jiang et al., 2014). Both antibodies demonstrated potent neutralizing activity against live and pseudotyped MERS-CoV, and a high level of synergy when used together. selected mutants, only the variant MERS-4V2 demonstrated a substantial improvement of productivity (>10-fold) while main- taining potency as strong as that of the original MERS-4 in neutralizing pseudotyped virus and binding the S trimer (Fig- ure S1A). Sequence analysis showed that the original CDR3 res- idues Ala-Gly-Asn-Asp (AGND) of MERS-4 were replaced by Thr-Asn-Thr-Tyr (TNTY) in MERS-4V2 (Figure S1B). y ( ) ( g ) To study the overall structure of antibodies bound to the RBD, we expressed MERS-4 and MERS-4V2 in HEK293F cells and obtained the corresponding Fab fragments. The antibody and RBD complexes were formed by mixing the MERS-4 or MERS-4V2 Fab with the RBD. However, despite our repeated efforts in optimizing and screening more than 200 crystals, we were only able to obtain X-ray diffraction data of RBD in complex with MERS-4 Fab at 4.5 A˚ and with MERS-4V2 Fab at 7 A˚ . Nevertheless, we solved the structure of the MERS-4 Fab/RBD complex (PDB: 5ZXV) and reﬁned it to Rwork and Rfree factors of 30.2% and 34.4%, respectively (Figure S2A; Table S1). We went further to construct an scFv version of MERS-4V2 and replaced the MERS-4V2 Fab during complex formation with RBD. The structure of the MERS-4V2 scFv/ RBD complex (PDB: 5YY5) was successfully solved to a reso- lution of 2.8 A˚ with Rwork and Rfree factors of 24.7% and 27.7%, respectively (Figure S2B; Table S1). Structural determination of antibodies and their epitope spec- iﬁcities provides a critical foundation for a better understanding of their mechanisms of neutralization. INTRODUCTION In particular, the short b5-b6 loop interacted with the HCDR1 and HCDR2 loops of the antibody heavy chain, and the long b6-b7 loop predominately interacted with the LCDR2 loop of the antibody light chain (Figures 1C and 1D). RESULTS We conducted a detailed analysis of structural features at the binding interface derived from the MERS-4V2 scFv/RBD com- plex (Figures 2 and S2C). At the binding interface, 16 MERS- 4V2 scFV residues from all 6 CDR except for HCRD3 formed contacts with 15 residues from the b5-b6, b6-b7, and b7-b8 loops of the receptor-binding subdomain of the RBD (Figure 2A; Table S2). Speciﬁcally, the RBD residues Leu507 and Ser508 from the b5-b6 loop interacted with Ser30, Asn31, and Tyr53 from the heavy chain (Figure 2B; Table S2). The RBD residues Gln516, Asn519, Asn521, Gln522, Tyr523, and Pro525 from the b6-b7 loop interacted with Tyr50, Trp51, Asp53, Gln54, Arg55, and Asp61 from the antibody light chain (Figures 2C and 2D; Table S2). Furthermore, the RBD residues Lys543, and Leu545 to Gly550 from the b7-b8 loop interacted with Ala33, Tyr35, Tyr53, and Tyr59 from the heavy chain, as well as Asn32, Tyr33, Tyr35, Trp51, and Trp92 from the light chain (Figures 2B Figure 1. Crystal Structures of MERS-CoV RBD in Complex with MERS-4 and Its Variant MERS-4V2 Figure 1. Crystal Structures of MERS-CoV RBD in Complex with MERS-4 and Its Variant MERS-4V2 (A) Overall structures of the RBD/MERS-4 Fab and the RBD/MERS-4V2 scFv (right) complexes. The RBD core subdomain was in blue, while the recep- tor-binding subdomain was in green, the MERS-4 light chain in magenta, the MERS-4 heavy chain in cyan, the MERS-4V2 VL in orange, and the MERS- 4V2 VH in red. (B) Structural superimposition of the RBD/MERS-4 and the RBD/MERS-4V2 complexes and schematic illustration of the MERS-CoV RBD (right). (B) Structural superimposition of the RBD/MERS-4 and the RBD/MERS-4V2 complexes and schematic illustration of the MERS-CoV RBD (right). (C and D) MERS-4 (C) and MERS-4V2 (D) interact with the b5-b6, b6-b7, and b7-b8 loops of the RBD. See also Figure S1 and Table S1. Structural Alterations in the RBD Bound to MERS-4 and MERS-4V2 We have previously shown that MERS-4 interfered with the interaction between soluble RBD and the cellular receptor DPP4 expressed on the surface of Huh7 cells (Jiang et al., 2014). Surface plasmon resonance (SPR) analysis showed that the binding of soluble RBD to chip-coupled DPP4 was reduced in the presence of increasing concentrations of MERS-4 in a dose-dependent manner (Fig- ure S3). However, structural superim- position demonstrated that the epitope targeted by MERS-4 and MERS-4V2 is located outside the DPP4 binding surface on the RBD (Figure 3). This apparent disconnect prompted us to compare all of the available RBD structures in the unbound (PDB: 4ZPW, 4KQZ, and 4L3N) (Chen et al., 2013; Lu et al., 2013; Wang et al., 2015a), DPP4-bound (PDB: 4L72 and 4KR0) (Lu et al., 2013; Wang et al., 2013), MERS-4-bound, and MERS-4V2- bound states (Figure 3A). While the overall structure of RBD remained relatively stable in all the different states, the MERS-4- or MERS-4V2-bound RBD was found to have a conformational change of the b5-b6 loop (Leu506 to Glu513) (Figure 3A). This particular change involved the folding of the b5-b6 loop toward a shallow groove on the RBD interface critical for accommodating a short helix of DPP4 (Figure 3A). The maximum distance change occurred at Asp510, whose Ca atom moved more than 3 A˚ into the groove (Figure 3B). Our previous study revealed that the b5-b6 loop participates in the formation of a shallow groove to accommo- date the docking of a short helix of DPP4 in patch 2 of the bind- ing interface (Figure 3C) (Wang et al., 2013). Residues Leu506, Asp510, and Glu513 from this loop were found to be involved in forming the core hydrophobic and peripheral hydrophilic inter- actions in patch 2 (Figure 3C), and mutations of these residues and 2E; Table S2). Among all interactions at the interface, one example of hydrophobic interactions involved Leu548 (RBD b7-b8 loop), Tyr35 (heavy chain), Tyr35 (light chain), and Trp92 (light chain) (Figures 2B and 2E; Table S2). Examples of hydrogen bonds included Ser508 (RBD b5-b6 loop) to Tyr53 (heavy chain), Asn519 (RBD b6-b7 loop) to Tyr50 (light chain), Asn521 (RBD b6-b7 loop) to Asp61 (light chain), and Gln522 (RBD b6-b7 loop) to Arg55 (light chain) (Figures 2B–2D; Table S2). As indi- cated above, the HCDR3 appeared not to be involved in the RBD interaction within a distance cutoff of 4.0 A˚ . Overall Structure of MERS-4 and Its MERS-4V2 Variant Bound to the RBD We previously reported that the human monoclonal antibody MERS-4 targets the RBD of the spike glycoprotein and exhibits strong neutralization activity against live and pseudotyped MERS-CoV infection (Jiang et al., 2014). However, the yield of MERS-4 from the transfected HEK293F cells was rather low (<1 mg/L), hampering our efforts toward detailed structural anal- ysis. To identify a variant with improved productivity, we gener- ated a library of mutant MERS-4 comprising random replace- ments in the 5-residue-long CDR3 region, displayed on the surface of yeast, and selected for binding to the RBD through ﬂuorescence-activated cell sorting (FACS)-based sorting as described previously (Jiang et al., 2014). Among a total of 17 442 Cell Reports 24, 441–452, July 10, 2018 and 2E; Table S2). Among all interactions at the interface, one example of hydrophobic interactions involved Leu548 (RBD b7-b8 loop), Tyr35 (heavy chain), Tyr35 (light chain), and Trp92 (light chain) (Figures 2B and 2E; Table S2). Examples of hydrogen bonds included Ser508 (RBD b5-b6 loop) to Tyr53 (heavy chain), Asn519 (RBD b6-b7 loop) to Tyr50 (light chain), Asn521 (RBD b6-b7 loop) to Asp61 (light chain), and Gln522 (RBD b6-b7 loop) to Arg55 (light chain) (Figures 2B–2D; Table S2). As indi- cated above, the HCDR3 appeared not to be involved in the RBD interaction within a distance cutoff of 4.0 A˚ . This may explain the unchanged neutralizing and binding activities of MERS-4V2 compared with the parental MERS-4 despite the found to have a c (Leu506 to Glu513) the folding of the b RBD interface crit DPP4 (Figure 3A). T Asp510, whose Ca (Figure 3B). Our pr participates in the f date the docking of ing interface (Figure Asp510, and Glu513 forming the core hy Conﬁrmation of the Unique Neutralizing Epitope q g p p To further conﬁrm the unique epitope in the context of binding and neutralization, we mutated a panel of the RBD residues to alanine, including Leu507, Ser508 in the b5-b6 loop, as well as Leu545, Ser546, Pro547, Leu548, and Glu549 in the b7-b8 loop. All of the mutants except for Leu548 could be expressed and puriﬁed in insect cells as the same way as the wild-type RBD. The SPR analysis showed that mutations at Leu507, Leu545, Ser546, Pro547, and Glu549 in the RBD signiﬁcantly decreased the binding afﬁnity to MERS-4 (Figures 5A and S4A) and MERS-4V2 (Figures 5A and S4B) by more than 10- fold. The mutation at Ser508 decreased the binding afﬁnity to both antibodies by less than 3-fold (Figures 5A and S4). We next measured the neutralization activity of MERS-4 against pseudotyped viruses bearing wild-type or mutant S glycopro- tein (Leu507Ala, Leu545Ala, Ser546Ala, and Pro547Ala) in the cell entry assay. Consistent with the binding changes, pseudo- typed viruses bearing these mutations became less sensitive to MERS-4 neutralization (Figure 5B). Those bearing Ser508Ala, Leu548Ala, or Glu549Ala mutations failed to produce detectable amounts of infectious viral particles and therefore were excluded from our experiments. Lastly, we went further to study whether the binding of MERS-4 to the mutant S glycoprotein was also reduced when the protein was expressed on the sur- face of cells. Speciﬁcally, transfected HEK293T cells expressing either the wild-type or mutant S glycoprotein were harvested, stained with MERS-4 or antibody 5F9 speciﬁc for the NTD of S glycoprotein as a control (Chen et al., 2017a), and analyzed by FACS. As shown in Figure 5C, MERS-4 demonstrated variable levels of reduction in its binding to the mutant S glyco- protein, while the control antibody 5F9 remained virtually unchanged. Furthermore, the L545A mutation completely abolished the binding activity of MERS-4, which was consistent with the results obtained using the pseudotyped viruses. These E D L545 S546 P547 L548 E549 K543 Y33 W51 Y35 N32 W92 E β7-β8 loop D Q522 R55 Y523 P525 Q54 D53 W51 β6-β7 loop Figure 2. The Binding Interface between MERS-CoV RBD and MERS-4V2 (A) Overall view of the interface showing the MERS-4V2 epitope consisting of residues from the b5-b6, b6-b7, and b7-b8 loops of the RBD. (B) Interactions between the RBD residues from the b5-b6 loop, the b7-b8 loop, and MERS-4V2 heavy chain. Structural Alterations in the RBD Bound to MERS-4 and MERS-4V2 This may explain the unchanged neutralizing and binding activities of MERS-4V2 compared with the parental MERS-4 despite the four-residue replacement in the HCDR3 (Figures S1A and S1B). Cell Reports 24, 441–452, July 10, 2018 443 is that all three RBDs in the ‘‘down’’ position keep the S trimer in an inactive, non-receptor-binding state, whereas at least one RBD in the ‘‘up’’ position is required for the S trimer to be in an activated, receptor-binding state (Pallesen et al., 2017; Yuan et al., 2017). To study which state MERS-4 could bind and access its epitope, we superimposed MERS-4 Fab/RBD crystal struc- tures onto the MERS-CoV S trimer structure in the two major conformational states. At the same time, we also analyzed the binding of receptor DPP4 and MERS-27 to the S trimer glycopro- tein. As shown in Figure 4, severe steric clashes would be ex- pected between MERS-4 Fab and the N-terminal domain (NTD) of the neighboring S monomer when all three RBD are in the ‘‘down’’ position, suggesting that the epitope of MERS-4 is covered and inaccessible in the inactivated state. By contrast, when there was one RBD in the ‘‘up’’ position, the epitope of MERS-4 became readily exposed and accessible. Similarly, the receptor DPP4 also favored RBD in the ‘‘up’’ over the one in the ‘‘down’’ position (Pallesen et al., 2017; Yuan et al., 2017). Howev- er, the MERS-27 epitope was readily accessible regardless of the state of the RBD. Taken together, these results indicate that MERS-4 binds to the RBD in the ‘‘up’’ position when the virus be- comes partially activated. MERS-27, however, binds to the RBD irrespective of the conformational states within the S trimer. β5-β6 loop β7-β8 loop β6-β7 loop S508 L507 G550 P547 S546 L548 E549 L545 K543 Q516 P525 N519 Q522 N521 Y523 A β5-β6 loop β7-β8 loop β6-β7 loop S508 L507 G550 P547 S546 L548 E549 L545 K543 Q516 P525 N519 Q522 N521 Y523 A C D B L507 S508 Y53 N31 S30 Y59 A33 Y35 P547 L548 G550 Q516 N519 N521 Y50 W51 R55 D61 Q522 R55 Y523 P525 Q54 D53 W51 L545 S546 P547 L548 E549 K543 Y33 W51 Y35 N32 W92 E β5-β6 loop β7-β8 loop β6-β7 loop β6-β7 loop β7-β8 loop Figure 2. Conﬁrmation of the Unique Neutralizing Epitope (C–E) Interactions between the RBD residues from the b6-b7 loop (C and D), b7-b8 loop (E), and the corresponding residues of MERS-4V2 light chain. See also Figure S2 and Table S2. (C–E) Interactions between the RBD residues from the b6-b7 loop (C and D), b7-b8 loop (E), and the corresponding residues of MERS-4V2 light chain. See also Figure S2 and Table S2. decreased the binding afﬁnity between RBD and DPP4 signiﬁ- cantly. The conformational change identiﬁed here is therefore expected to bring steric clashes between Asp510 from RBD and Ser292 and Arg317 from DPP4 (Figure 3D), thereby pre- venting the docking of the short helix of DPP4 into the shallow groove of RBD. Structural Alterations in the RBD Bound to MERS-4 and MERS-4V2 The Binding Interface between MERS-CoV RBD and MERS-4V2 (A) Overall view of the interface showing the MERS-4V2 epitope consisting of residues from the b5-b6, b6-b7, and b7-b8 loops of the RBD. (B) Interactions between the RBD residues from the b5-b6 loop, the b7-b8 loop, and MERS-4V2 heavy chain. (C–E) Interactions between the RBD residues from the b6-b7 loop (C and D), b7-b8 loop (E), and the corresponding residues of MERS-4V2 light chain. See also Figure S2 and Table S2. B L507 S508 Y53 N31 S30 Y59 A33 Y35 P547 L548 G550 β5-β6 loop β7-β8 loop C C Q516 N519 N521 Y50 W51 R55 D61 β6-β7 loop B Binding of MERS-4 to the Different Conformational States of RBD in the Context of the S Trimer (C) Patch 2 of the RBD/DPP4 binding interface in which residues Leu506, Asp510, and Glu513 from the RBD b5-b6 loop are critical for DPP4 binding. results suggest that reduced binding afﬁnity of MERS-4 to the mutant S glycoprotein is one of the major contributors to viral resistance. Collectively, we conﬁrmed the unique epitope tar- geted by MERS-4 and MERS-4V2 observed in the crystal struc- tures and highlighted the unusual mechanism of neutralization by these antibodies. (Pelikan et a (31% monom MERS-4/MER improved the ure 6), reveal sient behavio exclusion ch (D) The steric clashes in the red circle between the b5-b6 loop of the RBD and the DPP4 receptor upon antibody binding. See also Figure S3. See also Figure S3. Fab complex (Figure S6A), indicating that MERS-4 and MERS-27 could bind to the RBD at the same time. To conﬁrm this, we used small-angle X-ray scat- tering (SAXS) study on the puriﬁed ternary complex in solution. We initially built a monomeric model of the RBD/ MERS-4/MERS-27 ternary complex, which was generated by superimposing the RBD/MERS-4 and RBD/MERS-27 crystal structures. However, the mono- meric model ﬁts poorly to the SAXS data with a c value of 7.0 (Figure 6). To investigate the assembly of the complex, we built a dimeric model of the ternary complex using the interface observed in the RBD/MERS-27 crystal structure. To understand the dynamic assembly, MultiFoXS was applied to deﬁne the population of the models in solution (Pelikan et al., 2009). SAXS ﬁtting with multi-state models (31% monomer model, 18% dimer model of the RBD/ MERS-4/MERS-27, and 51% RBD/MERS-27) signiﬁcantly improved the goodness of the ﬁt with a c value of 2.0 (Fig- ure 6), revealing the transient complexation of Fabs. The tran- sient behavior was also observed from the SEC-SAXS (size exclusion chromatography in line with SAXS) (Figure S6). The broad distribution of radius of gyration (Rg) values across the SEC-SAXS peak, ranging from 47 to 37 A˚ (Figure S6B), suggests a multi-state mixture of the complex, which was further conﬁrmed by the good ﬁts obtained for various sections of the SEC-SAXS peak using ensemble models (Figures S6C and S6D). These results collectively showed that MERS-4 and MERS-27 are capable of binding to RBD at different epitopes to form a ternary complex. results suggest that reduced binding afﬁnity of MERS-4 to the mutant S glycoprotein is one of the major contributors to viral resistance. Binding of MERS-4 to the Different Conformational States of RBD in the Context of the S Trimer Collectively, we conﬁrmed the unique epitope tar- geted by MERS-4 and MERS-4V2 observed in the crystal struc- tures and highlighted the unusual mechanism of neutralization by these antibodies. Binding of MERS-4 to the Different Conformational States of RBD in the Context of the S Trimer We and others have recently demonstrated that MERS-CoV and SARS-CoV S trimers existed in various conformational states involving the ‘‘up’’ or ‘‘down’’ positions of RBD (Gui et al., 2017; Pallesen et al., 2017; Yuan et al., 2017). The working hypothesis 444 Cell Reports 24, 441–452, July 10, 2018 results suggest that reduced binding afﬁnity of MERS-4 to the mutant S glycoprotein is one of the major contributors to viral resistance Collectively we conﬁrmed the unique epitope tar Figure 3. Compar Site with the Ep MERS-4V2, and C the RBD b5-b6 Lo State (A) Structural super epitopes of MERS- distinct from the DP conformational differ b5-b6 loop between bound states. (B) Zoom-in view of t unbound (4KQZ: bl wheat) and DPP4-bo with either the MER 4V2-bound (green) (r (C) Patch 2 of the R which residues Leu5 the RBD b5-b6 loop (D) The steric clashes b5-b6 loop of the RB antibody binding. See also Figure S3. Fab complex (F that MERS-4 an to the RBD at the this, we used s tering (SAXS) s ternary complex built a monome MERS-4/MERS-2 which was gene the RBD/MERS crystal structure meric model ﬁt data with a c va investigate the as we built a dimer complex using th the RBD/MERS To understand MultiFoXS was population of t (Pelikan et al., 2009). SAXS ﬁtting (31% monomer model, 18% dime MERS 4/MERS 27 and 51% RBD results suggest that reduced binding afﬁnity of MERS-4 to the mutant S glycoprotein is one of the major contributors to viral resistance. Collectively, we conﬁrmed the unique epitope tar- geted by MERS-4 and MERS-4V2 observed in the crystal struc- tures and highlighted the unusual mechanism of neutralization by these antibodies. (Pelikan et a (31% monom MERS-4/MER improved the ure 6), reveali sient behavio exclusion chr Figure 3. Comparisons of the DPP4 Binding Site with the Epitopes of MERS-4 and MERS-4V2, and Conformational Change of the RBD b5-b6 Loop in the Antibody-Bound State (A) Structural superimposition showing that the epitopes of MERS-4 and MERS-4V2 (right) are distinct from the DPP4 binding site. A signiﬁcant conformational difference was found in the RBD b5-b6 loop between antibody-bound and DPP4- bound states. (B) Zoom-in view of the aligned RBD b5-b6 loops in unbound (4KQZ: blue; 4L3N: magenta; 4ZPW: wheat) and DPP4-bound (4L72: cyan; 4KR0: yellow) with either the MERS-4-bound (green) or MERS- 4V2-bound (green) (right) states. Binding of MERS-4 and MERS-27 to the RBD The epitope of MERS-4 is different from those of other re- ported antibodies including MERS-27, which we isolated and deﬁned previously (Figure S5). It prompted us to study the combined binding of MERS-4 and MERS-27 to the RBD. Gel ﬁltration analysis showed that the peak of the mixture comprising RBD, MERS-4 Fab, and MERS-27 Fab had a forward shift compared with that of the RBD/MERS-4 Cell Reports 24, 441–452, July 10, 2018 445 A MERS-4 Fab MERS-27 Fab RBD one protomer DPP4 B C D E F G H MERS-27 Fab one protomer RBD MERS-4 Fab Clashes with neighbouring NTD NTD DPP4 Clashes with neighbouring RBD Figure 4. Structural Superimpositions of the RBD/DPP4, RBD/MERS-4, and RBD/MERS-27 Crystal Structures onto the MERS-CoV S Trimer Glycoprotein in Receptor-Binding Inactivated and Activated States (A) MERS-CoV S trimer in receptor-binding inactivated state with all three RBDs in the ‘‘down’’ positions (PDB: 5w9j). The S trimer is shown with semi-transparent surface, in which one S protomer (S1 subunit in green and S2 subunit in orange) is shown as a cartoon. (B–D) Structural superimpositions of the RBD/DPP4 (B), RBD/MERS-4 (C), and RBD/MERS-27 (D) crystal structures onto the S trimer in receptor-binding in- activated state. DPP4 and MERS-4 Fab have steric clashes with the RBD and NTD of the neighboring S protomer, respectively. (E) MERS-CoV S trimer in receptor-binding activated state with one RBD in the ‘‘up’’ positions (PDB: 5w9h). (F–H) Structural superimpositions of the RBD/DPP4 (F), RBD/MERS-4 (G), and RBD/MERS-27 (H) crystal structures onto the S trimer in receptor-binding A B C D MERS-27 Fab one protomer RBD MERS-4 Fab Clashes with neighbouring NTD NTD DPP4 Clashes with neighbouring RBD A B one protomer RBD NTD DPP4 Clashes with neighbouring RBD C MERS-4 Fab Clashes with neighbouring N C D MERS-27 Fab MERS-4 Fab Clashes with neighbouring NTD D MERS-27 Fab th NTD A D C B MERS-4 Fab MERS-27 Fab G H MERS-4 Fab MERS-27 Fab RBD one protomer DPP4 E F G H RBD one protomer DPP4 E F MERS-4 Fab G G E H G F Fab MERS-27 Fab H RBD one protomer one protomer Figure 4. Binding of MERS-4 and MERS-27 to the RBD Structural Superimpositions of the RBD/DPP4, RBD/MERS-4, and RBD/MERS-27 Crystal Structures o Glycoprotein in Receptor-Binding Inactivated and Activated States positions of the RBD/DPP4, RBD/MERS-4, and RBD/MERS-27 Crystal Structures onto the MERS-CoV S Trimer ding Inactivated and Activated States Glycoprotein in Receptor-Binding Inactivated and Activated States (A) MERS-CoV S trimer in receptor-binding inactivated state with all three RBDs in the ‘‘down’’ positions (PDB: 5w9j). The S trimer is shown with semi-transparent surface, in which one S protomer (S1 subunit in green and S2 subunit in orange) is shown as a cartoon. (B–D) Structural superimpositions of the RBD/DPP4 (B), RBD/MERS-4 (C), and RBD/MERS-27 (D) crystal structures onto the S trimer in receptor-binding in- activated state. DPP4 and MERS-4 Fab have steric clashes with the RBD and NTD of the neighboring S protomer, respectively. (E) MERS-CoV S trimer in receptor-binding activated state with one RBD in the ‘‘up’’ positions (PDB: 5w9h). (F–H) Structural superimpositions of the RBD/DPP4 (F), RBD/MERS-4 (G), and RBD/MERS-27 (H) crystal structures onto the S trimer in receptor-binding activated state. The epitope is exposed and readily accessible for binding. y p p g (A) MERS-CoV S trimer in receptor-binding inactivated state with all three RBDs in the ‘‘down’’ positions (PDB: 5w9j). The S t surface, in which one S protomer (S1 subunit in green and S2 subunit in orange) is shown as a cartoon. (B–D) Structural superimpositions of the RBD/DPP4 (B), RBD/MERS-4 (C), and RBD/MERS-27 (D) crystal structures onto activated state. DPP4 and MERS-4 Fab have steric clashes with the RBD and NTD of the neighboring S protomer, respec (E) MERS-CoV S trimer in receptor-binding activated state with one RBD in the ‘‘up’’ positions (PDB: 5w9h). (F–H) Structural superimpositions of the RBD/DPP4 (F), RBD/MERS-4 (G), and RBD/MERS-27 (H) crystal structures on activated state. The epitope is exposed and readily accessible for binding. Synergistic Neutralization Effects of MERS-4 with Other Antibodies against RBD and NTD shown in Figure 7A, the percent neutralization obtained using combined MERS-4 and m336 demonstrated a 2.60-fold reduc- tion of half maximal inhibitory concentration (IC50) and 2.73- fold reduction of IC80 compared with that of either MERS-4 or m336 alone. Furthermore, the combination index (CI) values of combined MERS-4 and m336 at fractional effect values of effec- tive dose 50%, 75%, 90%, and 95% (ED50, ED75, ED90, and ED95) were 0.48, 0.38, 0.30, and 0.26, respectively. As a CI value of 1 indicates an additive effect, <1 indicates synergism, and >1 indicates antagonism, the combination of MERS-4 and m336 works in a clearly synergistic manner. Furthermore, the combi- nation of MERS-4 and 5F9 demonstrated better synergy in We have previously shown that MERS-4 and MERS-27 exhibited a synergistic effect by titrating the neutralizing potency of an equimolar mixture of the two antibodies and comparing the dose response with that of neutralization assays performed with the individual antibody alone (Chou, 2010; Chou and Tala- lay, 1984; Keck et al., 2013). The synergy between them was consistent with the unique epitopes of MERS-4 and MERS-27 on the RBD (Figure 7C). Here, we further tested whether MERS-4 could synergize with additional antibodies targeting either the RBD (m336) or NTD (5F9) of the S glycoprotein. As 446 Cell Reports 24, 441–452, July 10, 2018 Figure 5. Impact of Mutations in the RBD on Binding and Neutralization Sensitivity to MERS-4 and MERS-4V2 Figure 5. Impact of Mutations in the RBD on Binding and Neutralization Sensitivity to MERS-4 and MERS-4V2 (A) Binding afﬁnities of the wild-type RBD and its mutants (L507A, S508A, L545A, S546A, P547A, and E549A) to MERS-4 and MERS-4V2. (B) Neutralizing activity of MERS-4 against MERS-CoV pseudotyped with wild-type or mutant S glycoprotein (L507A, L545A, S546A, and P547A). The fold changes in IC50 of mutant viruses relative to the wild-type (WT) (>1: increase resistance and <1: increase sensitivity) (right). (C) MERS-4 staining of HEK293T cells expressing wild-type or mutant S glycoprotein. The fold changes in MFI of mutant viruses relative to the wild-type were listed in the table. MFI, median ﬂuorescence intensity. See also Figure S4. DISCUSSION We report the structural and functional anal- ysis of the potent neutralizing antibody MERS-4 and its variant MERS-4V2, which revealed a unique epitope speciﬁcity and novel mechanism of neutralization. The structure of MERS-4 Fab bound to RBD was determined at a resolution of 4.5 A˚ , and that of MERS-4V2 scFv bound to RBD was solved at 2.8 A˚ . MERS-4 and MERS- 4V2 demonstrated the same binding mode and epitope speciﬁcity. In contrast to all other RBD-targeting neutralizing anti- bodies, which directly compete with DPP4 for binding to the RBD, MERS-4 and MERS-4V2 approached the RBD outside the RBD-DPP4 binding interface by target- ing the b5-b6, b6-b7, and b7-b8 loops. MERS-4- and MERS-4V2-bound RBD demonstrated signiﬁcant conformational changes largely involving the folding of the b5-b6 loop toward a shallow groove on the RBD interface critical for accommodating a short helix of DPP4. In the context of the S trimer, MERS-4 prefers binding to the RBD in the ‘‘up’’ rather than the ‘‘down’’ position when virus becomes partially activated. Site-directed mutagenesis conﬁrmed the key residues critical for bind- ing and neutralizing activities of MERS-4 and MERS-4V2. Reduced afﬁnity for the RBD appeared to be the major contrib- utor to the compromised neutralizing activities against the mutant viruses. A synergistic effect was observed between MERS-4 and RBD-speciﬁc (m336) as well as NTD-speciﬁc (5F9) antibodies, although the exact mechanism remains un- known. Taken together, our study reveals that MERS-4 and MERS-4V2 recognize a unique neutralizing epitope with an un- usual mechanism of action. Such special features will enable We report the structural and functional anal- ysis of the potent neutralizing antibody MERS-4 and its variant MERS-4V2, which revealed a unique epitope speciﬁcity and novel mechanism of neutralization. The structure of MERS-4 Fab bound to RBD was determined at a resolution of 4.5 A˚ , and that of MERS-4V2 scFv bound to RBD was solved at 2.8 A˚ . MERS-4 and MERS- 4V2 demonstrated the same binding mode and epitope speciﬁcity. In contrast to all other RBD-targeting neutralizing anti- bodies, which directly compete with DPP4 for binding to the RBD, MERS-4 and MERS-4V2 approached the RBD outside the RBD-DPP4 binding interface by target- ing the b5-b6, b6-b7, and b7-b8 loops. MERS-4- and MERS-4V2-bound RBD demonstrated signiﬁcant conformational changes largely involving the folding of the b5-b6 loop toward a shallow groove on the RBD interface critical for accommodating a short helix of DPP4. Figure 6. Combination of MERS-4 and MERS-27 in Binding to the RBD Regardless of the exact process, the observed folding of the b5-b6 loop toward the binding interface would be ex- pected to disrupt the docking of the short helix of DPP4 onto the binding surface of RBD, thereby blocking virus entry. Of note, such unique mechanism of action has not been reported for other viruses. Antibodies against the receptor binding site (RBS) of inﬂuenza virus and the CD4 binding site (CD4bs) of HIV type I (HIV-1) exert their neutralizing activities largely through direct competition with their respective receptors (Wu and Kong, 2016; Ren and Zhou, 2016). Perhaps the closest scenario to MERS-4-mediated inhibition is found in antibodies targeting the V3 region of the HIV-1 envelope where binding may capture or induce conformational changes that block the subsequent interaction between the V3 region and the co-receptor CCR5 or CXCR4. Certainly, such a hypothesis would have to be veriﬁed in the future (Barnes et al., 2018; Mouquet et al., 2012; Pejchal et al., 2011). MERS-4 to synergize with other antibodies and provide a valu- able addition for the combined use of antibodies against MERS-CoV infection. Since the identiﬁcation of the highly pathogenic MERS-CoV in 2012 (Bermingham et al., 2012; Zaki et al., 2012), great efforts have been made to develop prophylactic and therapeutic inter- ventions against this virus. In particular, monoclonal antibodies and vaccines targeting the S glycoprotein are a major research focus due to its critical role in mediating viral entry and its great potency in inducing protective antibody response in infected and naive individuals (Chen et al., 2009; Zumla et al., 2016). Among close to 20 reported neutralizing antibodies, the large majority (MERS-27, m336, D12, 4C2, MCA1, CDC-C2, and JC57-14) were shown to directly compete with the cellular receptor DPP4 for binding to the RBD (Chen et al., 2017b; Li et al., 2015; Wang et al., 2015a, 2018; Ying et al., 2015; Yu et al., 2015). This dominant mechanism of action is further supported by structural studies in which their epitopes were found to clearly, although variably, overlap with the DPP4 binding surface (Figure S7). Because MERS-4 shares many biochemical and bio- logical features with the abovementioned antibodies, it was ex- pected that its mechanism of neutralization would follow the same suit. However, a novel epitope speciﬁcity and unusual mechanism of action came as a pleasant surprise. Figure 6. Combination of MERS-4 and MERS-27 in Binding to the RBD Figure 6. Combination of MERS-4 and MERS-27 in Binding to the RBD 31% monomer model of the RBD/MERS-4/MERS-27 18% dimer model of the RBD/MERS-4/MERS-27 MERS-CoV RBD MERS-27 Fab MERS-4 Fab experiment monomer model multi-state model 51% model of the RBD/MERS-27 I(q) q(Å-1) q(Å-1) le d o m /t n e m ir e p x e 31% monomer model of the RBD/MERS-4/MERS-27 18% dimer model of the RBD/MERS-4/MERS-27 MERS-CoV RBD MERS-27 Fab MERS-4 Fab 51% model of the RBD/MERS-27 experiment monomer model multi-state model I(q) q(Å-1) Comparison of the experimental SAXS data (black dots) with the theoretical scattering curve calcu- lated from the full-atomic RBD/MERS-4/MERS-27 monomer model (red line) and the theoretical scat- tering curve calculated from an ensemble consisting of 31% monomer model, 18% dimer model of the RBD/MERS-4/MERS-27, and 51% RBD/MERS-27 (green line). Residuals calculated as I (q) experi- mental/I (q) model are shown below the scattering curves. MERS-4 Fab I(q) 18% dimer model of the RBD/MERS-4/MERS-27 51% model of the RBD/MERS-27 18% dimer model of the RBD/MERS-4/MERS-27 See also Figure S6. q(Å-1) le d o m /t n e m ir e p x e 51% model of the RBD/MERS-27 in the context of the S trimer, it is plausible that some of the loop structures in the RBD could also be quite ﬂexible, providing op- portunities for antibody binding during the structural transformation. The other is that the antibody itself triggers such a structural alteration. If this were the case, the heavy chain of MERS-4 or MERS-4V2 would likely push and fold the b5-b6 loop toward the receptor binding interface in or- der to achieve optimal binding. This hy- pothesis is supported by atomic analysis of the interaction be- tween MERS-4V2 and the RBD. Among a total of 16 interactive binding residues, 14 from the b6-b7 and b7-b8 loops interacted with the light chain, whereas 2 in the b5-b6 loop participated in binding with the heavy chain. The interactions between the b6-b7 and b7-b8 loops of the RBD and the light chain of MERS-4V2 are therefore the most likely the driving force of anti- gen-antibody binding. However, optimal binding of MERS-4 would require the heavy chain to overcome the steric obstruction by pushing and folding the b5-b6 loop toward the binding inter- face, resulting in a distorted conformation as shown in the crystal structure. DISCUSSION In the context of the S trimer, MERS-4 prefers binding to the RBD in the ‘‘up’’ rather than the ‘‘down’’ position when virus becomes partially activated. Site-directed mutagenesis conﬁrmed the key residues critical for bind- g activities of MERS-4 and MERS-4V2. the RBD appeared to be the major contrib- omised neutralizing activities against the synergistic effect was observed between -speciﬁc (m336) as well as NTD-speciﬁc though the exact mechanism remains un- ether, our study reveals that MERS-4 and ze a unique neutralizing epitope with an un- of action. Such special features will enable particular at relatively lower concentrations (Figure 7B). The percent neutralization of combined MERS-4 and 5F9 demon- strated a 15.21-fold reduction in IC50 and 52.7-fold reduction in IC80 compared with that of either antibody alone. Furthermore, the CI values of combined MERS-4 and 5F9 at fractional effect values of ED 50%, 75%, 90%, and 95% (ED50, ED75, ED90, and ED95) were 0.06, 0.05, 0.06, and 0.06, respectively. These results showed that MERS-4 can act in synergy with RBD-spe- ciﬁc m336 as well as NTD-speciﬁc 5F9 antibodies. Cell Reports 24, 441–452, July 10, 2018 447 Figure 6. Combination of MERS-4 and MERS-27 in Binding to the RBD Regardless of the exact process, the observed folding of the b5-b6 loop toward the binding interface would be ex- pected to disrupt the docking of the short helix of DPP4 onto the binding surface of RBD, thereby blocking virus entry. Of note, such unique mechanism of action has not been reported for other viruses. Antibodies against the receptor binding site (RBS) of inﬂuenza virus and the CD4 binding site (CD4bs) of HIV type I (HIV-1) exert their neutralizing activities largely through direct competition with their respective receptors (Wu and Kong, 2016; Ren and Zhou, 2016). Perhaps the closest scenario to MERS-4-mediated inhibition is found in antibodies targeting the V3 region of the HIV-1 envelope where binding may capture or induce conformational changes that block the subsequent interaction between the V3 region and the co-receptor CCR5 or CXCR4. Certainly, such a hypothesis would have to be veriﬁed in the future (Barnes et al., 2018; Mouquet et al., 2012; Pejchal et al., 2011). 51% model of the RBD/MERS-27 in the context of the S trimer, it is plausible that some of the loop structures in the RBD could also be quite ﬂexible, providing op- portunities for antibody binding during the structural transformation. The other is that the antibody itself triggers such a structural alteration. If this were the case, the heavy chain of MERS-4 or MERS-4V2 would likely push and fold the b5-b6 loop toward the receptor binding interface in or- der to achieve optimal binding. This hy- pothesis is supported by atomic analysis of the interaction be- tween MERS-4V2 and the RBD. Among a total of 16 interactive binding residues, 14 from the b6-b7 and b7-b8 loops interacted with the light chain, whereas 2 in the b5-b6 loop participated in binding with the heavy chain. The interactions between the b6-b7 and b7-b8 loops of the RBD and the light chain of MERS-4V2 are therefore the most likely the driving force of anti- gen-antibody binding. However, optimal binding of MERS-4 would require the heavy chain to overcome the steric obstruction by pushing and folding the b5-b6 loop toward the binding inter- face, resulting in a distorted conformation as shown in the crystal structure. Figure 6. Combination of MERS-4 and MERS-27 in Binding to the RBD This not only reveals a new vulnerable site on the RBD, but also provides a novel target for future vaccine design and development. To the best of our knowledge, this is the ﬁrst report demon- strating that antibody binding can indirectly disrupt the interac- tion between RBD with DPP4. While the exact process requires further study, there are at least two possibilities. One is that the antibody captures and ﬁxes one of the RBD conformational states when the b5-b6 loop spontaneously bends toward the re- ceptor binding interface. Given the highly dynamic nature of RBD The MERS-CoV spike glycoprotein showed limited sequence variation among different strains, especially in the RBD that is responsible for receptor binding. However, this does not mean the spike glycoprotein will remain unchanged as the virus con- tinues to spread among multiple animal species and to probe 448 Cell Reports 24, 441–452, July 10, 2018 gure 7. Effects of MERS-4 Combined with m336, 5F9, and MERS-27, Respectively, in Neutralizing Pseudotyped MERS-CoV ) Effects of MERS-4 combined with m336 in neutralizing pseudotyped MERS-CoV. Percent neutralization was calculated for serial 3-fold dilutions of eac ntibody alone and in combination at constant ratios in a range of concentrations from 27 times to 1/81 of IC50s. The constant ratios of the combined antibodie ere their IC50s. On the x axis, a dose of 1 was at the IC50 concentration. Fractional effect (FA) plots generated by the CompuSyn program for MERS-4, m336, an eir combination showing dosage versus effect. Median effect plot of calculated CI values (logarithmic) versus FA values, in which a log CI of <0 is synergism an og CI of >0 is antagonism. Data shown are average values from four independent experiments. and C) The percent neutralization, fractional effect, and CI values for MERS-4 combined with 5F9 (B) and MERS-4 combined with MERS-27 (C) were calculate nd generated using the same method. ee also Figures S5 and S7. Figure 7. Effects of MERS-4 Combined with m336, 5F9, and MERS-27, Respectively, in Neutralizing Pseudotyped MERS-CoV ects of MERS-4 Combined with m336, 5F9, and MERS-27, Respectively, in Neutralizing Pseudotyped MERS-Co Figure 7. Effects of MERS-4 Combined with m336, 5F9, and MERS-27, Respectively, in Neutralizing Pseud (A) Effects of MERS-4 combined with m336 in neutralizing pseudotyped MERS-CoV. Figure 6. Combination of MERS-4 and MERS-27 in Binding to the RBD Percent neutralization was calculated for serial 3-fold dilutions of each antibody alone and in combination at constant ratios in a range of concentrations from 27 times to 1/81 of IC50s. The constant ratios of the combined antibodies were their IC50s. On the x axis, a dose of 1 was at the IC50 concentration. Fractional effect (FA) plots generated by the CompuSyn program for MERS-4, m336, and their combination showing dosage versus effect. Median effect plot of calculated CI values (logarithmic) versus FA values, in which a log CI of <0 is synergism and a log CI of >0 is antagonism. Data shown are average values from four independent experiments. (B and C) The percent neutralization, fractional effect, and CI values for MERS-4 combined with 5F9 (B) and MERS-4 combined with MERS-27 (C) were calculated and generated using the same method. S l Fi r S5 d S7 and adapt in human population. For stronger and broader pro- tective effect against MERS-CoV, a combined use of two or more antibodies will provide a superior alternative (Wang et al., 2018). However, any effective combination would require the candidate antibodies to recognize distinct epitopes for additive or synergistic effect. The unique epitope of MERS-4 and MERS-4V2 therefore makes them good candidates for combina- tion use with those reported elsewhere (Chen et al., 2017b; Li et al., 2015; Wang et al., 2015a, 2018; Ying et al., 2015; Yu et al., 2015). Indeed, combinations of MERS-4 and MERS-27, MERS-4 and m336, and MERS-4 and 5F9 demonstrated impres- sive synergy in the pseudotyped MERS-CoV assay. The exact mechanism in achieving the synergy, however, is uncertain, particularly for those sharing the same mechanism in disrupting interaction between RBD and DPP4 (MERS-4 and MERS-27, and MERS-4 and m336). Presumably, the two antibodies may preferentially act on RBD at the different spatial and temporal points during interaction with the receptor DPP4, allowing better Cell Reports 24, 441–452, July 10, 2018 449 10 mM sodium acetate (pH 4.5) was immobilized to 600 response units on the ﬂow cell. For the collection of data, MERS-CoV RBD alone and its various complex with MERS-4 Fab were injected in a buffer of 10 mM HEPES (pH 7.2), 150 mM NaCl, and 0.005% (v/v) Tween 20 over the ﬂow cells. exposure of otherwise less accessible epitopes. Cell Surface Staining HEK293T cells were transfected by wild-type or mutant MERS-CoV spike expression plasmids. After 48 hr, cells were harvested and washed using PBS and incubated with monoclonal antibodies (mAbs) for 30 min at room temperature. Cells were then washed and stained with ﬂuorescently labeled anti-human IgG-PE or anti-mouse IgG-FITC secondary antibody. Cells were then washed by PBS and analyzed with FACSCalibur and FACSComp soft- ware (BD Biosciences). Cooperativity of the Two Neutralizing mAbs for Virus Neutralization Synergistic, additive, and antagonistic interaction between MERS-4 and 5F9, MERS-4 and m336, and MERS-4 and MERS-27 for virus neutralization were evaluated by the median effect analysis method by the CompuSyn software as previously reported. The measured neutralization values were input to the program as fractional effects (FA) ranging between 0.01 and 0.99 for each of the two antibodies and for both in combination. CI values were calculated in relation to FA values. A logarithmic CI value of 0 indicates an additive effect, <0 indicates synergism, and >0 indicates antagonism. Structural Determination and Reﬁnement The structure was determined by molecular replacement with the crystallo- graphic software PHASER (McCoy et al., 2007). The search models are the MERS-CoV RBD structure (PDB: 4L72) and the structures of the variable and constant domain of the heavy and light chains available in the PDB with the highest sequence identities. Iterative reﬁnement with the program PHENIX and model building with the program COOT were performed to complete the structure reﬁnement (Adams et al., 2002; Emsley and Cowtan, 2004). Structure validation was performed with the program PROCHECK (Laskowski et al., 1993). All structure reﬁnement statistics are listed in Table S1. SAXS Data Collection and Analysis SAXS data were collected at the SIBLYS beamline 12.3.1 of the Advanced Light Source at the Lawrence Berkeley National Laboratory using 1.0 A˚ wave- length and Pilatus 2M detector at a 1.5-m sample-to-detector distance (Classen et al., 2013), resulting in scattering vectors ranging from 0.01 to 0.5 A˚ 1. The scattering vector is deﬁned as q = 4p sinq/l, where 2q is the scat- tering angle. SEC in line with SEC-SAXS was performed to ensure the aggre- gation-free state of the sample. The SEC column was equilibrated with running buffer (50 mM Tris-HCl [pH 7.3], 100 mM NaCl, 3% glycerol, and 0.01% so- dium azide) with a ﬂow rate of 0.5 mL/min. The 50-mL sample was run through the SEC and 2-s X-ray exposures were collected continuously during an 25-min elution. The SAXS frames recorded prior to the protein elution peak were used to subtract all other frames. The subtracted frames were investi- gated by Rg and scattering intensity at q = 0 A˚ 1 (I(0)) derived by the Guinier approximation I(q) = I(0) exp(q2Rg 2/3) with the limits qRg < 1.5 (Guinier and Fournet, 1955). I (0) and Rg values were compared for each collected SAXS curve across the entire elution peak. The elution peak was mapped by plotting the scattering intensity at q = 0 A˚ 1 (I (0)) relative to the recorded frame. Grad- uate decreasing of Rg values across an elution peak indicates transient sample behavior. SAXS was also acquired in the high-throughput modality at sample concentrations between 1 and 5 mg/mL to compare with the SEC-SAXS pro- ﬁle (Hura et al., 2009). The full atomic model was built with the program MODELER to construct the missing loops and linkers (Sali and Blundell, 1993). The theoretical SAXS proﬁle and the corresponding ﬁt to the experi- mental data were calculated using the program FoXS (Schneidman-Duhovny et al., 2013). A multistate model of complexes coexisting in solution was selected by MultiFoXS (Schneidman-Duhovny et al., 2016). The size of the multistate model was selected based on the level of improvement in the SAXS ﬁt. Crystallization and Data Collection Crystals of the RBD/MERS-4 Fab complex were successfully grown at 18C using the sitting drop vapor diffusion method, which involved mixing equal vol- ume of protein and reservoir solution containing 2% v/v tacsimate (pH 5.0), 0.1 M sodium citrate tribasic dihydrate (pH 5.6), 16% w/v polyethylene glycol 3350, and 2 M sodium thiocyanate. Crystals of the RBD/MERS-4V2 scFv com- plex were successfully grown at 18C using the sitting drop vapor diffusion method, which involved mixing equal volumes of protein and reservoir solution containing 0.1 M Tris (pH 7.5), 10% w/v polyethylene glycol 8000, and 8% (v/v) ethylene glycol. Diffraction data were collected on the BL17U beamline at Shanghai Synchrotron Research Facility (Wang et al., 2015b) and processed with HKL2000 (Otwinowski and Minor, 1997). All data collection and process- ing statistics are listed in Table S1. MERS-CoV Pseudotyped Virus Production and Neutralization Assay S Co seudotyped us oduct o a d eut a at o ssay The MERS-CoV pseudotyped virus was generated by cotransfecting pcDNA 3.1 expression vectors encoding the wild-type or mutant MERS-CoV S glyco- protein, with a pNL4-3R-Eluciferase viral backbone plasmid into 293T cells as described previously (Shang et al., 2011). The viral titers of the pseudotyped virus were measured as luciferase activity in relative light units 48 hr after trans- fection. The mutant S glycoprotein expression vector was generated by the site-directed mutagenesis kit and conﬁrmed by sequencing. Neutralization as- says were performed by incubating 100 TCID50 (median tissue culture infec- tious dose) of pseudotyped virus with 16 serial 1:3 dilutions of puriﬁed antibody at 37C for 1 hr; then Huh7 cells (about 1.5 3 104 per well) were added. Infec- tivity was quantiﬁed by the luciferase activity 48 hr after infection. IC50s were calculated with the dose-response inhibition model in GraphPad Prism (GraphPad Software). Protein Expression and Puriﬁcation The MERS-CoV RBD (residues 367–588) and the ectodomain of human DPP4 (residues 39–766) were expressed in Sf9 insect cells. The puriﬁed RBD was di- gested with endoglycosidase F1 and F3 at room temperature overnight and was then further puriﬁed through gel-ﬁltration chromatography. The MERS-4 and MERS-4V2 IgG were expressed in HEK293F cells. The puriﬁed MERS-4 and MERS-4V2 were digested with endoproteinase Lys-C at 37C, and the Fab and Fc fragments were separated by loading samples onto a diethylami- noethyl (DEAE) ion-exchange column. The genes encoding the scFv of MERS- 4V2 were cloned with a C-terminal His-tag in the heavy chain variable region and light chain variable region (VH-VL) orientation, linked together by a (G4S)3. The MERS-4V2 scFv was expressed in HEK293F cells through tran- sient transfection. The scFv was captured by nickel beads and puriﬁed through gel-ﬁltration chromatography. Figure 6. Combination of MERS-4 and MERS-27 in Binding to the RBD If this is the case, the observed synergy would be most likely derived from the recognitions of distinct epitopes rather than the same neutralization mechanism. Obviously, antibodies with distinct mechanisms and binding at disparate epitopes would be more likely to have synergy than those shared mechanism and over- lapped epitopes. Synergy showed here between MERS-4 and 5F9 is a good example. Nevertheless, the exact mechanisms un- derlying synergy must be complex and should be treated differ- ently from case to case. The preliminary results presented here do offer some rationales for MERS-4 as a valuable addition for the combined use of antibodies against MERS-CoV infection. SPR Analysis Real-time binding and analysis by SPR were conducted on a BIAcore T200 in- strument. MERS-4 IgG (20 mg/mL) and MERS-4V2 (20 mg/mL) in 10 mM so- dium acetate (pH 5.0) was immobilized to 600 response units on the ﬂow cell. For the collection of data, RBD and its mutants were injected in a buffer of 10 mM HEPES (pH 7.2), 150 mM NaCl, and 0.005% (v/v) Tween 20 over the ﬂow cells at various concentrations. Data were analyzed with the BIAcore T200 evaluation software by ﬁtting to a 1:1 Langmuir binding model. For the DPP4 binding inhibition assay, RBD (100 nM) and MERS-4 Fab were mixed in advance at a molar ratio 1:1, 1:2, 1:5, and 1:10. DPP4 (10 mg/mL) in 450 Cell Reports 24, 441–452, July 10, 2018 AUTHOR CONTRIBUTIONS Corti, D., Zhao, J., Pedotti, M., Simonelli, L., Agnihothram, S., Fett, C., Fernan- dez-Rodriguez, B., Foglierini, M., Agatic, G., Vanzetta, F., et al. (2015). Prophy- lactic and postexposure efﬁcacy of a potent human monoclonal antibody against MERS coronavirus. Proc. Natl. Acad. Sci. USA 112, 10473–10478. S.Z., P.W., P.Z., and L.J. expressed, puriﬁed, and crystallized the protein, and carried out the SPR analysis with help from D.W.; P.Z., Y.L., W.J., H.W., and X.S. carried out the pseudotyped virus entry and antibody neutralization ana- lyses; S.Z. and P.W. collected and processed the diffraction data; X.W. carried out structural determination and reﬁnement; M.H. collected and analyzed the SAXS data with input from S.W. and X.F.; X.W. and L.Z. supervised the project and wrote the manuscript with help from S.Z., P.Z., P.W., and A.F. de Wit, E., van Doremalen, N., Falzarano, D., and Munster, V.J. (2016). SARS and MERS: recent insights into emerging coronaviruses. Nat. Rev. Microbiol. 14, 523–534. Du, L., Zhao, G., Yang, Y., Qiu, H., Wang, L., Kou, Z., Tao, X., Yu, H., Sun, S., Tseng, C.T., et al. (2014). A conformation-dependent neutralizing monoclonal antibody speciﬁcally targeting receptor-binding domain in Middle East respi- ratory syndrome coronavirus spike protein. J. Virol. 88, 7045–7053. ACKNOWLEDGMENTS Choi, J.Y. (2015). An outbreak of Middle East respiratory syndrome coronavi- rus infection in South Korea, 2015. Yonsei Med. J. 56, 1174–1176. We thank Dr. Jianhua He and staff scientists at the SSRF BL17U beam line and Dr. Shilong Fan at the X-ray crystallography platform of the Tsinghua University Technology Center for assistance in diffraction data collection. We thank Prof. Tianlei Ying and Prof. Shibo Jiang (Fudan University) for providing the m336. We thank Prof. Wenjie Tan (Chinese Center for Disease Control and Prevention) for providing the 5F9. This work was supported by the National Key Plan for Sci- entiﬁc Research and Development of China (grants 2016YFD0500307 and 2016YFC1200902), the National Natural Science Foundation of China (grants 31470751, U1405228, 81530065, and 81471929), and the Grand Challenges China (grant 81661128042). Chou, T.C. (2010). 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Chen, Y., Rajashankar, K.R., Yang, Y., Agnihothram, S.S., Liu, C., Lin, Y.-L., Baric, R.S., and Li, F. (2013). Crystal structure of the receptor-binding domain The accession numbers for the atomic coordinates reported in this paper are PDB: 5ZXV and 5YY5. from newly emerged Middle East respiratory syndrome coronavirus. J. Virol. 87, 10777–10783. Chen, Y., Lu, S., Jia, H., Deng, Y., Zhou, J., Huang, B., Yu, Y., Lan, J., Wang, W., Lou, Y., et al. (2017a). A novel neutralizing monoclonal antibody targeting the N-terminal domain of the MERS-CoV spike protein. Emerg. Microbes Infect. 6, e37. SUPPLEMENTAL INFORMATION Supplemental Information includes seven ﬁgures and two tables and can be found with this article online at https://doi.org/10.1016/j.celrep.2018.06.041. Chen, Z., Bao, L., Chen, C., Zou, T., Xue, Y., Li, F., Lv, Q., Gu, S., Gao, X., Cui, S., et al. (2017b). 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https://openalex.org/W4210786322	https://www.researchsquare.com/article/rs-1294111/latest.pdf	English	null	Low-Intensity Pulsed Ultrasound Promotes Cell Viability And Inhibits Apoptosis of H9C2 Cardiomyocytes In 3D Bioprinting Scaffolds Via PI3K-Akt And ERK1/2 Pathways	Research Square (Research Square)	2,022	cc-by	7,089	Yugang Hu Yuanting Yang Renmin Hospital of Wuhan University Yuanting Yang Renmin Hospital of Wuhan University Research Article Keywords: low-intensity pulse ultrasound, 3D bio-printing, cell viability, gelatin methacryloyl, apoptosis Posted Date: May 26th, 2022 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/22 Page 1/22 Abstract Objective: The aim of this study was to investigate whether low-intensity pulsed ultrasound (LIPUS) promotes myocardial cell viability in three-dimensional (3D) cell-laden gelatin methacryloyl (GelMA) scaffolds. Methods: Cardiomyoblasts (H9C2s) were mixed in 6% (w/v) GelMA bio-inks and printed using extrusion- based 3D bioprinter. These scaffolds were exposed to LIPUS with different parameters or sham-irradiated to optimize the LIPUS treatment. The viability of H9C2s was measured using Cell Counting Kit-8 (CCK8), cell cycle, and live and dead cell double staining assays. Western blot analysis was performed to determine the protein expression levels. Results: We successfully fabricated 3D bio-printed cell-laden GelMA scaffolds. CCK8 and live and dead cell double-staining assays indicated that the optimal conditions for LIPUS were a frequency of 0.5 MHz and exposure time of 10 min. Cell cycle analysis showed that LIPUS promoted the entry of cells into the S and G2/M phases from the G0/G1 phase. Western blot analysis revealed that LIPUS promoted the phosphorylation and activation of ERK1/2 and PI3K-Akt. The ERK1/2 inhibitor (U0126) and PI3K inhibitor (LY294002) significantly reduced LIPUS-induced phosphorylation of ERK1/2 and PI3K-Akt, respectively, which in turn reduced the LIPUS-induced viability of H9C2s in 3D bio-printed cell-laden GelMA scaffolds. Conclusion: A frequency of 0.5 MHz and exposure time of 10 min for LIPUS exposure can be adapted to achieve optimized culture effects on myocardial cells in 3D bio-printed cell-laden GelMA scaffolds via the ERK1/2 and PI3K-Akt signaling pathways. Introduction Ischemic heart disease (IHD) is one of the most common leading causes of morbidity and mortality worldwide [1-3]. Because of the limited capability to regenerate cardiac tissues, the treatment options available for patients with end-stage heart failure caused by myocardial infarction typically involve grafting tissues from donors [4-6]. However, the critical shortage of donor organs, immune rejection, limited durability, and high healthcare costs may prevent the wide use of grafting in clinical practice [7-9], thus, researches on tissue engineering in order to grow tissue directly from cells have been widely motivated nowadays. Among them, as the key element for tissue engineering, a fully satisfactory cell- scaffold-microenvironment need stimultaneously support the growth of different cell types, each with specific mechanical properties, cell populations, and geometric structures. Hence, there are two challenges for tissue engineering, fabrication methods and biomaterials. However, traditional fabrication methods for scaffolds, electrospinning, freeze-drying and so on, lack precise control of internal structural features and topology. three-dimensional (3D) bioprinting, an attached fabricating mechanics that applies a layer-by-layer method, has been widely proposed as an additive method for creating functional cardiac tissue [10-12], due to its higher cell density, moreaccurate and controllable for the fabrication of multifunctional scaffolds. Biomaterials also play an important role on the cardiac tissue Page 2/22 Page 2/22 engineering because they provide the necessary mechanical support and physical structure for cardiomyocytes to attach, grow and maintain their physiological functions. Among the synthesized biomaterials, gelatin methacryloyl (GelMA) has received great attention because of its good biocompatibility, low immunogenicity, and ability to promote cell growth. Nevertheless, various major challenges related to 3D bioprinting with GelMA for cardiac tissues on a macroscopic scale remain to be addressed, including ensuring cell viability and function and the multiple cell type usage, composition, and physical properties required to simulate the complex structure of the extracellular matrix [13, 14]. Maintaining sufficient cell viability and functionality for a long duration may be the most difficult task when the cells are transferred from 2D culture to a 3D environment.As a mechanical stimulus, low- intensity pulsed ultrasound (LIPUS) is considered as a secure and practical therapy for fracture healing and has been demonstrated to promote the proliferation of different cell types in vitro [15-18]. Guo et al. Introduction combined chondrocytes and alginate to construct a 3D scaffold with or without LIPUS exposure and found that LIPUS significantly enhanced the porosity and permeability of the 3D alginate scaffold, which is significant for cell growth [19]. Similar results were also demonstrated in 3D scaffolds containing human bone marrow mesenchymal stem cells [20, 21]. However, Wang et al. showed that a relatively low dose of LIPUS (70 mW/cm2) increased human omental adipose-derived mesenchymal stem cell viability, whereas a high dose of LIPUS (210 mW/cm2) promoted apoptosis [22]. LIPUS was also reported to inhibit the proliferation of osteosarcoma cells (30 mW/cm2) [23] and promote apoptosis of osteoclasts (30 mW/cm2) [24]. Thus, the optimal dose of LIPUS for various cell types and intensities must be determined. In addition, the effect of LIPUS on cardiac muscle cells in a 3D scaffold is unclear. Moreover, ERK1/2 and PI3K-Akt signaling pathways are important pathways that mediate cell cycle progression and promote cell survival and proliferation and LIPUS also had close relationship with these pathways in previous studies.Therefore, in the present study, a 3D bio-printed gelatin methacryloyl (GelMA) scaffold containing H9C2 cells was fabricated using a desktop 3D bioprinter. The effect of LIPUS on the apoptosis and viability of H9C2s was determined, and the roles of the ERK1/2 and PI3K-Akt signaling pathways were also examined. Our results support the applied of LIPUS to promote the viability of cardiac cells and may improve the application of LIPUS to generate functional cardiac tissues. Optimization of LIPUS treatment protocol and dosage The LIPUS exposure device (Chongqing Haifu Medical Technology Co., Ltd., Chongqing, China) comprises an array of five transducers (34.8 mm in diameter), which is specifically drafted for a 6-well culture plate. A 6-well plate was deposited on the transducers with a thin layer of ultrasound coupling agent between them. The parameters of the LIPUS device were an intensity of 1.0 W/cm2 (temporal average intensity), duty cycle of 20%, and pulse repetition frequency of 1 kHz; four frequencies (0.5, 1.0, 1.5, and 2.0 MHz) were used. All Scaffolds in the LIPUS group were exposed to LIPUS, whereas those in the control group received sham irradiation (LIPUS was turned off). GelMA bioink preparation Cardiomyoblasts (rat heart myoblast H9C2 cells) obtained from Procell (Wuhan, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, NY, USA) complemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 100 IU/mL penicillin (Servicebio, Wuhan, China), and 100 mg/mL streptomycin (Servicebio, Wuhan, China) at 37 °C in a 5% CO2 atmosphere. Lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP), GelMA powder, and the UV light source (405 nm) were purchased from the SuZhou Intelligent Manufacturing Research Institute (SuZhou, China). LAP was dissolved in phosphate-buffered saline (PBS, Servicebio, Wuhan, China) to a concentration of 0.25% (w/v) and heated Page 3/22 Page 3/22 at 55 °C for 15 min to ensure complete dissolution. GelMA powder was dissolved in LAP solution at a concentration of 6% at 70 °C for 30 min and filtered through a filter membrane (0.22 µm, Millipore, Billerica, MA, USA) before use. All of these processes were conducted in the bechtop. Cardiomyoblasts were collected from the culture dish and homogeneously dispersed into the GelMA solution (37 °C) to form a single-cell suspension at a cell density of approximately 5 × 106 cells/mL. The cell-laden GelMA solution was then loaded into a 5-mL syringe equipped with a 25 G needle (SUNP BIOTECH, China), which was fixed at the extrusion printhead. 3D-bioprinting Scaffolds were fabricated using a customized extrusion-based 3D printer BioMaker (SUNP BIOTECH, China). Computer-aided design software was used to produce a G-code file for printing. A 3D scaffold model with dimensions of 10 mm width, 10 mm length, and 1 mm height was designed with a 1-mm filament grid and 0.2-mm layer thickness. Before bioprinting, the cell-laden GelMA bio-ink was placed at 4 °C for approximately 30 s until it changed from liquid to semi-solid, and then placed in the 3D printer for bio-printing. The cell-laden GelMA bio-ink was extruded onto a moving platform based on the model described above, and Biomarker Software Suite 1.0.7 (SUNP BIOTECH, China) was applied to monitor and manage the printing process. The 3D GelMA scaffolds containing H9C2 cells were printed into a Petri dish and immersed in DMEM at 37 °C with 5% CO2 after UV light exposure (wavelength: 405 nm) for 30 s to photo-crosslink the bio-inks to ensure their long-term stability. All printing processes were performed in the bechtop at room temperature with the nozzle set at a moving speed of 10 mm/s and the pressure was 0.5MPa. Moreover, everytime before bioprinting, we wiped the bioprinter with 75% alcohol totally and exposed it to the UV irradiation for 30 min for sterility. Cytocompatibility tests A Cell Counting Kit-8 (CCK-8; Servicebio, Wuhan, China) assay was performed to measure the proliferation of H9C2 cells. After 1, 4, and 7 days of treatment with LIPUS or sham irradiation, scaffolds with H9C2 cells were incubated in CCK-8 solution for 4 h. Next, 10 µL of the supernatant was transferred into a 96- Page 4/22 Page 4/22 well culture plate, and its absorbance at 450 nm was measured using a microplate reader (EnSight; Perkin Elmer, Waltham, MA, USA). To reduce the effect of non-clay adsorbing the staining solution, scaffolds without seeded cells were used as the blank group. well culture plate, and its absorbance at 450 nm was measured using a microplate reader (EnSight; Perkin Elmer, Waltham, MA, USA). To reduce the effect of non-clay adsorbing the staining solution, scaffolds without seeded cells were used as the blank group. Cell viability was evaluated using the Live and Dead Cell Double Staining Kit (EFL-CLD-001, SuZhou) after treatment with LIPUS or sham irradiation for 1, 4, and 7 days. The scaffolds were washed twice with PBS, and then immersed in live/dead staining solution of 10 mL PBS containing 5 µL calcein-AM and 5 µL propidium iodide (PI) solution according to the manufacturer’s instructions. After incubation at 37 °C for 45 min, the staining solution was removed, and the scaffolds were washed with PBS. Fluorescence staining was analyzed using confocal laser scanning microscopy (ECLIPSE TI; Nikon, Tokyo, Japan). Cell viability was calculated from the number of green (representing live cells) and red points (representing dead cells) by randomly five images of each groups. Flow cytometry For the cell cycle assay, after LIPUS or sham irradiation treatment, cell-laden GelMA scaffolds were cut into small pieces and lysed using GelMA Lysis Buffer (EFL-GM-LS-001, SuZhou, China), harvested, and fixed with pre-cooled 75% ethanol at 4 °C overnight. After centrifugation, the cells were incubated with PI and RNase A at 37 °C for 30 min in the dark. The cell cycle stages were analyzed at an excitation wavelength of 488 nm using flow cytometry (BD Biosciences, San Jose, USA) and the data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA). Apoptosis of H9C2 cells was determined using an FITC Annexin V and PI double staining kit (Servicebio, Wuhan, China). After different treatments, the cells were collected, resuspended, and incubated with 5 µL of Annexin V and 5 µL of PI. After incubation for 20 min in the dark at room temperature, the cells were stained for 15 min, and a flow cytometer (BD Biosciences, San Jose, USA) was used to detect apoptosis. The data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA). Western blot analysis H9C2s were pre-treated with U0126 (20 µmol/L) (specific inhibitors of the ERK1/2 pathway) or LY294002 (50 µmol/L) (specific inhibitors of the PI3K-Akt pathway) for 1 h before 3D bioprinting. After bioprinting, the scaffolds were treated with LIPUS or sham irradiation and then lysed with GelMA Lysis Buffer for 2 h. The H9C2 cells were isolated by centrifugation and lysed in RIPA lysis buffer, after which the proteins were obtained using centrifugation. Protein concentrations were determined using the Bradford protein assay kit. The protein samples were separated using SDS-PAGE and then electro-transferred onto polyvinylidene fluoride membranes (Millipore). After washing, the membranes were blocked with 5% skim milk for 1 h, and then incubated overnight at 4 °C with specific primary antibodies for Bcl2, Bax, Caspase- 3 (all from ASPEN, Wuhan, China), ERK1/2, phospho-ERK1/2, PI3K, phospho-PI3K, Akt, and phospho-Akt (all from ASPEN). After washing three times, the membranes were incubated for 1 h at room temperature Page 5/22 Page 5/22 with secondary antibodies (ASPEN). The BeyoECL Plus kit was used for color development according to the manufacturer’s instructions (Beyotime, Shanghai, China). The protein bands were quantified by densitometry scanning using the AlphaEaseFC 4.0 software (Alpha Innotech Corp, San Leandro, CA, USA). The quantitative densitometry values of all proteins were normalized to those of GAPDH. with secondary antibodies (ASPEN). The BeyoECL Plus kit was used for color development according to the manufacturer’s instructions (Beyotime, Shanghai, China). The protein bands were quantified by densitometry scanning using the AlphaEaseFC 4.0 software (Alpha Innotech Corp, San Leandro, CA, USA). The quantitative densitometry values of all proteins were normalized to those of GAPDH. Statistical analysis At least three independent experiments were performed for all tests. All data were analyzed using SPSS 25.0 (SPSS, Inc., Chicago, IL, USA) and expressed as the mean ± standard deviation. A two-sample t-test or one-way analysis of variance was used to analyze continuous variables across groups. All statistical tests were two-tailed, and a value of P < 0.05 was considered to indicate statistically significant results. Determination of the optimal frequency and exposure time of LIPUS After successful bio-fabrication of the GelMA scaffolds (Figure 1), the scaffolds were transferred into 6- well plates for LIPUS exposure or sham irradiation. To determine the optimal frequency and exposure time of LIPUS for ensuring H9C2s viability, we first performed a CCK-8 assay. As shown in Figure 2A–C and Table 1, the mean optical density values of the LIPUS groups were much higher than those of the control group after 4 and 7 days of LIPUS exposure. The linear increase in absorbance indicated that cell viability was increased in both groups over 7 days. However, the trends in cell viability were not entirely consistent in the LIPUS and control groups; the absorbance of the former was much higher, indicating that performing LIPUS at different frequencies is beneficial for the proliferation of H9C2 cells when using the same cell number of cells in all groups. Moreover, GelMA scaffolds with H9C2 cells treated at a frequency of 0.5 MHz for 10 min showed significantly higher viability compared to that in the control group (P < 0.05) and other frequencies and exposure time groups; based on these results, the optimal conditions were determined. We then performed flow cytometry to analyze the cell cycle distribution of the LIPUS-treated and control groups to confirm our results. As shown in Figure 3 and Table 2, the number of cells in G0/G1 phase after LIPUS treatment was significantly decreased (P < 0.05) compared to that in the control group, whereas cells in the S and G2/M phases were increased significantly (P < 0.05) after one day of LIPUS treatment or sham irradiation. The proportion of cells in the S and G2/M phases reached a maximum at a frequency of 0.5 MHz and exposure time of 10 min. These results demonstrate that LIPUS can induce H9C2 cells in GelMA scaffolds to enter the S and G2/M phases from the G0/G1 phase, indicating H9C2 cell proliferation. Thus, LIPUS at a frequency of 0.5 MHz and exposure time of 10 min was determined to be the optimal conditions and applied in the following experiments. Page 6/22 We confirmed our results by performing live/dead cell double staining, in which living cells and dead cells were labeled as green and red, respectively. Determination of the optimal frequency and exposure time of LIPUS At one day after LIPUS exposure or sham irradiation, the viabilities of cells in the LIPUS and control groups were 75.8 ± 1.1% and 66.8 ± 1.8%, respectively (P < 0.05). After seven days of culture, the viability of cells in the LIPUS group increased by 90.8 ± 2.1%, whereas that in the control group slightly increased to 69.8 ± 1.2% (P < 0.05, Figure 2D–E). 3D images of the cell-laden GelMA scaffolds were also obtained after culture for 7 days using Z-stack scanning to confirm these results. Effects of LIPUS on apoptosis of H9C2s To investigate the protective effects of LIPUS on cardiomyocyte apoptosis in GelMA scaffolds, Annexin V/PI analysis was performed after 24 h of LIPUS exposure or sham irradiation. As shown in Figure 4A and B, compared with the control group, cardiomyocytes in the LIPUS groups at all exposure times exhibited a significantly decreased apoptotic rate; the lowest rate was observed at 10 min of exposure time. Furthermore, the expression of apoptosis-related proteins was evaluated. LIPUS increased Bcl-2 levels and decreased the Bax and cleaved caspase-3 levels compared to that in the control group (Figure 4C–G). These results suggest that LIPUS inhibits the apoptotic signaling pathway. Activation of ERK1/2 and PI3K-Akt in LIPUS-induced GelMA scaffolds Extracellular signal-regulated kinases (ERK1/2) and PI3K-Akt signaling pathways have been reported to play important roles in cell proliferation. Western blotting was conducted to determine whether the ERK1/2 and PI3K-Akt signaling pathways are involved in protective effects of LIPUS exposure. The phosphorylation of ERK1/2, PI3K, and Akt protein expression was markedly increased by LIPUS (Figure 5A, P < 0.01). The protein levels of phospho-ERK1/2, phospho-PI3K, and phospho-Akt significantly increased and peaked in 0.5MHz-10min LIPUS treatment group.; therefore, this treatment time was selected for subsequent western blot analysis. These results demonstrate that ERK1/2 and PI3K-Akt signaling is activated following LIPUS treatment. Discussion 3D bioprinting is among the most advanced therapeutic strategies for fabricating complicated implants with biomimetic features, which exhibit both the native physiochemical and biomechanical characteristics of humans [25, 26]. Although the bioprinting technique is in its infancy and analogs with full biological functionality have not yet been generated, 3D bioprinting is widely used to create bone, cartilage, neural, and cardiovascular tissues and is employed in studies of the molecular basis of cardiac function, as well as to explore related signaling pathways [27-30]. Moreover, preparing myocardial cells with high viability and that function for a longer time in a 3D environment, which will enable 3D- bioprinting to be translated from the experimental stage to clinical practice, remains difficult. In this study, we first combined 3D bioprinting with LIPUS to investigate the impact of LIPUS on the proliferation of H9C2s in 3D printed scaffold-based cardiac patches and found that LIPUS promoted cell viability and inhibited apoptosis of cardiomyocytes via the PI3K-AKT and ERK1/2 pathways in 3D bioprinted scaffolds. A simplified process of printing and tolerant conditions of shaping can reduce the time in which cells are not in the culture environment to prevent the loss of cell viability [31]. Thus, we performed this study using a desktop 3D-bioprinter with an acceptable speed range to shorten the printing time. Moreover, GelMA, which forms covalently crosslinked hydrogels under UV light exposure in the presence of a photoinitiator, has received attention in the field of biomedical applications for building constructs with various 3D architectures because of its good biocompatibility, low immunogenicity, and ability to promote cell growth [32-34]. Nevertheless, one of the main challenges to using GelMA in engineered scaffolds is to balance the physical printability and biological biocompatibility, as a high concentration of GelMA (≥10% w/v) has excellent printability but decreased cell viability. In contrast, low levels of GelMA (≤5% w/v) show high cell stability and viability and low viscosity of GelMA bio-inks, leading to a slow change from liquid to semi-solid. Because of this, low-concentration GelMA bio-inks exhibit poor processability during the 3D printing process [35, 36]. In this study, we selected 6% w/v as the final concentration of GelMA for 3D bioprinting. Inhibition of ERK1/2 and PI3K-Akt signaling pathways reduces LIPUS-induced proliferation of H9C2s in GelMA scaffolds Inhibition of ERK1/2 and PI3K-Akt signaling pathways reduces LIPUS-induced proliferation of H9C2s in GelMA scaffolds To determine whether the ERK1/2 and PI3K-Akt signaling pathways contributed to the proliferative effect of LIPUS, H9C2s were pretreated with the inhibitors U0126 (10 µmol/L) or LY294002 (50 µmol/L) respectively, for 1 h before 3D-bioprinting. The protein levels of phospho-ERK1/2 and phospho-Akt in the LIPUS-treated group significantly decreased after pretreatment with U0126 and LY294002, respectively (Figure 5B–C, P < 0.01), indicating that these agents effectively blocked activation of ERK1/2 and Akt in LIPUS-treated H9C2s. Cell proliferation was assessed using CCK-8 and live/dead cell double staining assays. Cell viability in the LIPUS-treated group was significantly higher than that in the LIPUS group pretreated with U0126 or LY294002 (LIPUS + inhibitor groups) and the control group after 1 day of LIPUS treatment (P < 0.01, Page 7/22 Page 7/22 Figure 6A). The live/dead cell double staining assays also showed that the viable ratio of H9C2s in 3D- printed GelMA scaffolds increased significantly after LIPUS treatment compared to that in the control group, and that this increase was blocked by pre-treatment with U0126 or LY294002 (P < 0.01, Figure 6B– C). These results demonstrate that the ERK1/2 and PI3K-Akt signaling pathways are involved in promoting H9C2S cell proliferation following LIPUS of 3D-printed GelMA scaffolds. Figure 6A). The live/dead cell double staining assays also showed that the viable ratio of H9C2s in 3D- printed GelMA scaffolds increased significantly after LIPUS treatment compared to that in the control group, and that this increase was blocked by pre-treatment with U0126 or LY294002 (P < 0.01, Figure 6B– C). These results demonstrate that the ERK1/2 and PI3K-Akt signaling pathways are involved in promoting H9C2S cell proliferation following LIPUS of 3D-printed GelMA scaffolds. Discussion To improve printability, this material was placed at 4 °C for approximately 30 s to accelerate the change in state from liquid to semi-solid before 3D bioprinting and immediately after 3D bioprinting, and the GelMA scaffolds were exposed to UV light for 30 s, which is a relatively long time, to maintain the structural integrity and precision after printing. The cell-laden GelMA scaffolds maintained their structural integrity for more than 30 days, which may ensure the consistency of the cell growth space and provides a promising material for in vivo studies. Page 8/22 Page 8/22 As a non-toxic and noninvasive form of mechanical stimulation, LIPUS has been widely used in fracture healing, wound healing, drug delivery, thrombolysis, and chemotherapy [37, 38]. Moreover, in vivo and in vitro studies demonstrated that LIPUS, depending on the irradiated parameters, promotes cell proliferation, osteogenic differentiation, and extracellular matrix formation [39]. Xie et al. found that LIPUS had a positive effect on the proliferation of human bone marrow mesenchymal stem cells at an optimal intensity of 50 or 60 mW/cm2 (frequency = 1.5 MHz, exposure time = 5 min) [40]. Another study showed that the optimal working parameters for LIPUS were 150 mW/cm2 when combined with 3D bioprinting tissue scaffolds [41]. Furthermore, Gao et al. verified the impact of LIPUS on different sources of mesenchymal stem cells and showed that both 250 and 750 mW/cm2 had the same effect on cell proliferation; additionally, different types of mesenchymal stem cells responded differently to various LIPUS treatment regimens, with the ultrasound exposure duration less important than the intensity [42]. In addition to promoting cell proliferation, LIPUS was reported to inhibit cell growth. Katiyar et al. found that the optimal intensity of LIPUS for promoting proliferation was approximately 75 mW/cm2; when the intensity was increased to 465 mW/cm2, LIPUS significantly reduced the proliferation of MC3T3-E1 cells [43]. Taken together, these studies suggest that different types of cells under varying conditions may respond differently to different LIPUS stimulation. In this study, we combined LIPUS and cell-laden 3D- bioprinted GelMA scaffolds and found that the optimal frequency was 0.5 MHz (intensity = 1.0 W/cm2, exposure time = 10 min) in H9C2s. Bcl-2 is a member of the Bcl-2 family of proteins that inhibits pro-apoptotic molecules and plays a vital role in cell survival [44]. Discussion Bax is also a member of the Bcl-2 family and induces apoptosis via cytochrome C- mediated cleavage of caspase-3 [45]. In this study, the effects of LIPUS exposure on the expression levels of Bcl-2, Bax, and cleaved caspase-3 were evaluated. Increased expression of Bcl-2 and decreased expression of Bax and cleaved caspase-3 were observed in H9C2s in response to LIPUS irradiation.We also found that LIPUS-activated ERK1/2 and PI3K-Akt signaling pathways induced H9C2s to enter the S and G2/M phases from the G0/G1 phase. ERK1/2 and PI3K-Akt signaling pathways are important pathways that mediate cell cycle progression and promote cell survival and proliferation. We investigated the roles of the ERK1/2 and PI3K-Akt signaling pathways in LIPUS-induced cell proliferation. The levels of p-ERK1/2 and p-Akt increased significantly after LIPUS irradiation compared to that in the control group. In addition, when LIPUS-treated H9C2s were pre-treated with the inhibitors U0126 and LY294002, the amounts of p-ERK1/2 and p-Akt were significantly decreased, thus inhibiting LIPUS-induced H9C2s proliferation in 3D-printed GelMA scaffolds. Therefore, LIPUS likely promotes H9C2s proliferation via phosphorylation and activation of ERK1/2 and PI3K-Akt. Although the precise mechanism by which LIPUS activates these signaling pathways remains unclear, it has been hypothesized that, initially, these acoustic radiation forces can sensitize the H9C2s in 3D-printed GelMA scaffolds through its mechanosensitive surface structure and, consequently, induce morphological deformation [46]. 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Author Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by HYG,JY, CQ, WH, YYT,ZYX, TTT, ZX and ZQ. The first draft of the manuscript was written by HYG and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Availability of Data and Material The data that support this study are available from the corresponding author upon reasonable request. Availability of Data and Material The data that support this study are available from the corresponding author upon reasonable request. Ethics approval: Not applicable. References Coaxial extrusion bioprinting of 3D microfibrous constructs with cell- favorable gelatin methacryloyl microenvironments[J]. Biofabrication, 2018,10(2):24102. [34] Liu W, Zhang Y S, Heinrich M A, et al. Rapid Continuous Multimaterial Extrusion Bioprinting[J]. Adv Mater, 2017,29(3). 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Tables Table 2 Effects of LIPUS on the cell cycle phase distribution of H9C2 cells in 3D-printed GelMA scaffolds he cell cycle phase distribution of H9C2 cells in 3D-printed GelMA scaffolds le 2 Effects of LIPUS on the cell cycle phase distribution of H9C2 cells in 3D Tables Page 13/22 ptical density (OD) values at 450 nm of each group at different days after LIPUS treatm Groups Frequency Exposure time 5min 10min 15min Day 0 Control group 0 0.296±0.025 0.290±0.021 0.306±0.032 LIPUS groups 0.5MHz 0.289±0.044 0.309±0.033 0.312±0.041 1.0MHz 0.301±0.043 0.319±0.037 0.291±0.033 1.5MHz 0.300±0.037 0.294±0.031 0.305±0.032 2.0MHz 0.299±0.027 0.305±0.034 0.309±0.036 Day 1 Control group 0 0.351±0.016 0.351±0.016 0.351±0.016 LIPUS groups 0.5MHz 0.389±0.036 0.416±0.028* 0.410±0.028 1.0MHz 0.375±0.022 0.384±0.021 0.396±0.030 1.5MHz 0.362±0.040 0.376±0.014 0.383±0.027 2.0MHz 0.370±0.023 0.376±0.031 0.400±0.032 Day 4 Control group 0 0.695±0.109 0.695±0.109 0.695±0.109 LIPUS groups 0.5MHz 1.201±0.029* 1.426±0.039* 1.367±0.062* 1.0MHz 1.153±0.157* 1.126±0.068# 1.145±0.076# 1.5MHz 1.129±0.146* 1.133±0.073# 1.157±0.073# 2.0MHz 1.132±0.086* 1.166±0.071# 1.228±0.079 Day 7 Control group 0 1.051±0.129 1.051±0.129 1.051±0.129 LIPUS groups 0.5MHz 1.483±0.065* 1.628±0.056* 1.480±0.100* 1.0MHz 1.466±0.087* 1.413±0.066# 1.427±0.073 1 5MHz 1 456±0 033* 1 473±0 081* 1 473±0 089* Table 1 Optical density (OD) values at 450 nm of each group at different days after LIPUS treatment (n=6) LIPUS: low-intensity pulsed ultrasound. Data were mean (SD) unless otherwise stated. For all analyses, Kruskal-Wallis test were used in this study and when the overall p values greater than 0.05, we did not perform group comparisons, p values for all analyses were adjusted for multiple comparisons to control the false-discovery rate using the adaptive Benjamini-Hochberg procedure. Compared with control group, <0.05 and compared with 0.5MHz group, #<0.05. able 2 Effects of LIPUS on the cell cycle phase distribution of H9C2 cells in 3D-printed GelMA scaffolds Group Frequency/ET Cycle phase distribution (%, n = 3) G0/G1 S G2/M S+G2/M Control Sham irradiation 75.49±2.06 18.73±2.51 5.79±0.58 24.51±2.92 LIPUS groups 0.5MHz/5min 62.37±1.53 26.81±1.15* 10.81±1.36 37.62±1.89* 0.5MHz/10min 56.14±2.21* 31.57±2.98* 12.29±1.74* 43.86±1.52* 0.5MHz/15min 60.94±1.57* 26.56±1.34* 12.50±1.78* 39.06±1.66* 1.0MHz/5min 63.66±2.50* 24.35±2.68* 11.99±1.18* 36.34±2.08* 1.0MHz/10min 65.28±2.24 24.32±2.91* 10.39±1.65 34.72±1.53# 1.0MHz/15min 64.70±2.49 24.30±2.49 10.99±1.17* 35.30±2.16* 1.5MHz/5min 66.38±1.96 23.02±1.15 10.59±1.89 33.62±1.49# 1.5MHz/10min 66.11±2.11 24.44±1.58* 9.44±1.09 33.88±1.96# 1.5MHz/15min 68.04±2.08# 23.45±1.76 8.51±2.19# 31.96±2.89# 2.0MHz/5min 72.13±2.78# 20.08±1.49# 8.12±1.73# 28.40±2.63# 2.0MHz/10min 70.33±1.51# 20.18±2.60# 9.43±3.21# 29.61±1.94# 2.0MHz/15min 71.61±6.78# 19.75±2.08# 8.71±3.21# 28.57±2.11# PUS: low-intensity pulsed ultrasound. Data were mean (SD) unless otherwise stated. For all analysis, ruskal-Wallis test were used in this study and p values were adjusted for multiple comparisons to ontrol the false-discovery rate using the adaptive Benjamini-Hochberg procedure. Compared with contro oup, <0.05 and compared with 0.5MHz/10min group, #<0.05. Figures Page 15/22 Figure 1 Schematic illustration of bio-ink formulation, scaffold printing and LIPUS treatment. From left to right: images of GelMA and H9C2s and their formulation process; simple illustration of extrusion-based 3D printing working principle and UV light for photo-crosslink; the photo and optical microscopic image of 3D-bioprinted GelMA scaffolds; scale bar=100μm; Apparatus and diagram of LIPUS-exposure system. Figure 1 Schematic illustration of bio-ink formulation, scaffold printing and LIPUS treatment. From left to right: images of GelMA and H9C2s and their formulation process; simple illustration of extrusion-based 3D printing working principle and UV light for photo-crosslink; the photo and optical microscopic image of 3D-bioprinted GelMA scaffolds; scale bar=100μm; Apparatus and diagram of LIPUS-exposure system. Page 16/22 Fi 2 Figure 2 Figure 2 Figure 2 Effects of LIPUS on the proliferation of H9C2s in 3D-bioprinted cell-laden GelMA scaffolds. The LIPUS group and the control group were treated with different frequencies of LIPUS (frequency=0.5, 1.0, 1.5 and 2.0MHz) and sham irradiation (control) for 5 minutes (A), 10 minutes (B) or 15 minutes (C) and the proliferation of H9C2s in the LIPUS group and the control group were measured using a Cell Counting Kit- Effects of LIPUS on the proliferation of H9C2s in 3D-bioprinted cell-laden GelMA scaffolds. The LIPUS group and the control group were treated with different frequencies of LIPUS (frequency=0.5, 1.0, 1.5 and 2.0MHz) and sham irradiation (control) for 5 minutes (A), 10 minutes (B) or 15 minutes (C) and the proliferation of H9C2s in the LIPUS group and the control group were measured using a Cell Counting Kit- Page 17/22 Page 17/22 8 assay before and 1, 4 and 7days after LIPUS treatment (n = 6). P < 0.05 and *P < 0.01 vs Control; mean ± SEMSD. (D) Cell 8 assay before and 1, 4 and 7days after LIPUS treatment (n = 6). P < 0.05 and *P < 0.01 vs Control; mean ± SEMSD. (D) Cell viability of H9C2s in LIPUS group and control group after 1, 4 and 7 days for LIPUS treatment (frequency=0.5MHz, time=10min) or sham irradiation (n = 3). (E) 2D and 3D confocal images of live (green, calcein-AM)/dead (red, PI) stained cells in 3D-bioprinted cell-laden GelMA scaffolds after 1, 4 and 7days for LIPUS treatment (frequency=0.5MHz, time=10min) or sham irradiation, scale bar=100μm. Page 18/22 g re 3 Figure 3 Effects of LIPUS on cell cycle phase distribution measured by flow cytometry in the control and LIPUS groups with different exposure parameters. Cells harvested were fixed and stained with propidium iodide, and their DNA contents were analyzed by flow cytometry. The result of one representative assay from three similar independent experiments is shown. x- and y-axes denote DNA content and cell number, respectively. Each phase was calculated by using the cell ModFit software. Page 19/22 Figure 4 Effect of LIPUS on cell apoptosis in 3D-bioprinted cell-laden GelMA scaffolds. (A) Representa of the flow cytometry analysis of cell apoptosis after the treatment of LIPUS for different expo (n=3). (B) The statistical results of the flow cytometry analysis of cell apoptosis. (C) and (D) W blotting of apoptosis-related factors (n=3). (E-G) Quantitative analysis of Bcl-2, Bax and cleav Fi 4 Figure 4 Effect of LIPUS on cell apoptosis in 3D-bioprinted cell-laden GelMA scaffolds. (A) Representative graphs of the flow cytometry analysis of cell apoptosis after the treatment of LIPUS for different exposure time (n=3). (B) The statistical results of the flow cytometry analysis of cell apoptosis. (C) and (D) Western blotting of apoptosis-related factors (n=3). (E-G) Quantitative analysis of Bcl-2, Bax and cleaved Caspase Page 20/22 Page 20/22 3 in different groups. Error bars represent the SD of the mean. Compared with control group, P<0.05, P<0.01. P<0.01. Figure 5 Phosphorylation of extracellular signal-regulated kinases (ERK1/2) and PI3K-Akt in H9C2s in 3D- bioprinted cell-laden GelMA scaffolds. (A) H9C2s were stimulated by LIPUS (frequency=0.51MHz, ET=10 min). Cells were collected for Western blot analysis at the indicated time point after LIPUS treatment. Phosphorylation of ERK1/2 and PI3K-Akt induced by LIPUS were analyzed by Western blot (n=3). (B, C) H9C2s were stimulated by LIPUS (frequency=1MHz, ET=10 min) in the presence of the specific inhibitors of ERK1/2 (U0126, 10 μmol/L) and PI3K-Akt (LY294002, 50 μmol/L). ERK1/2, phospho-ERK1/ 2, PI3K, Figure 5 Phosphorylation of extracellular signal-regulated kinases (ERK1/2) and PI3K-Akt in H9C2s in 3D- bioprinted cell-laden GelMA scaffolds. (A) H9C2s were stimulated by LIPUS (frequency=0.51MHz, ET=10 min). Cells were collected for Western blot analysis at the indicated time point after LIPUS treatment. Phosphorylation of ERK1/2 and PI3K-Akt induced by LIPUS were analyzed by Western blot (n=3). (B, C) H9C2s were stimulated by LIPUS (frequency=1MHz, ET=10 min) in the presence of the specific inhibitors of ERK1/2 (U0126, 10 μmol/L) and PI3K-Akt (LY294002, 50 μmol/L). ERK1/2, phospho-ERK1/ 2, PI3K, phospho-PI3K, Akt and phospho-Akt were analyzed by Western blot at 10 min after LIPUS treatment and sham irradiation (n=3). P <0.05 and P <0.01. Page 21/22 Figure 6 Effects of the two inhibitors (U0126 and LY294002) on LIPUS-induced proliferation of H9C2s in 3D- bioprinted cell-laden GelMA scaffolds. (A) Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay before LIPUS irradiation and at 7 days after irradiation in the presence or absence of U0126 and LY294002 (n=6). Cell proliferation was further detected by live and dead cell double staining assays in the Figure 6 Effects of the two inhibitors (U0126 and LY294002) on LIPUS-induced proliferation of H9C2s in 3D- bioprinted cell-laden GelMA scaffolds. (A) Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay before LIPUS irradiation and at 7 days after irradiation in the presence or absence of U0126 and LY294002 (n=6). Cell proliferation was further detected by live and dead cell double staining assays in the presence or absence of (B and D) LY294002 and (C and E) UO126 (n=3). P <0.05 and *P <0.01. Scale bar=100μm. Effects of the two inhibitors (U0126 and LY294002) on LIPUS-induced proliferation of H9C2s in 3D- bioprinted cell-laden GelMA scaffolds. (A) Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay before LIPUS irradiation and at 7 days after irradiation in the presence or absence of U0126 and LY294002 (n=6). Cell proliferation was further detected by live and dead cell double staining assays in the presence or absence of (B and D) LY294002 and (C and E) UO126 (n=3). P <0.05 and **P <0.01. Scale bar=100μm. Page 22/22
https://openalex.org/W4312185590	https://e-journal.upr.ac.id/index.php/jpm-upr/article/download/7971/4201	Indonesian	null	Meningkatkan Pendapat Keluarga Melalui Pengolahan Buah Pepaya Muda Pada Kelompok Ibu PKK di Kelurahan Kalampangan Kota Palangka Raya	Pengabdian Kampus/Pengabdian Kampus: Jurnal Informasi Kegiatan Pengabdian Pada Masyarakat	2,022	cc-by	2,314	Jurnal Pengabdian Kampus Vol 9. No.2, 121-126, Desember 2022 Jurnal Pengabdian Kampus Vol 9. No.2, 121-126, Desember 2022 ISSN 2252-8628(p); 2776-091X(e) Abstrak Kegiatan pengabdian pada masyarakat telah dilaksanakan di Jalan Doho Kelurahan Kalampangan Kecamatan Sebangau Kota Palangka Raya, sasaran kegiatan adalah Kelompok ibu-ibu PKK Kelurahan Kalampangan Kecamatan Sebangau Kota Palangka Raya yang kesehariannnya sebagai ibu rumah tangga atau membantu suami di kebun. Tujuan dari kegiatan ini adalah mengolah buah pepaya muda menjadi produk olahan makanan yang mempunyai nilai jual untuk membangun usaha baru bagi ibu-ibu PKK. Tahapan kegiatan terdiri atas: 1) Sosialisasi dan Penyuluhan pembuatan abon dan keripik dari buah pepaya, 2) Pelatihan dan demontrasi pembuatan abon pepaya dan keripik pepaya, 3) pembinaan/pendampingan dan monitoring selama kegiatan berlangsung melalui tinjauan lapang maupun media telepon. Hal yang perlu diperhatikan dalam pengolahan buah papaya adalah: 1) Ada perbedaan kematangan buah papaya muda untuk bahan untuk pembuatan abon dan keripik, 2) ketipisan irisan buah, 3) pembuatan abon memerlukan perendaman dengan air garam, pencucian, dan penirisan, 5) Pencampuran irisan bahan abon dengan tepung dilakukan bertahap, dan 6) digoreng dengan minyak cukup banyak dan api sedang supaya tingkat kematangan seragam dan produk hasil olahan benar-benar kering merata. Kelurahan Kalampangan merupakan jalan utama provinsi yang menghubungkan Kalimantan Tengah dengan Kalimantan Selatan, memiliki akses jalan darat yang memungkinkan masyarakatnya untuk berwirausaha meningkatkan pendapatan karena letaknya yang strategis. Hal ini memberi peluang bagi warga sekitar untuk berwirausaha jajanan ringan seperti abon dan keripik pepaya. Apabila dijual segar, hasil yang didapat tidak seberapa dibandingankan apabila pepaya dijual dalam produk olahan makanan ringan siap saji yang bisa dinikmati oleh pengguna jalan. Kata kunci: abon, keripik, pepaya muda yang memiliki harga jual tinggi sangat membantu meningkatkan perekonomian masyarakat pedesaaan terutama kelompok ibu-ibu PKK Kelurahan Kalampangan yang umumnya berprofesi sebagai ibu rumah tangga atau membantu suami di kebun. Berdasarkan hasil survei pendahuluan ke Kelurahan Kalampangan sebagai lokasi mitra, umumnya pemanfaatan tanaman pepaya masih sebatas untuk konsumsi buah segar maupun pemanfaatan daun pepaya dan buah pepaya muda hanya untuk sayuran. Kegiatan pelatihan ini diharapkan dapat membuka peluang usaha baru sehingga menambah penghasilan dan pendapatan bagi ibu rumah tangga. Pengolahan buah papaya menjadi berbagai jenis olahan adalah merupakan salah satu solusi untuk memanfaatkan buah pepaya menjadi produk olahan yang lebih beragam, sehingga buah papaya tidak hanya untuk dimakan dalam keadaan segar. Daging buah pepaya muda selain dapat dimasak sebagai Kata kunci: abon, keripik, pepaya muda Metode Pelaksanaan Bahan yang digunakan adalah: Bahan yang digunakan adalah: a. 3 buah pepaya muda ukuran sedang b. 250 gr tepung beras c. 250 gr tepung tapioka dicampur jadi d. 50 gr tepung bumbu satu e. 1 sdt merica f. 2 sdm ketumbar bubuk g. Penyedap rasa Bumbu yang dihaluskan: a. 1 sdm garam b. 4 siung bawang merah c. 5 siung bawah putih d. 1 ruang jahe e. 2 buah serai f. minyak goreng Pendahuluan Pepaya (Carica papaya L.) merupakan tanaman yang banyak dibudidaya dan hidup di sekitar pekarangan rumah. Buah dari tanaman ini tergolong populer dan digemari oleh masyarakat karena selain harganya relatif murah juga kandungan gizinya cukup tinggi. Buah pepaya mengandung kalori, karbohidrat, protein, lemak, serat, antioksidan, vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, asam folat, vitamin C, vitamn E dan vitamin K (Almatsier dan Sunita, 2010). Ditambahkan oleh Kemenkes (2022), bahwa buah pepaya juga dapat melancarkan pencernaan, meningkatkan imunitas tubuh, baik untuk kesehatan jantung, menyehatkan kulit dan rambut, sebagai sun protection (melindungi kulit dari sinar UV) serta dapat menyembuhkan luka. Harga jual pepaya adalah sangat murah, sehingga diversifikasi produk buah pepaya menjadi bentuk olahan 121 Jurnal Pengabdian Kampus Vol 9. No.2, 121-126, Desember 2022 Jurnal Pengabdian Kampus Vol 9. No.2, 121-126, Desember 2022 ISSN 2252-8628(p); 2776-091X(e) sayuran dan diolah juga menjadi berbagai sajian produk olahan seperti keripik dan abon. Tempat dan Waktu Kegiatan Kegiatan Pengabdian Kepada Masyarakat ini dilaksanakan di rumah kediaman Ketua Kelompok Mitra yaitu ibu Rukayah dan bapak Jaini yang diikuti oleh 19 orang ibu-ibu PKK. Pelaksanaan kegiatan pada bulan Desember 2020 di Jl. Doho No.03 Kelurahan Kalampangan Kecamatan Sabangau, Kota Palangka Raya. Bumbu yang dihaluskan: Tahapan Kegiatan Tahapan kegiatan pengabdian pada masyarakat rmeliputi : b. Pelatihan dan Demonstrasi Pelatihan dan demonstrasi yang telah dilaksanakan meliputi pembuatan abon pepaya dan keripik pepaya. Respon peserta sangat antusias mengikutisetiap tahapan kegiatan pembuatan abon dan keripik pepaya muda. Adapun tujuan dari kegiatan pengabdian ini adalah mengolah buah pepaya muda menjadi produk olahan makanan yang mempunyai nilai jual untuk wirausaha baru bagi ibu-ibu PKK. 2. Pembuatan keripik pepaya Alat dan bahan-bahan yang digunakan adalah: Alat dan bahan-bahan yang digunakan adalah: Alat dan bahan-bahan yang digunakan adalah: a. Sosialisasi dan Penyuluhan Langkah-langkah pembuatannya: Langkah-langkah pembuatannya: Sosialisasi dan penyuluhan dilakukan pada awal kegiatan, meliputi penyampaian materi yang dilakukan oleh tim dosen agar kegiatan demonstrasi dapat berjalan sesuai dengan harapan, yaitu dengan menjelaskan rangkaian kegiatan pembuatan abon dan keripik pepaya muda serta memotivasi kalayak sasaran ibu-ibu PKK untuk berwirausaha skala home industry. Pada sesi penyuluhan berapa peserta juga bertanya secara aktif. a. Kulit pepaya muda dikupas sampai bersih, kemudian pepaya dibelah menjadi empat bagian b. Biji-biji halus berwarna putih yang menempel pada daging buah dibuang dan dibersihkan c. Semua bumbu dihaluskan. d. Pepaya diparut kasar (diameter ± 2-3 mm dan panjang ± 4 cm) dengan menggunakan alat kemudian direndam dengan air garam, setelah itu dicuci sampai bersih dan ditiriskan sampai kandungan airnya rendah e. Mencampurkan parutan pepaya dengan bumbu sampai rata f. Mencampurkan adonan pepaya secukupnya sesaat sebelum digoreng dengan tepung untuk setiap kali pengorengan, jangan dicampur sekaligus untuk menghindari parutan pepaya yang lengket dan mengumpal saat digoreng sehingga hasil abon kurang bagus g. Lalu digoreng sampai kering dan tiriskan. 122 ISSN 2252-8628(p); 2776-091X(e) Jurnal Pengabdian Kampus Vol 9. No.2, 121-126, Desember 2022 Jurnal Pengabdian Kampus Vol 9. No.2, 121-126, Desember 2022 e. Irisan buah pepaya yang sudah bersih dimasukkan kedalam adonan tepung secukupnya sesaat sebelum pengorengan e. Irisan buah pepaya yang sudah bersih dimasukkan kedalam adonan tepung secukupnya sesaat sebelum pengorengan f. Digoreng sampai kering dengan tanda kandungan air pada minyak goreng sudah berkurang (gelembung – gelembung air pada minyak goreng mulai hilang g. Saat menggoreng menggunakan minyak yang banyak dengan suhu ± 1650C selama ± 15 menit, setelah itu ditiriskan hingga dingin h. Pengemasan, keripik pepaya dapat disimpan ditoples atau plastik kemasan. h. Pengemasan, keripik pepaya dapat disimpan ditoples atau plastik kemasan. h. Pengemasan, keripik pepaya dapat disimpan ditoples atau plastik kemasan. 2. Pembuatan keripik pepaya c. Pembinaan/Pendampingan dan Monitoring a. Buah pepaya yang mengkal dan masih keras sebanyak 5 kg Pembinaan serta pendampingan dilakukan pada saat kegiatan berlangsung, demikian juga sesudah kegiatan apabila ada peserta yang perlu pendampingan. Monitoring selama kegiatan berlangsung dapat dilakukan melalui tinjauan lapang ataupun lewat media telepon. b. Pisau dan alat untuk mengupas dan memotong c. Alat untuk mengiris c. Alat untuk mengiris Hasil Yang Dicapai Areal pemukiman mitra merupakan daerah pedesaan di sekitar hutan yang merupakan desa binaan transmigrasi asal Jawa Tengah dan Yogyakarta. Penduduk Kelampangan sangat gigih untuk memperbaiki taraf hidup, sekarang mereka berhasil membuat hasil pertanian menjadi produk unggulan, sebagai pemasok sayuran Bumbu yang dihaluskan : Cara membuat keripik pepaya : a. Kupas kulit buah pepaya dan potong, lalu bersihkan biji-bijinya b. Iris buah pepaya tipis-tipis ±1 mm (mudah kering saat digoreng dan krispy) dengan menggunakan alat, lalu rendam dengan air garam dan bilas hingga bersih, lalu tiriskan Hasil Yang Dicapai Areal pemukiman mitra merupakan daerah pedesaan di sekitar hutan yang merupakan desa binaan transmigrasi asal Jawa Tengah dan Yogyakarta. Penduduk Kelampangan sangat gigih untuk memperbaiki taraf hidup, sekarang mereka berhasil membuat hasil pertanian menjadi produk unggulan, sebagai pemasok sayuran Hasil Yang Dicapai Hasil Yang Dicapai c. Mencampur tepung tapioka dan tepung beras dengan perbandingan 1 : 1 aduk hingga rata, kemudian masukkan 1 butir telur dan santan kental sekitar 3 sdm dan beri sedikit penyedap rasa, lalu masukkan bumbu yang telah dihaluskan, aduk - aduk sampai rata hingga kalis d. Tambahkan air secukupnya sampai didapatkan kekentalan yang pas 123 Jurnal Pengabdian Kampus Vol 9. No.2, 121-126, Desember 2022 ISSN 2252-8628(p); 2776-091X(e) untuk Kota Palangka Raya dan sekitarnya (Sunaryati, 2021). untuk Kota Palangka Raya dan sekitarnya (Sunaryati, 2021). b. Untuk mendapatkan rasa abon yang enak dan gurih, maka irisan bahan direndam dahulu dengan air garam untuk beberapa saat (± 20 menit) untuk menghilangkan rasa pahit dari getah papain buah pepaya muda Produk olahan buah pepaya muda berupa abon pepaya dan keripik pepaya dibuat dengan teknologi yang sederhana sehingga mudah dilakukan oleh ibu-ibu PKK. Ada beberapa bantuan dari tim pelaksana kegiatan pengabdian yaitu berupa alat-alat sederhana, seperti : alat pemotong dan pengiris buah pepaya muda untuk hasil olahan yang lebih tipis dan seragan sehingga hasil penggorengan menjadi lebih crispy, juga beberapa peralatan dapur untuk memasak serta sealer untuk pengemasan produk olahan abon dan keripik. c. Setelah proses pencucian, sebaiknya irisan bahan segera ditiriskan untuk beberapa saat, guna menurunkan kadar air bahan (diusahakan kandungan air serendah mungkin), dengan cara dikering- anginkan agar hasil abon kering dan krispy d. d. Pencampuran irisan bahan dengan tepung (tepung tapioka, tepung beras dan tepung bumbu) tidak boleh sekaligus (dicampur langsung sumuanya), melainkan bertahap/ secukupnya sesaat sebelum proses pengorengan bahan abon. Hal ini bertujuan untuk menghindari saling lengket antara irisan bahan yang cukup tipis, karena apabila irisan bahan saling lengket maka kualitas hasil abon akan jelek yaitu bagian dalam pada gumpalan bahan masih belum kering sekali sehingga hasil abon kurang krispy dan tidak tahan disimpan terlalu lama Tahapan kegiatan terdiri atas 1) Sosialisasi dan penyuluhan pembuatan abon dan keripik dari buah pepaya, 2) Pelatihan dan demontrasi pembuatan abon pepaya dan keripik pepaya, 3) pembinaan/ pendampingan dan monitoring selama kegiatan berlangsung melalui tinjauan lapang maupun telp. Kontribusi mendasar pada khalayak sasaran adalah pembuatan dua produk makanan yang terbuat dari buah pepaya muda dengan harapan menjadi wirausaha baru bagi ibu-ibu PKK guna menambah pendapatan keluarga. e. Saat penggorengan menggunakan api sedang dengan diaduk supaya menghasilkan tingkat kematangan abon yang merata dan kering Pada pembuatan keripik buah pepaya muda, ada hal – hal yang perlu dicermati, yaitu antara lain : 1. Saran a. Saat penggorengan sebaiknya menggunakan api sedang dengan minyak cukup banyak. Kelurahan Kalampangan memiliki akses jalan darat yang memungkinkan masyarakatnya untuk berwirausaha meningkatkan pendapatan karena merupakan jalan utama provinsi yang menghubungkan Kalimantan Tengah dengan Kalimantan Selatan. b. Sebelum dikemas sebaiknya hasil penggorengan abon dan keripik ditiriskan kandungan minyaknya. Kesimpulan Kelurahan Kalampangan merupakan jalan utama provinsi yang menghubungkan Kalimantan Tengah dengan Kalimantan Selatan, memiliki akses jalan darat yang memungkinkan masyarakatnya untuk berwirausaha meningkat-kan pendapatan karena letaknya yang strategis. Hal ini memberi peluang bagi warga sekitar untuk berwirausaha jajanan ringan seperti abon dan keripik pepaya. Apabila dijual segar, hasil yang didapat tidak seberapa dibandingankan apabila pepaya dijual dalam produk olahan makanan ringan siap saji yang bisa dinikmati oleh pengguna jalan. Ada perbedaan bahan untuk pembuatan abon dan keripik buah pepaya muda, yaitu untuk pembuatan abon sebaiknya menggunakan buah pepaya yang masih muda kulit buah berwarna hijau, daging buah berwarna putih kehijauan, sedangkan untuk bahan pembuatan keripik, sebaiknya menggunakan buah pepaya mengkal (daging buah pepaya mulai berwarna pink kemerahan). Pemilihan bahan tersebut berkaitan dengan rasa (kualitas hasil), yaitu untuk abon identik dengan rasa gurih sedangkan untuk keripik kecenderungan memiliki rasa gurih dan manis dengan warna hasil penggorengan keripik coklat kemerahan. Pada pembuatan abon dan keripik dari buah pepaya muda, ada beberapa hal yang perlu diperhatikan yaitu antara lain : a. Pada pembuatan abon sebaiknya menggunakan buah pepaya yang masih muda, sedangkan untuk pembuatan keripik, sebaiknya menggunakan buah pepaya mengkal. b. Saat mengiris buah pepaya sebaiknya menggunakan alat. c. Agar rasa abon pepaya enak dan gurih, maka irisan buah pepaya direndam dahulu dengan air garam untuk beberapa saat (± 20 menit). d. Setelah proses pencucian, irisan buah pepaya segera ditiriskan untuk menurunkan kadar air. Saat penggorengan diusahakan minyak cukup banyak supaya tingkat kematangan seragam dan merata. Saat penggorengan sebaiknya api jangan terlalu besar karena akan menyebabkan bagian luar bahan sudah matang namun bagian dalam masih basah (belum kering) sehingga menurunkan kualitas hasil (tidak renyah dan krispy). e. Pencampuran irisan bahan abon dengan tepung tidak boleh sekaligus, melainkan bertahap dan secukupnya sesaat sebelum proses pengorengan. Hasil Yang Dicapai Pengirisan bahan keripik harus seragam (lebar tapi tipis) untuk mendapatkan hasil keripik yang kering dan renyah, sebaiknya menggunakan alat pemotong Yang perlu diperhatikan pada saat kegiatan pelaksanaan kegiatan praktek pembuatan abon pepaya muda adalah sebagai berikut: 2. Tingkat kekentalan adonan tepung harus pas, jangan terlau kental atau encer, kaarena sangat mempengaruhi kualitas hasil penggorengan keripik, yaitu apabila terlalu kental maka hasil keripik tidak akan kering/ renyah dan mudah melempem sehingga tidak tahan disimpan terlalu lama a. Saat pengiris bahan abon harus menggunakan alat pasrah/serut, untuk menghasilkan keseragaman bentuk irisan dengan ukuran dan ketebalan yang sama supaya tingkat kematangan saat penggorengan sama dengan hasil bahan abon krispy 3. Sebelum dikemas sebaiknya hasil penggorengan abon dan keripik ditiriskan 124 ISSN 2252-8628(p); 2776-091X(e) Jurnal Pengabdian Kampus Vol 9. No.2, 121-126, Desember 2022 kandungan minyaknya supaya tidak tengik apabila disimpan lama kandungan minyaknya supaya tidak tengik apabila disimpan lama Daftar Pustaka Ana, W., Tri, C., Ateng, S., dkk (2019). Pelatihan Inovasi Produk Pangan Abon 125 ISSN 2252-8628(p); 2776-091X(e) Jurnal Pengabdian Kampus Vol 9. No.2, 121-126, Desember 2022 Pepaya Muda di Dusun Pamagersari Desa Tanjungsari Sumedang. Jurnal Pengabdian Kepada Masyarakat. Vol. 3 No.1. Anonim, 2015. Profil Desa dan Kelurahan Kalampangan. Direktorat Jenderal Pemberdayaan Masyarakat dan Desa. Departemen Dalam Negeri 2009. https://sang- innovator.blogspot.com/2017/11/ aneka-produk-olahan-dari-buah- pepaya.html Kemenkes, 2022. Buah Pepaya yang Kaya Manfaat. Direktorat Jenderal Pencegahan dan Pengendalian Penyakit. Kementerian Kesehatan Republik Indonesia Jakarta. Ramli Redi, dan Hamzah Faizah. 2017. Pemanfaatan Buah Pepaya (Carica papaya L.) dan Tomat (Lycopersicum esculentum Mill) dalam Pembuatan Fruit Leather. Program Studi Teknologi Hasil Pertanian. Jurusan Teknologi Pertanian Fakultas Pertanian Universitas Riau. Jorn Faperta Vol 4 No.1. Ramli Redi, dan Hamzah Faizah. 2017. Pemanfaatan Buah Pepaya (Carica papaya L.) dan Tomat (Lycopersicum esculentum Mill) dalam Pembuatan Fruit Leather. Program Studi Teknologi Hasil Pertanian. Jurusan Teknologi Pertanian Fakultas Pertanian Universitas Riau. Jorn Faperta Vol 4 No.1. Sunaryati, R. 2021. Usahatani Sayuran Berkelanjutan di Kelurahan Kalampangan Kecamatan Sabangau Kota Palangka Raya. Penerbit Lembaga Literasi Dayak. 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https://openalex.org/W2587427429	https://www.beilstein-journals.org/bjnano/content/pdf/2190-4286-8-43.pdf	English	null	Methods for preparing polymer-decorated single exchange-biased magnetic nanoparticles for application in flexible polymer-based films	Beilstein journal of nanotechnology	2,017	cc-by	5,803	Methods for preparing polymer-decorated single exchange-biased magnetic nanoparticles for application in flexible polymer-based films Full Research Paper Open Access Address: Université Paris Diderot, Sorbonne Paris Cité, CNRS UMR 7086 ITODYS, Case 7090, 5 rue Thomas Mann, Paris, France Email: Souad Ammar* - ammarmer@univ-paris-diderot.fr; Fayna Mammeri* - fayna.mammeri@univ-paris-diderot.fr * Corresponding author ‡ Equal contributors Keywords: assembly; ATRP; magnetic nanoparticle; exchange-bias; films; functionalization; polymerization; poly(methyl methacrylate); polystyrene; seed-mediated growth; surface Beilstein J. Nanotechnol. 2017, 8, 408–417. doi:10.3762/bjnano.8.43 Received: 12 October 2016 Accepted: 19 January 2017 Published: 09 February 2017 This article is part of the Thematic Series "Hybrid nanomaterials: from the laboratory to the market". Guest Editor: A. Taubert © 2017 Ourry et al.; licensee Beilstein-Institut. License and terms: see end of document. Introduction nano-building-blocks for the aforementioned devices. To reduce mutual magnetic attraction and aggregation as far as possible in the first stage of polymer grafting, mechanical stirring and dilute suspensions of reactants were used, even if the functio- nalization of large amounts of particles becomes difficult. We specifically graft poly(methyl methacrylate) (PMMA) and poly- styrene (PS) chains around CoxFe3−xO4@CoO. Several key pa- rameters have to be taken into account to realize a controlled polymerization reaction and especially when one aims to graft an ATRP initiator at the surface of particles: the nature of the surface (e.g., oxide or metal) and the interface between the components of the resulting hybrid, namely polymer chains and inorganic NPs (e.g., covalent, ionic, van der Waals). Several halogenated coupling agents can be used to covalently graft an organic group onto the surface of oxide NPs, e.g., organo- silanes [21-23], carboxylate [24,25] or phosphonate/phosphate molecules [26,27]. Polymer-based hybrid materials are opening the way for engi- neering new, multifunctional, flexible materials exhibiting novel properties (e.g., mechanical, magnetic, electrical, optical) due to the synergy between the two components, polymer and inorganic nanoparticles (NPs) [1]. In the case of magnetic hybrids, one of the main challenges is to avoid NP aggregation. The magnetic response of NPs to external magnetic stimulus depends strongly on their intrinsic properties (composition, size, and shape) but also on their spatial arrangement (self-assembly, dispersion in organic media, compatibility with polymers) [2,3]. If the interparticle distances are decreased too much, a collec- tive magnetic glass state is set up, reducing magnetization and coercivity because of inhomogeneous and frustrated macrospin cluster freezing [4-6]. Ideally, flexible magnetic devices require dense but well-separated magnetic NPs to decrease interparticle interactions, particularly dipolar ones [7,8]. The general strategy for such a purpose consists of forming core–shell hybrid structures in which the shell consists of a corona of polymer chains grafted onto the inorganic NP surface. Among the available polymer grafting processes, living-radical polymerization (e.g., atom-transfer radical polymerization (ATRP), reversible addition–fragmentation chain transfer (RAFT) or nitroxide-mediated polymerization (NMP)) makes it possible to establish robust polymer–particle bonds and then grow polymer brushes of controlled molecular weight and poly- dispersity with a satisfactory grafting density. The polymer chains also stabilize the inorganic NPs with respect to the ambient atmosphere and provide compatibility with the result- ing polymer matrix. Abstract Background: Magnetic nanoparticles (NPs) must not only be well-defined in composition, shape and size to exhibit the desired properties (e.g., exchange-bias for thermal stability of the magnetization) but also judiciously functionalized to ensure their stability in air and their compatibility with a polymer matrix, in order to avoid aggregation which may seriously affect their physical proper- ties. Dipolar interactions between NPs too close to each other favour a collective magnetic glass state with lower magnetization and coercivity because of inhomogeneous and frustrated macrospin cluster freezing. Consequently, tailoring chemically (through sur- face functionalization) and magnetically stable NPs for technological applications is of primary importance. Results: In this work, well-characterized exchange-biased perfectly epitaxial CoxFe3−xO4@CoO core@shell NPs, which were isotropic in shape and of about 10 nm in diameter, were decorated by two different polymers, poly(methyl methacrylate) (PMMA) or polystyrene (PS), using radical-controlled polymerization under various processing conditions. We compared the influence of the synthesis parameters on the structural and microstructural properties of the resulting hybrid systems, with special emphasis on sig- nificantly reducing their mutual magnetic attraction. For this, we followed two routes: the first one consists of the direct grafting of bromopropionyl ester groups at the surface of the NPs, which were previously recovered and redispersed in a suitable solvent. The second route deals with an “all in solution” process, based on the decoration of NPs by oleic acid followed by ligand exchange with the desired bromopropionyl ester groups. We then built various assemblies of NPs directly on a substrate or suspended in PMMA. Conclusion: The alternative two-step strategy leads to better dispersed polymer-decorated magnetic particles, and the resulting nanohybrids can be considered as valuable building blocks for flexible, magnetic polymer-based devices. 408 408 Beilstein J. Nanotechnol. 2017, 8, 408–417. Introduction Organosilanes present the disadvantage of condensing after hydrolysis and leading to a thin shell of polysiloxane whose structure and thickness cannot be well controlled. Carboxylates can be degrafted in solution [28], and are, consequently, not the best candidates for the desired applications. Phosphates and phosphonates are suitable for functionalizing iron and silicon oxide surfaces [29,30] through covalent bonds, in mono-, bi- or tridentate modes; but to date, they have not been used for cobalt oxide and ferrite surfaces. In the case of PMMA, we followed two routes: the first one consists of a direct grafting of bromopropionyl ester molecules ob the surface of ENPs that were previously recovered and redispersed in a suitable solvent. The second uses an “all in solution” process, based on a prior decoration of ENPs by oleic acid ligands, which were subsequently exchanged by the desired bromopropionyl ester species. In the case of PS, we fol- lowed exclusively the first route, but in all the cases, the polymer chains were grown by ATRP (see the general synthe- sis scheme summarized in Figure 1), acting on the NP/mono- mer weight ratio and on the polymerization time parameters. The resulting nanohybrids were then characterized with special emphasis on the effect of the reaction parameters on their main microstructural properties and taking into account the fact that PS polymerizes more slowly than PMMA. This strategy has been widely investigated with magnetic NPs. To date, most NPs studied were of iron oxide [9-14]. Exchange- biased NPs (ENPs) have been scarcely considered [15] despite their improved magnetic properties. These particles consist of ferro- or ferrimagnetic (F) cores coated with nanocrystalline antiferromagnetic (AF) layers, and exhibit exchange coupling at the F–AF interface (see for instance [16-18]), leading to an en- hanced effective magnetic anisotropy constant (Keff) and a higher temperature of transition from a magnetically blocked state to a superparamagnetic one (TB) [19,20]. Focusing on such particles, in this work, we propose various material processing routes to prepare weakly interacting and densely arranged hybrid ENPs. The ENPs used were prepared by seed-mediated growth in a polyol medium; they consist of ferrimagnetic CoxFe3−xO4 single crystals, almost isotropic in shape and of about 10 nm in diameter, coated in a perfectly epitaxial fashion with an antiferromagnetic CoO polycrystalline shell about 1 nm thick, as described in previous work [16]. We then controlled their surface functionality to tentatively design well-tailored Results and Discussion We chose to work on two very common thermoplastic poly- mers: PMMA and PS, which have very similar properties (e.g., specific temperatures, mechanical properties, density) but rather different side chains; PMMA has an aliphatic ester group while PS has a more rigid aromatic ring. However, despite their struc- 409 Beilstein J. Nanotechnol. 2017, 8, 408–417. Figure 1: Polymer-decorated exchange-biased CoxFe3−xO4@CoO magnetic nanoparticles synthesis. Figure 1: Polymer-decorated exchange-biased CoxFe3−xO4@CoO magnetic nanoparticles synthesis. 2-phosphonooxy-2-bromo-2-methylpropanoate, was grafted onto the ENP surface. Under the conditions used (see Experi- mental), the grafting density of the initiator molecules is 2.8 molecules nm−2, in good agreement with previous studies [27]. Then, the PMMA chains were grown by ATRP at 30 °C in the presence of cuprous bromide and N,N,N′,N′,N′′- pentamethyldiethylenetriamine (PMDTA) to form the tural and reactivity differences, we followed the same reaction pathway to elaborate ENPs decorated by the two polymers (Figure 1). Growth of PS chains Styrene polymerizes more slowly than methyl methacrylate. Yousi et al. [34] demonstrated that the propagation rate, for similar conversion yields, can be increased by catalysts. Masson et al. [35] reported an increase in the styrene polymerization rate, using malonitrile as a catalyst and from initiator molecules anchored on iron oxide NPs. However, it appears that bonding the initiator to the surface through a phosphonate group limits the rate, reducing the effect of the catalyst. The length of the carbon backbone of the initiator (between phosphate and α-bromo-ester functions) is very important to the ATRP poly- merization rate when initiators are directly anchored to the par- ticle surface. Sunday et al. [36] reported that long alkyl chains (16 carbon atoms) or short ones (3 carbon atoms) lead to higher polymerization rates and grafting densities than intermediate chains (e.g., 11 carbon atoms). Masson et al. used an 11-carbon- long initiator. That which we used, the same as for PMMA (2-phosphonooxy-2-bromo-2-methylpropanoate), has only two carbon atoms, and might be expected to give good polymeriza- tion rates. Figure 2: XPS survey spectra of (a) ENP-PS-18, (b) ENP-PS-24. Table 1: XPS-determined atomic composition of as-prepared CoxFe3−xO4-CoO nanoparticles (ENP) and PS-based hybrids. Sample Elemental atomic composition (%) Fe Co O C P Br ENP (CoxFe3−xO4@CoO)* 14.5 24.3 40.2 21.0 – – ENP-PS-18 7.7 10.5 37.6 41.5 2.3 0.4 ENP-PS-24 7.4 11.6 37.7 41.8 1.2 0.3 Reproduced in part with permission from [15]. Copyright 2016 The Royal Society of Chemistry. Table 1: XPS-determined atomic composition of as-prepared CoxFe3−xO4-CoO nanoparticles (ENP) and PS-based hybrids. Sample Elemental atomic composition (%) Fe Co O C P Br ENP (CoxFe3−xO4@CoO) 14.5 24.3 40.2 21.0 – – ENP-PS-18 7.7 10.5 37.6 41.5 2.3 0.4 ENP-PS-24 7.4 11.6 37.7 41.8 1.2 0.3 *Reproduced in part with permission from [15]. Copyright 2016 The Royal Society of Chemistry. The C 1s peak prior to functionalization indicates that organic residues (polyol and acetate molecules) are present at the core–shell NP surface. The Co 2p and Fe 2p peaks are much weaker after polymerization due the presence of a significant polymer coating. The decomposition of the C 1s peak for poly- styrene (PS) was difficult since there are many sources of organic matter (PS, initiator, residual polyol and acetates); how- ever, unfortunately, styrene contains no other element suitable for XPS analysis. Growth of PMMA chains We previously developed a two-step pathway to produce the hybrid polymer-decorated ENPs [15]. First, an ATRP initiator, 410 Beilstein J. Nanotechnol. 2017, 8, 408–417. catalytic Cu-PMDETA complex, with polymerization times of 1 to 3 h. The polymer coating was characterized by X-ray photo- electron spectroscopy (XPS) and thermogravimetric analysis (TGA) [15]. For all samples the grafted chain density was 1.4 chain nm−2, i.e., significantly higher than the results previ- ously reported [15]. A chain density ranging between 0.1 and 1 chain nm−2 is commonly reported [13,26,31,32]. In the present case, a higher grafting density means better protection of the magnetic particles against oxidation and more stable magnetic properties over time [33]. Indeed, the aim here is not necessarily to grow very long polymer chains which are diamagnetic, but to functionalize ENPs efficiently in order to increase their compatibility with polymer matrices. Compari- son of the grafting densities obtained here with those reported elsewhere suggests that, for ENPs of similar size, phosphates and phosphonates are better grafted and in greater quantity to the surface of ENPs than carboxylates and organosilanes. during the washing and purification steps. Chemical composi- tions (provided by XPS data) of as-produced NPs and ENP-PS are listed in Table 1. Figure 2: XPS survey spectra of (a) ENP-PS-18, (b) ENP-PS-24. Growth of PS chains However, in the XPS chamber (under ultravacuum conditions), the polymer chains tend to collapse. Therefore, one must consider that XPS measurements do not allow the exact chain length to be determined but that of a polymer in a random coil conformation. Nevertheless, we can conclude that more polystyrene is present around the ENP when the polymeriza- tion time increases. TGA thermograms of PS-decorated ENPs are presented in Figure 4. PS is generally decomposed at about 380 °C. It can be seen that, although the polymer is thicker at the ENP surface in the case of PS, the weight losses of PS (6% and 14% for NP-PS-18 and NP-PS-24, respectively) are lower described previously [15]. However, electrons pass only through 0.5 nm of the Fe-rich core after having penetrated the 1 nm thick CoO shell, leading to a difference in depth analysis of the core and the shell. Then, we assumed that cobalt and car- bon are representative of the CoO shell [30] and the PS brushes, respectively, to determine the PS thickness from the C 1s and Co 2p peaks and their relative intensities. The thicknesses were found to be 2.5 ± 0.5 nm for ENP-PS-18 and 3.0 ± 0.5 nm for ENP-PS-24. However, in the XPS chamber (under ultravacuum conditions), the polymer chains tend to collapse. Therefore, one must consider that XPS measurements do not allow the exact chain length to be determined but that of a polymer in a random coil conformation. Nevertheless, we can conclude that more polystyrene is present around the ENP when the polymeriza- tion time increases. TGA thermograms of PS-decorated ENPs are presented in Figure 4. PS is generally decomposed at about 380 °C. It can be seen that, although the polymer is thicker at the ENP surface in the case of PS, the weight losses of PS (6% and 14% for NP-PS-18 and NP-PS-24, respectively) are lower TEM images presented in Figure 5 depict well-dispersed nano- particles, although the surface of the grid was not totally covered with ENPs. Similar images have been obtained for the two samples. There are spaces between the ENPs that are close to each other, suggesting that they are separated by PS coatings. Growth of PS chains However, IR spectra of PS-decorated ENPs (Figure 3) exhibit several peaks characteristic of polystyrene: 3024 cm−1 (νas(CH2_arom)), 2950 cm−1 (νs(CH_aliph)) and 1600 cm−1 (νC=C) when compared to a commercial reference. Working under the same conditions previously used for PMMA growth on ENPs, we prepared a series of samples by varying the polymerization time from 18 to 24 h; the resulting hybrids will be referred to as ENP-PS-h (where h corresponds to the polymerization time in hours). The survey XPS spectra of the resulting hybrids evidence all the characteristic signals of ENPs and initiator species (Figure 2). Hence, the O 1s (531.0 eV), Fe 2p3/2 (711.5 eV), Co 2p3/2 (781.7 eV), P 2p (133.5eV), and Br 3p3/2 (190.7 eV) peaks can be assigned to CoxFe3−xO4@CoO and PO(OH)2O(CH2)2OCOC(CH3)2Br phases, respectively. The absence of Cu signals in the ENP-PS spectra suggests that all metallic Cu complexes were eliminated Similar to the PMMA-based nanohybrids, the amount of cobalt was found to be higher than that of iron for PS-functionalized ENPs. The mean free paths of 1.5 nm for iron in CoO and 1.4 nm for cobalt in CoO were calculated following the method 411 Beilstein J. Nanotechnol. 2017, 8, 408–417. Figure 4: TGA curves of (a) ENP-PS-18 and (b) ENP-PS-20. Figure 3: IR spectrum of ENP-PS-18 and those of free PS and ENPs functionalized by the ATRP initiator only (ENP-Br). Figure 4: TGA curves of (a) ENP-PS-18 and (b) ENP-PS-20. Figure 4: TGA curves of (a) ENP-PS-18 and (b) ENP-PS-20. Figure 4: TGA curves of (a) ENP-PS-18 and (b) ENP-PS-20. Figure 3: IR spectrum of ENP-PS-18 and those of free PS and ENPs functionalized by the ATRP initiator only (ENP-Br). than those of PMMA in ENP-PMMA-1 and ENP-PMMA-3 [15], meaning that PS collapses less than PMMA. This is doubtful due to the presence of aromatic rings. described previously [15]. However, electrons pass only through 0.5 nm of the Fe-rich core after having penetrated the 1 nm thick CoO shell, leading to a difference in depth analysis of the core and the shell. Then, we assumed that cobalt and car- bon are representative of the CoO shell [30] and the PS brushes, respectively, to determine the PS thickness from the C 1s and Co 2p peaks and their relative intensities. The thicknesses were found to be 2.5 ± 0.5 nm for ENP-PS-18 and 3.0 ± 0.5 nm for ENP-PS-24. Ligand exchange for improved separation of magnetic NPs SEM images (Figure 10) present well-dispersed ENPs in both cases, suggesting that the ENP-PMMA NPs are very well sepa- rated and can be dispersed and assembled in a controlled manner, thanks to the polymer grafted on the ENP surface. ENPs and small aggregates or isolated ENPs at higher dilutions. SEM images (Figure 10) present well-dispersed ENPs in both cases, suggesting that the ENP-PMMA NPs are very well sepa- rated and can be dispersed and assembled in a controlled manner, thanks to the polymer grafted on the ENP surface. Figure 8 presents the TEM images. Better-separated ENPs are recovered when oleic acid is used, and they are, consequently, more adapted for applications in flexible polymer devices. The production of such nanoparticles thus opens the way to various applications such as: (i) the fundamental study of the magnetic properties of the various assemblies of these magnet- ic ENPs. There is still intensive research to be done on the control of the magnetic properties of magnetic films, either comprised of nanocomposites or not [37], via the spatial ENP orientation [28,38] to be combined with polymer processing [39,40]. Ligand exchange for improved separation of magnetic NPs In the previous sections, we demonstrated the feasibility of pre- paring polymer (PMMA or PS) functionalized magnetic parti- cles with improved polymer chain grafting densities than those previously reported. However, it is still a serious challenge to separate all the particles and avoid a few aggregates. Hence, we replaced the centrifugation of the ENPs after the polyol synthe- sis by direct ligand exchange between adsorbed polyol mole- cules and oleic acid (OA) in the reaction mixture (see Experi- Figure 5: TEM images of ENP-PS-18 (left) and ENP-PS-24 (right). Figure 5: TEM images of ENP-PS-18 (left) and ENP-PS-24 (right). 412 Beilstein J. Nanotechnol. 2017, 8, 408–417. Figure 7: Size distributions of ENPs, in polyol solvent (diethylene glycol) and functionalized by oleic acid, dispersed in tetrahydrofuran solvent (THF), obtained by DLS. mental). Oleic acid is known to cap oxide nanoparticles by ionic bonding [24,25]. Moreover, it bears a double bond C=C, induc- ing a degree of structural rigidity and a kink in the chain, promoting the spacing of the particles during their organization. TEM images (Figure 6) clearly show a better separation of the ENPs previously coated with OA instead of being recovered simply by centrifugation of the polyol mixture. Figure 7 shows the variation of the size distribution of the mag- netic particles as a function of the surface state (with or without oleic acid), determined by dynamic light scattering (DLS) mea- surements. The distribution is found to be quite polydisperse when ENPs are purified by centrifugation from the polyol and narrower when they are coated with OA. Moreover, the aver- age diameter is found to be ≈100 nm for the former and 25 nm for the latter, which shows that the functionalization by OA through ligand exchange leads to a better separation of exchange-biased magnetic particles. Then, PMMA chains were grown from the surface of ENP-Br by the experimental proce- dure described previously [15]. Figure 7: Size distributions of ENPs, in polyol solvent (diethylene glycol) and functionalized by oleic acid, dispersed in tetrahydrofuran solvent (THF), obtained by DLS. Figure 7: Size distributions of ENPs, in polyol solvent (diethylene glycol) and functionalized by oleic acid, dispersed in tetrahydrofuran solvent (THF), obtained by DLS. ENPs and small aggregates or isolated ENPs at higher dilutions. Assembly of PMMA-decorated magnetic nanoparticles Finally, we assembled PMMA-functionalized ENPs prepared by the ligand exchange procedure and dispersed in THF. Thin films were prepared by drop casting on silicon wafers and SEM images were recorded. Interestingly, Figure 9 depicts an incom- plete monolayer of ENPs (with a scheme presented in the insert). As a preliminary result, focusing on these PMMA-decorated ENP assemblies, a net decrease of the blocking temperature value, defined as the critical temperature at the relaxed/blocked magnetic states transition, was observed when the ENP dilution ratio was increased (Figure 11). Such behaviour is Then, ENP-PMMA was introduced in a solution of PMMA in THF, where the ENP/PMMA ratio was varied from 1:1 to 1:9. Large aggregates were obtained at higher concentrations of Figure 6: TEM images of a) ENP-Br obtained after centrifugation of as-prepared ENPs and direct grafting of ATRP initiator; b) ENP-Br obtained by two-step ligand exchange by oleic acid followed by grafting of ATRP initiator. Figure 6: TEM images of a) ENP-Br obtained after centrifugation of as-prepared ENPs and direct grafting of ATRP initiator; b) ENP-Br obtained by two-step ligand exchange by oleic acid followed by grafting of ATRP initiator. 413 Beilstein J. Nanotechnol. 2017, 8, 408–417. Figure 8: TEM images of magnetic ENPs decorated with PMMA brushes: (a,b) ENP-Br obtained through direct grafting of ATRP initiator after 3 h polymerization; (c,d) ENP-Br obtained by ligand exchange (oleic acid/ATRP initiator) under the same conditions. Figure 8: TEM images of magnetic ENPs decorated with PMMA brushes: (a,b) ENP-Br obtained through direct grafting of ATRP initiator after 3 h polymerization; (c,d) ENP-Br obtained by ligand exchange (oleic acid/ATRP initiator) under the same conditions. quite common for superparamagnetic single-domain nanoparti- cles, and it is here respected in the case of exchange-biased nanoparticles. Figure 9: Thin films prepared by drop casting of a suspension of ENP- PMMA in THF. It is clear that the material processing approach we proposed here, namely, the controlled surface polymerization of oxide- based ENPs, is able to tune their magnetic properties. Further magnetic measurements are still in progress to fully appreciate the dipolar interaction effect on the exchange bias and the influ- ence of NP assembly on the exchange field. Conclusion We described the functionalization of 10 nm exchange-biased CoxFe3−xO4@CoO core@shell NPs by two different polymers, poly(methyl methacrylate) (PMMA) and polystyrene (PS), Figure 9: Thin films prepared by drop casting of a suspension of ENP- PMMA in THF. Figure 10: SEM images of various assemblies of ENP-PMMA, obtained by drop casting from THF suspension with ENP/PMMA ratios of 1:1, 1:2 and 1:9. Figure 10: SEM images of various assemblies of ENP-PMMA, obtained by drop casting from THF suspension with ENP/PMMA ratios of 1:9 Figure 10: SEM images of various assemblies of ENP-PMMA, obtained by drop casting from THF suspension with ENP/PMMA ratios of 1:1, 1:2 and 1 9 Figure 10: SEM images of various assemblies of ENP-PMMA, obtained by drop casting from THF suspension with ENP/PMMA ratios of 1:1, 1:2 and 1:9. 414 Beilstein J. Nanotechnol. 2017, 8, 408–417. Figure 11: Thermal variation of the normalized dc magnetic magneti- zation measured in zero-field cooling (ZFC) conditions for the assem- bled PMMA-decorated ENP sample series (see Figure 10). Details around the maximum of the magnetization is given in the inset. PS chain growth (direct ligand exchange for ATRP initiator grafting) proceeded by first dispersing 80 mg of ENP-Br in 37 mL of toluene before adding 8.2 μL of PMDTA, 4.8 mL of styrene and 11.4 mL of malonitrile in order to arrive at the ratio styene/initiator/malonitrile/PMDETA/CuBr 1000:1:4:1:1. The mixture was degassed with argon and mechanically stirred for at least 1 hour. 5.7 mg of CuBr were added and the solution was heated at 90 °C and mechanically stirred under argon. The poly- merization time was varied from 18 to 24 h. Finally, the reac- tion was quenched by opening the system to the atmosphere. The resulting hybrids were referred to as ENP-PS-h, where h corresponds to the polymerization time in hours. PMMA chain growth (two-step ATRP initiator grafting, ligand exchange by phase transfer) was accomplished using 150 mg of ENPs, dispersed in 45 mL of diethylene glycol, which was added to 75 mL of a solution of oleic acid in toluene (10% v/v) and sonicated for 15 min and left overnight. The OA-functional- ized ENPs were recovered from the toluene layer and 375 mg of 2-phosphonooxy-2-bromo-2-methylpropanoate were added to the suspension and sonicated for 24 h. Toluene was then re- moved and the functionalized ENPs were recovered after several washings with THF. Hybrid synthesis PMMA chain growth by ATRP (direct ligand exchange for ATRP initiator grafting) was used as described in [15]: ENPs and 2-phosphonooxy-2-bromo-2-methylpropanoate were intro- duced in THF and sonicated at room temperature (RT). The re- sulting nanohybrids were then dispersed in acetonitrile before adding N,N,N′,N′,N′′-pentamethyldiethylenetriamine (PMDTA) methyl methacrylate. The mixture was degassed with argon and mechanically stirred for 1 h. CuBr was added and the solution was heated around 30 °C and sonicated under inert atmosphere. The polymerization time was tuned from 1 to 3 h to influence the length of the polymer chains. The reaction was quenched by opening the system and diluting the solution with THF and hexane. Hairy, hybrid ENPs were recovered by centrifugation and washing with THF. They were referred to as ENP-PMMA- h, where h corresponds to the polymerization time in hours. Conclusion Figure 11: Thermal variation of the normalized dc magnetic magneti- zation measured in zero-field cooling (ZFC) conditions for the assem- bled PMMA-decorated ENP sample series (see Figure 10). Details around the maximum of the magnetization is given in the inset. using radical-controlled polymerization under various process- ing conditions. We evidenced through TGA, IR spectroscopy and XPS measurements that polymer chains were efficiently grafted onto the nanoparticles using either the direct grafting or the “all in solution” process. In the TEM images we have also observed that very little aggregation occurs. Nevertheless, the alternative two-step strategy leads to better dispersed polymer- decorated magnetic particles, and the resulting nanohybrids can be considered as valuable building blocks for flexible, magnet- ic polymer-based devices. We also developed various assem- blies by varying the dilution of the ENP suspension in THF, where the assemblies varied from small aggregates to isolated ENPs at the surface of silicon substrates. These ENPs allow for the preparation of flexible, functional, hybrid PMMA-ENP films with enhanced properties (e.g., magnetic, mechanical). Hybrid characterization The different reaction steps were monitored by ATR-FTIR on a Thermo Nicolet 8700 spectrometer equipped with a diamond crystal (50 scans, 4 cm−1 resolution). Thermogravimetric analyses (TGA) were performed in air on a Labsys-Evo device with a heating rate of 10 °C min−1. X-ray photoelectron spec- troscopy (XPS) measurements were performed on as-prepared and functionalized ENPs using a Thermo VG ESCALAB 250 instrument equipped with a micro-focused, monochromatic Al Kα X-ray source (1486.6 eV) and a magnetic lens. The X-ray spot size was 500 µm (15 kV, 150 W). The spectra were acquired in the constant analyser energy mode with pass ener- gies of 150 and 40 eV for the general survey and the narrow scans, respectively. The samples were fixed on sample holders and out-gassed in the fast entry airlock (2 × 10−7 mbar). The Avantage software package was used for data acquisition and processing. 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https://openalex.org/W2004169086	https://europepmc.org/articles/pmc4278212?pdf=render	English	null	Characterisation of Non-Autoinducing Tropodithietic Acid (TDA) Production from Marine Sponge Pseudovibrio Species	Marine drugs	2,014	cc-by	9,987	Received: 15 September 2014; in revised form: 5 November 2014 / Accepted: 12 November 2014 / Published: 10 December 2014 Mar. Drugs 2014, 12, 5960-5978; doi:10.3390/md12125960 Mar. Drugs 2014, 12, 5960-5978; doi:10.3390/md12125960 Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 One new antibiotic to emerge from these initiatives is the antibacterial compound tropodithietic acid (TDA). The aim of this study was to provide insight into the bioactivity of and the factors governing the production of TDA in marine Pseudovibrio isolates from a collection of marine sponges. The TDA produced by these Pseudovibrio isolates exhibited potent antimicrobial activity against a broad spectrum of clinical pathogens, while TDA tolerance was frequent in non-TDA producing marine isolates. Comparative genomics analysis suggested a high degree of conservation among the tda biosynthetic clusters while expression studies revealed coordinated regulation of TDA synthesis upon transition from log to stationary phase growth, which was not induced by TDA itself or by the presence of the C10-acyl homoserine lactone quorum sensing signal molecule. One new antibiotic to emerge from these initiatives is the antibacterial compound tropodithietic acid (TDA). The aim of this study was to provide insight into the bioactivity of and the factors governing the production of TDA in marine Pseudovibrio isolates from a collection of marine sponges. The TDA produced by these Pseudovibrio isolates exhibited potent antimicrobial activity against a broad spectrum of clinical pathogens, while TDA tolerance was frequent in non-TDA producing marine isolates. Comparative genomics analysis suggested a high degree of conservation among the tda biosynthetic clusters while expression studies revealed coordinated regulation of TDA synthesis upon transition from log to stationary phase growth, which was not induced by TDA itself or by the presence of the C10-acyl homoserine lactone quorum sensing signal molecule. Keywords: TDA; Pseudovibrio; marine; antimicrobial; clinical Keywords: TDA; Pseudovibrio; marine; antimicrobial; clinical Characterisation of Non-Autoinducing Tropodithietic Acid (TDA) Production from Marine Sponge Pseudovibrio Species Catriona Harrington 1, F. Jerry Reen 1, Marlies J. Mooij 1,†, Fiona A. Stewart 1, Jean-Baptiste Chabot 1, Antonio F. Guerra 2, Frank O. Glöckner 2,3, Kristian F. Nielsen 4, Lone Gram 4, Alan D. W. Dobson 5, Claire Adams 1 and Fergal O’Gara 1,6,* 1 BIOMERIT Research Centre, School of Microbiology, University College Cork—National University of Ireland, Cork, Ireland; E-Mails: catriona.harrington@gmail.com (C.H.); J.Reen@ucc.ie (F.J.R.); marlies.mooij@mumc.nl (M.J.M.); fiona.stewart204@gmail.com (F.A.S.); jeanba-cha@hotmail.fr (J.-B.C.); c.adams@ucc.ie (C.A.) 2 Microbial Genomics and Bioinformatics Research Group, Max Planck Institute for Marine Microbiology, Bremen D-28359, Germany; E-Mails: afernand@mpi-bremen.de (A.F.G.); fog@mpi-bremen.de (F.O.G.) 2 Microbial Genomics and Bioinformatics Research Group, Max Planck Institute for Marine Microbiology, Bremen D-28359, Germany; E-Mails: afernand@mpi-bremen.de (A.F.G.); fog@mpi-bremen.de (F.O.G.) 3 Jacobs University Bremen gGmbH, Bremen 28759, Germany 3 Jacobs University Bremen gGmbH, Bremen 28759, Germany 4 Department of Systems Biology, Technical University of Denmark, DK-2800 Kgs. Lyngby, 4 Department of Systems Biology, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark; E-Mails: kfn@bio.dtu.dk (K.F.N.); gram@bio.dtu.dk (L.G.) Denmark; E-Mails: kfn@bio.dtu.dk (K.F.N.); gram@bio.dtu.dk (L.G.) 5 Environmental Research Institute, University College Cork—National University of Ireland, Cork, Ireland; E-Mail: a.dobson@ucc.ie 6 Curtin University, School of Biomedical Sciences, Perth, WA 6845, Australia † Current Address: Maastricht University Medical Centre, 6211 LK Maastricht, The Netherlands * Author to whom correspondence should be addressed; E-Mail: f.ogara@ucc.ie; Tel.: +353-21-4901315; Fax: +353-21-4275934. * Author to whom correspondence should be addressed; E-Mail: f.ogara@ucc.ie; Tel.: +353-21-4901315; Fax: +353-21-4275934. Received: 15 September 2014; in revised form: 5 November 2014 / Accepted: 12 November 2014 / Published: 10 December 2014 Abstract: The search for new antimicrobial compounds has gained added momentum in recent years, paralleled by the exponential rise in resistance to most known classes of current antibiotics. While modifications of existing drugs have brought some limited clinical success, there remains a critical need for new classes of antimicrobial compound to which key clinical pathogens will be naive. This has provided the context and impetus to marine biodiscovery programmes that seek to isolate and characterize new activities from the aquatic ecosystem. 5961 1. Introduction Due to the misuse and overuse of antibiotics, the emergence of pathogens that are resistant to virtually all of the currently available antibiotics has reached a critical stage. For this reason, there has been an urgent drive towards the discovery of new antimicrobial compounds. The marine environment provides a relatively untapped source of these unique natural products. In particular, marine sponges are emerging as a goldmine of antimicrobial activity, with many potential bioactive compounds produced by symbiotic bacteria associated with marine sponges [1]. As sessile organisms, sponges rely on an arsenal of metabolites, generally produced by their associated microorganisms, to defend against disease and to gain a competitive advantage within the marine ecosystem [2]. This symbiotic relationship is essential for sponge efficiency and survival [3]. Consequently, more novel bioactive metabolites are retrieved from sponges each year than any other marine organism [2], with approximately 3500 novel marine compounds isolated from marine sponges since 1985 [4]. Recent evidence suggests that the majority of bioactive compounds isolated from sponges are likely to be produced by associated microbiota [5]. Pseudovibrio species are ubiquitous in the marine environment, and in particular within marine sponges. They were first isolated and described in 2004 from seawater in Taiwan [6] but have since been isolated from ascidians [7] tunicates [8], algae [9], coral [10], tube worms [11] and from a plethora of marine sponges [12–17]. Pseudovibrio is in fact believed to be one of the few confirmed vertically transmitted sponge symbionts, due to its association with sponge larvae [18]. Antimicrobial activity within Pseudovibrio sp. has been documented a number of times [12,15–17,19,20]. Moreover, Pseudovibrio species produce specific bioactive compounds, such as heptylprodigiosin [20] and biosurfactant compounds [11]. More recently, the antibacterial compound tropodithietic acid (TDA) was recovered from Pseudovibrio sp. D323, isolated from the red alga Delisea pulchra [9], which correlated with the previous prediction of TDA production by Pseudovibrio sp. strain JEO62 based on the presence of the tdaA-tdaF biosynthetic cluster in its genome [5,21]. TDA was initially isolated from several species within the Roseobacter clade including Phaeobacter inhibens and Ruegeria sp. [21–24]. TDA is a suphur containing compound with a unique structure consisting of a dithiet moiety fused to tropone-2-carboxylic acid, which is believed to co-exist with its tautomer, thiotorpocin [25], previously 5962 Mar. Drugs 2014, 12 identified in Pseudomonas sp. [26,27]. 1. Introduction TDA has been shown to have a strong inhibitory activity against a range of marine bacteria, such as Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes, the fish pathogens Vibrio anguillarum and Vibrio splendidus [28] as well as marine algae [21] and a range of human pathogenic bacteria [24]. It has been proposed as a potential fish larval probiotic, as it has been shown not only to reduce mortality in the larvae of fish such as turbot and cod [29], but also that it has no toxic effects against the eukaryotic models C. elegans and Artemia sp. [30]. Here, we identify TDA production in Pseudovibrio species isolated from the marine sponges Axinella dissimilis, Polymastia boletiformis and Haliclona simulans and investigate phenotypic characteristics, bioactivity and molecular mechanisms of its production. Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 a direct association between pigment production and the antimicrobial bioactivity. Four Pseudovibrio isolates which produced the brown pigment and displayed particularly potent levels of bioactivity—namely W64, W69, W74 and W89—were selected for further analysis. Table 1. Antimicrobial activity of the 33 RAPD group representatives against a range of human and fish pathogens. human and fish pathogens. Strain Antagonistic Activity against Pathogen Y. ruckerri E. tarda V. anguillarum E. coli MUH E. coli NCIMB 949 M. morganii S. Typhimurium LT2 S. Typhimurium C5369 P. sporunum S. arizonae S. Mar. Drugs 2014, 12 aureus NCDO 949 JIC5 +++ +++ +++ ++ + +++ - ++ * + * + * ++ JIC6 + ++ + - + * + * - - + * - ++ JIC17 +++ ++ + - + * ++ + * - + * + * + W10 +++ ++ - + - + * - +* + * - + W19 - - - - - - - - - - - W62 +++ ++ ++ + + * +++ - + * ++ * - ++ W63 +++ +++ - - - - - ++ * +++ * + * ++ W64 +++ +++ +++ + + +++ ++ * ++ * ++ ++ ++ W65 +++ ++ +++ ++ + +++ + * ++ * + * + ++ W69 +++ +++ ++ + + ++ ++ * ++ * ++ * ++ * ++ W71 +++ ++ ++ + + +++ + * ++ * + * + + W89 +++ +++ +++ + ++ ++ ++* ++ * ++ * + ++ W99 +++ +++ +++ ++ + +++ + * ++ * + * + ++ WC43 ++ - ++ + * + + * + * + * + * + ++ W74 +++ ++ + + ++ +++ + * ++ * + * ++ * + W85 +++ ++ +++ ++ + * + + * + * + * - ++ W78 ++ + ++ + * + * +++ +* + * + * - + W94 ++ + ++ + * + * ++ + * + * + * + * ++ W96 +++ ++ ++ + * + * ++ + * ++ * - ++ * + WM31 +++ ++ +++ + * + * + + * + * - - ++ WM33 ++ + + + * + * - + - - - + WM34 +++ ++ +++ + * + * +++ + * + * + * + * ++ WM40 +++ ++ ++ + + ++ - ++ ++ * ++ * ++ WM50 - - - - - - - - - - - WC13 +++ ++ ++ + + * ++ - + * - + * ++ WC15 + - + * - - + * - - - - + WC21 ++ + * ++ + + * ++ - + * - + * ++ WC22 +++ + + + + * + - - - + * ++ WC30 +++ + ++ + + * + - + * - + * + WC32 +++ ++ ++ ++ + * ++ - ++ * + * + * ++ WC41 +++ ++ ++ + + * ++ - ++ * - ++ * ++ HC6 +++ ++ +++ ++ * + +++ + * ++ * + * + * ++ HMMA3 ++ + ++ + * + * + * + * - - - + Diameter of growth inhibition (mm): + ≥1 mm; ++ ≥2 mm; +++ ≥4 mm. ure Conditions Enhance the Production of Bioactive Compounds by Pseudovibrio Specie A collection of 72 Pseudovibrio isolates from the marine sponges Axinella dissimilis, Polymastia boletiformis and Haliclona simulans, which were previously classified into 33 groups based on their RAPD profiles [31], was further investigated for antimicrobial activity. A representative strain from each RAPD group was tested for bioactivity via a spot-plate overlay assay on both SYP-SW and marine (MA) agar, against the indicator strain S. aureus NCDO 949. Zones of inhibition were produced by 26 of the 33 representatives of the RAPD groups on SYP-SW. Interestingly, all these strains displayed higher levels of inhibition when grown on MA than on SYP-SW (Figure 1), while 5 strains displayed antimicrobial bioactivity only when grown on MA. Two strains displayed no bioactivity against S. aureus NCDO 949 on either media (Supplementary Table S1). Figure 1. Overlay assay with tropodithietic acid (TDA) producing Pseudovibrio isolate WC22—representative of most Pseudovibrio isolates. S. aureus NCDO949 was used as an indicator strain and was strongly inhibited when the Pseudovibrio colony had turned brown. (a) Marine agar; and (b) starch-yeast extract-peptone–sea water (SYP-SW)_agar. In order to establish the spectrum of bioactivity produced by the marine isolates, each individual isolate was tested against a range of clinical and fish pathogens, as well as a number of laboratory strains (Supplementary Table S2). High levels of inhibition were seen in 31 of the 33 strains against both clinical and fish pathogens (Table 1). The vast majority of the isolates displayed antibacterial activity against V. anguillarum (88%), E. tarda (88%), Y. ruckerri (94%), E. coli MUH (82%), E. coli NCIMB (85%), M. morganii (88%), S. Typhimurium LT2 (52%), S. Typhimurium C5369 (76%), P. sputonum (64%), S. arizonae (67%) and S. aureus NCDO 949 (94%). Interestingly, each of the 31 isolates showing antimicrobial bioactivity produced a characteristic brown pigment when grown on MA. This brown pigment was not produced by the two strains which showed no inhibitory activity. This strongly suggested 5963 meter of growth inhibition (mm): + ≥1 mm; ++ ≥2 mm; +++ ≥4 mm. * partial inhibition. Blue: fish hogen, yellow: laboratory strain, red: human pathogen. Mar. Drugs 2014, 12 * partial inhibition. Blue: fish pathogen, yellow: laboratory strain, red: human pathogen. Diameter of growth inhibition (mm): + ≥1 mm; ++ ≥2 mm; +++ ≥4 mm. * partial inhibition. Blue: fish pathogen, yellow: laboratory strain, red: human pathogen. Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 5964 2.2. Pseudovibrio Species Derived from Marine Sponge Produce Tropodithietic Acid (TDA) Drugs 2014, 12 similar environment or whether these bacteria would have some level of resistance against TDA, due to their close association. Therefore, the antimicrobial activity of W74 was tested against a range of marine bacteria isolated from 2 deep sea sponges, namely Hexactinellida and Poecillastra species. W74 was spot-plated and overlaid with 136 different marine isolates to screen for tolerance against TDA. Of the 136 isolates, 126 (93%) displayed tolerance to TDA. Tolerant species included Psychrobacter, Alteromonas, Salinibacter, Alcanivorax, Flavobacterium and Micrococcus species. Strains displaying sensitivity towards TDA included Staphlococcus species, as well as species of the genera Idiomarina and Rhodococcus. The tolerant strains were subsequently tested for TDA production. No brown pigment production was observed in the TDA resistant marine isolates grown for 72 h from a starting OD600 of 0.01, suggesting that the isolates tested were not TDA producers. For confirmation of this, 12 resistant isolates were selected for compound extraction and tested for the presence of TDA using TLC spot-plate assays (Supplementary Table S3). All isolates tested negative for TDA production. These findings were interesting, as resistance to TDA has previously only been observed in TDA-producers alone [9,22,35]. A selection of 4 TDA sensitive isolates was also compound extracted and tested for TDA production, 3 of the 4 isolates tested negative for TDA production (Supplementary Table S3). Interestingly, the Pseudovibrio strain W19, isolated from Axinella dissimilis and previously shown to have limited antimicrobial activity was found not to produce TDA using TLC analysis and was resistant to TDA extracted from W74. The resistance of non-producing W19 to TDA further suggests that the prevalence of TDA resistance mechanisms may be more widespread within marine communities than previously thought. 2.2. Pseudovibrio Species Derived from Marine Sponge Produce Tropodithietic Acid (TDA) Pseudovibrio Species Derived from Marine Sponge Produce Tropodithietic Acid (TDA Previously the production of a brown pigment associated with antimicrobial activity by Tropodithietic Acid (TDA) producing marine isolates on MA has been reported [28,32], and high levels of activity of TDA against fish pathogens has been documented a number of times [23,30,33] as has activity against human pathogens [24]. While chemical analysis of the pigment has yet to be reported, it has been shown to rely on the same biosynthetic genes as TDA itself, suggesting a direct link to the antimicrobial compound [34]. Thus, the pigmented antimicrobial activity of the Pseudovibrio isolates may be due at least in part to TDA. To establish that the Pseudovibrio isolates were indeed producing TDA, a method adapted from Porsby et al. [24] for the isolation of TDA was employed to extract compounds from the four Pseudovibrio isolates with high levels of antimicrobial activity. TLC was performed on the Pseudovibrio extracts and plates were overlaid with S. aureus NCDO 949 and tetrazolium salt. A large zone of inhibition was observed at the position correlating to an Rf value of 0.71, the same as the TDA control, suggesting that the compound produced by the Pseudovibrio isolates W64, W69, W74 and W89 was the secondary metabolite TDA (Figure 2a). To further confirm the production of TDA by these isolates, the bioactive compounds extracted from the Pseudovibrio isolates were analysed via UHPLC-DAD-qTOFMS. This analysis confirmed the presence of TDA in the extract based on an identical retention time as an authentic reference standard, and the unique accurate mass of the [M + H]+ ion (212.9674 ± 0.005) as well as correct isotopic pattern of C8H4O3S2 (Figure 2b), unambiguously identifying TDA. Figure 2. (a) TLC-overlay assay of extracts from Pseudovibrio isolates. S. aureus NCD0 949 was used as an indicator strain. The concentration of the TDA control was 0.01 mg/mL; (b) UHPLC-DAD-qTOFMS data, showing the extracted ion chromatograms (212.9674 ± 0.005) of the [M + H]+ ion of TDA. Analysis demonstrated that the bioactive compound produced by 4 Pseudovibrio isolates was tropodithietic acid. 2.3. TDA-Producing Isolate W74 has Limited Activity against Marine Isolates 2.3. TDA-Producing Isolate W74 has Limited Activity against Marine Isolates Having established that the TDA-producing isolates were active against a range of clinical and fish pathogens, we sought to investigate if TDA had an inhibitory effect against bacteria isolated from a 5965 Mar. 2.4. TDA Extracted from Pseudovibrio Displays Bioactivity against Cystic Fibrosis (CF) Clinical Isolates Following the observed high levels of antimicrobial activity Pseudovibrio isolates displayed against a range of human pathogens, both previously [24] and this study, as well as the inhibitory effect displayed against the CF isolate P. sputorum, the effect of TDA extracted from the Pseudovibrio strain W74 was tested against a range of clinical isolates collected from the sputum of paediatric patients with CF from Cork University Hospital. TDA displayed antimicrobial activity against a number of CF isolates, namely Bacillus cereus, Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus and Streptococcus haemolyticus. Preventing chronic infection is essential in the maintenance of health in CF patients; hence it is of the outmost importance that suitable antibiotics are used to treat infection successfully. S. aureus has been shown to be the most common Gram positive organism found in the lungs of patients with cystic fibrosis, as well as being the second most persistent pathogen after P. aeruginosa [36]. Its sensitivity to TDA is all the more significant due to the fact that the above study revealed that S. aureus was not eliminated completely by oral antibiotics. The sensitivity of CF isolates to TDA was particularly interesting, as very little is known about the effect of TDA against clinical isolates. However, while a recent study by Neu et al. revealed that TDA has no toxicity when tested on the eukaryotic models Artemia sp. and Caenorhabditis elegans [30], further tests on animal models would be required before TDA could be considered as a suitable antibiotic for clinical use. Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 5966 2.5. Genomic Pathway The TDA biosynthetic cluster was found in the draft genome sequences from the three marine isolates W64, W74 and WM33 after a NCBI BLAST+ 2.2.29 homology search using the TDA genes from Ruegeria sp. TM1040 [37] as query. The gene sequence identifier for each tdaA-F homolog found in the draft genome sequences is reported in Table 2. All-vs-all homology comparison of the 6 genes involved in the TDA pathway for the different species [18,33,38,39] (Table 2) and the graphical representation was conducted by Circoletto [40] (Figure 3). The results from the all-vs-all comparative analysis indicate that the tda genes show a high degree of homology in all five marine Pseudovibrio isolates (>94% of similarity). Interestingly, of the 3 isolates tested in this study, we observed that W74 consistently produced higher levels of TDA when compared with other isolates under the same extraction conditions that cannot be fully explained by the sequence similarity. This higher level of TDA production was independent of any growth effects and may be due to differences in the regulation or production of TDA among marine isolates. Figure 3. Comparative analysis of the TDA genes from Pseudovibrio compared to Ruegeria sp. TM1040. As expected, Pseudovibrio isolates exhibited the highest degree of sequence similarity to each other. Ribons represent the degree of sequence similarity with red being most similar and green signifying least similarity. Outer rings represent the coverage of sequence similarity in each gene as indicated by each coloured connecting line. See Supplementary Tables S4–S9 for the detailed similarity values between each pair of genes. As the synthesis of TDA in Pseudovibrio isolates has not been characterised, we undertook to investigate the factors governing production of this important antimicrobial compound. Given that W74 produced the highest amounts of TDA under culture conditions, this isolate was selected for further analysis. As the synthesis of TDA in Pseudovibrio isolates has not been characterised, we undertook to investigate the factors governing production of this important antimicrobial compound. Given that W74 produced the highest amounts of TDA under culture conditions, this isolate was selected for further analysis. As the synthesis of TDA in Pseudovibrio isolates has not been characterised, we undertook to investigate the factors governing production of this important antimicrobial compound. Given that W74 produced the highest amounts of TDA under culture conditions, this isolate was selected for further analysis. 2.5. Genomic Pathway As the synthesis of TDA in Pseudovibrio isolates has not been characterised, we undertook to investigate the factors governing production of this important antimicrobial compound. Given that W74 produced the highest amounts of TDA under culture conditions, this isolate was selected for further analysis. Mar. Drugs 2014, 12 5967 Mar. Drugs 2014, 12 Table 2. Accession number for the six genes involved in the TDA pathway from the different species used in the comparative genomic analyses. Table 2. Accession number for the six genes involved in the TDA pathway from the different species used in the comparative genomic analyses. species used in the comparative genomic analyses. Strain Accession Numbers of tda Genes tdaA tdaB tdaC tdaD tdaE tdaF W64 W64_g2177 W64_g2176 W64_g2175 W64_g2174 W64_g2173 W64_g2160 W74 W74_g3196 W74_g3195 W74_g3194 W74_g3193 W74_g3192 W74_g3180 WM33 WM33_g4179 WM33_g4178 WM33_g4176 WM33_g4175 WM33_g4174 WM33_g4161 FO-BEG1 [38] PSE_2264 PSE_2263 PSE_2261 PSE_2260 PSE_2259 PSE_2247 JE062 [18] JE062_g1641 JE062_g1639 JE062_g1638 JE062_g1637 JE062_g1636 JE062_g1624 TM1040 [37] EF139200 EF139201 EF139202 EF139203 EF139204 EF139205 Phaeobacter gallaeciensis DSM 17395 [33] PGA1_262p00980 PGA1_262p00970 PGA1_262p00960 PGA1_262p00950 PGA1_262p00940 PGA1_262p00810 Phaeobacter gallaeciensis 2.10 [33] PGA2_239p0970 PGA2_239p0960 PGA2_239p0950 PGA2_239p0940 PGA2_239p0930 PGA2_239p0800 Phaeobacter gallaeciensis 2.10 [39] Pden_1600 Pden_1599 Pden_1615 Pden_1614 Pden_1613 Pden_1605 2.6. TDA Expression Occurs during Logarithmic Growth 2.6. TDA Expression Occurs during Logarithmic Growth In order to determine at what point in the life cycle of Pseudovibrio TDA production occurs, W74 was grown under shaking conditions over a period of 48h. The growth was measured and production of the brown pigment was noted at 0, 6, 12, 24, 30, 36, and 48 h (Figure 4A,B). An aliquot of culture was collected for compound extraction at each time point, and bioactivity was tested by spotting extracted TDA on a TLC plate overlaid with the indicator strain S. aureus NCDO 949. The colour change of W74 from opaque to brown occurred between 12 and 24 h, during the logarithmic stage of growth. From this point until the assay was completed at 48 h—i.e., from logarithmic stage until late stationary phase—strong bioactivity was seen in the extract. Clear zones of inhibition of S. aureus NCDO 949 occurred where extracts were spotted (Figure 4C). This demonstrates that TDA production is initiated during the logarithmic growth phase. Microscopy of samples demonstrated that after 6 h (before colour change) cells were rod-shaped and existed in a planktonic form. 2.5. Genomic Pathway However, subsequent to colour change (after 24 h), cells aggregated together in a rosette formation. This cell aggregation has previously been shown to be associated with the production of TDA in the Silicibacter strain TM1040 [37] and strains of Ruegeria and Phaeobacter [22,23,28] (Supplementary Figure S1). It is interesting to note that, although production of TDA occurred in both shaking and static cultures of W64 and W74, the amount of pigment production was much larger in the shaking cultures compared to those grown under static conditions. Furthermore, bacterial density was over 2-fold higher for isolates grown in shaking rather than static conditions. This is inconsistent with the findings of Bruhn and co-workers [35], who found that while cell density was up to 10-fold higher in shaking cultures, pigment production was only present in static cultures. It also contrasts markedly with the observations of Belas and colleagues [21,41] who demonstrated than TDA production was minimal in shaking cultures, and that of D’Alvise et al. [29] who showed that TDA expression in the Roseobacter clade species Ruegeria mobilis was only associated 5968 Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 with attached or biofilm associated cells. This provides further evidence suggesting that differences exist in the regulation of TDA production in marine isolates. Figure 4. Timecourse experiment (0–48 h) for the production of TDA by Pseudovibrio strain W74. (A) Growth curve assay; (B) Pigment production; and (C) TLC-overlay assay of ethyl-acetate extracts obtained from W74 at the respective timepoints. TDA is included at a concentration of 0.02 μg/mL. y p p at a concentration of 0.02 μg/mL. 2.7. Induction of TDA Genes in Pseudovibrio is Linked to Bioactivity Gene expression profiles were generated by Real Time RT-PCR on samples of Pseudovibrio isolate W74 at 0, 6, 12, 24, 30, 36, and 48 h time points in order to examine the kinetics of expression governing TDA production. The expression of 6 genes involved in the TDA pathway—namely tdaA, -B, -C, -D, -E and -F, normalized to gyrB, was investigated. All genes were expressed by 24 h, with induction occurring almost simultaneously, demonstrating that the expression levels of genes involved in the TDA pathway was directly correlated to brown pigment formation and bioactivity. In all cases, expression levels dropped by 48 h—i.e., late stationary phase (Figure 5). Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 suggested to act as a QS signal [21]. Therefore, both auto-induction and QS-regulation of TDA were investigated in Pseudovibrio W74. Figure 5. RT-PCR Data. TdaA and tdaB genes involved in the production of TDA were expressed at 24 h—directly correlating to both bioactivity and brown pigment production in cultures. Figure 5. RT-PCR Data. TdaA and tdaB genes involved in the production of TDA were expressed at 24 h—directly correlating to both bioactivity and brown pigment production in cultures. 0 5 10 15 20 6 12 24 30 36 48 Transcript Expression Relative to gyrB Time (hours) tdaAExpression 0 20 40 60 80 100 120 6 12 24 30 36 48 Transcript Expression Relative to gyrB Time (hours) tdaBExpression -5 0 5 10 15 6 12 24 30 26 48 Transcript Expression Relative to gyrB Time (hours) tdaCExpression 0 200 400 600 800 1000 1200 6 12 24 30 36 48 Transcript Expression Relative to gyrB Time (hours) tdaD Expression -50 0 50 100 6 12 24 30 36 48 Transcript Expression Relative to gyrB Time (hours) tdaE Expression 0 10 20 30 40 6 12 24 30 36 48 Transcript Expression Realtive to gyrB Time (hours) tdaF Expression 2.8. TDA is Not Autoinduced, or Induced by C10-AHL In order to determine if TDA is auto-induced or induced by the QS molecule 3-OH-C10-HSL, W74 was grown in the presence or absence of TDA (1 μM), or 3-OH-C10-HSL (100 nM) respectively. Growth dynamics were identical in control and test cultures over 36 h (Figure 6A). In all cases brown pigment was produced at the same time interval (between 24 and 36 h) (Figure 6B). Previously, we had shown induction at 24 h, and the delay observed here is likely due to the presence of MeOH and DMSO in the media, resulting in delayed entry into logarithmic stage of growth. Cultures were compound extracted at each time point. Mar. Drugs 2014, 12 All extracts—including a control of W74 grown in MB—showed TDA production 0 5 10 15 20 6 12 24 30 36 48 Transcript Expression Relative to gyrB Time (hours) tdaAExpression 0 20 40 60 80 100 120 6 12 24 30 36 48 Transcript Expression Relative to gyrB Time (hours) tdaBExpression -5 0 5 10 15 6 12 24 30 26 48 Transcript Expression Relative to gyrB Time (hours) tdaCExpression 0 200 400 600 800 1000 1200 6 12 24 30 36 48 Transcript Expression Relative to gyrB Time (hours) tdaD Expression -50 0 50 100 6 12 24 30 36 48 Transcript Expression Relative to gyrB Time (hours) tdaE Expression 0 10 20 30 40 6 12 24 30 36 48 Transcript Expression Realtive to gyrB Time (hours) tdaF Expression 2.8. TDA is Not Autoinduced, or Induced by C10-AHL 0 200 400 600 800 1000 1200 6 12 24 30 36 48 Transcript Expression Relative to gyrB Time (hours) tdaD Expression 0 5 10 15 20 6 12 24 30 36 48 Transcript Expression Relative to gyrB Time (hours) tdaAExpression -50 0 50 100 6 12 24 30 36 48 Transcript Expression Relative to gyrB Time (hours) tdaE Expression 0 20 40 60 80 100 120 6 12 24 30 36 48 Transcript Expression Relative to gyrB Time (hours) tdaBExpression tdaBExpression tdaE Expression 0 10 20 30 40 6 12 24 30 36 48 Transcript Expression Realtive to gyrB Time (hours) tdaF Expression -5 0 5 10 15 6 12 24 30 26 48 Transcript Expression Relative to gyrB Time (hours) tdaCExpression 2.8. TDA is Not Autoinduced, or Induced by C10-AHL In order to determine if TDA is auto-induced or induced by the QS molecule 3-OH-C10-HSL, W74 was grown in the presence or absence of TDA (1 μM), or 3-OH-C10-HSL (100 nM) respectively. Growth dynamics were identical in control and test cultures over 36 h (Figure 6A). In all cases brown pigment was produced at the same time interval (between 24 and 36 h) (Figure 6B). Previously, we had shown induction at 24 h, and the delay observed here is likely due to the presence of MeOH and DMSO in the media, resulting in delayed entry into logarithmic stage of growth. Cultures were compound extracted at each time point. 2.5. Genomic Pathway Induction and subsequent decline in expression of the genes encoding TDA strongly suggests that production of TDA is tightly controlled in Pseudovibrio isolates, as would be expected for an energy expensive process. A previous study has shown TDA production in Phaeobacter gallaeciensis (inhibens) to be under the control of quorum sensing 3OHC(10)-HSL (QS) [34], thus, the induction of TDA production in logarithmic growth, coupled with the reduction in gene expression in late stationary phase, suggested that quorum sensing may be involved in controlling TDA production in Pseudovibrio and warranted further investigation. Furthermore, studies have shown TDA to be auto induced, whereby TDA induced its own synthesis, in Silicibacter (Ruegeria) sp. and has been 2.7. Induction of TDA Genes in Pseudovibrio is Linked to Bioactivity 2.7. Induction of TDA Genes in Pseudovibrio is Linked to Bioactivity Gene expression profiles were generated by Real Time RT-PCR on samples of Pseudovibrio isolate W74 at 0, 6, 12, 24, 30, 36, and 48 h time points in order to examine the kinetics of expression governing TDA production. The expression of 6 genes involved in the TDA pathway—namely tdaA, -B, -C, -D, -E and -F, normalized to gyrB, was investigated. All genes were expressed by 24 h, with induction occurring almost simultaneously, demonstrating that the expression levels of genes involved in the TDA pathway was directly correlated to brown pigment formation and bioactivity. In all cases, expression levels dropped by 48 h—i.e., late stationary phase (Figure 5). Induction and subsequent decline in expression of the genes encoding TDA strongly suggests that production of TDA is tightly controlled in Pseudovibrio isolates, as would be expected for an energy expensive process. A previous study has shown TDA production in Phaeobacter gallaeciensis (inhibens) to be under the control of quorum sensing 3OHC(10)-HSL (QS) [34], thus, the induction of TDA production in logarithmic growth, coupled with the reduction in gene expression in late stationary phase, suggested that quorum sensing may be involved in controlling TDA production in Pseudovibrio and warranted further investigation. Furthermore, studies have shown TDA to be auto induced, whereby TDA induced its own synthesis, in Silicibacter (Ruegeria) sp. and has been 5969 3.1. Growth Conditions All Pseudovibrio strains were isolated from the sponges Axinella dissimilis Polymastia boletiformis and Haliclona simulans as previously described [16]. A selection of marine isolates from the UCC marine culture collection, previously isolated from the deep sea sponges of Hexactinellida and Poecillastra species, were used as indicator strains for TDA sensitivity assays. All marine sponge isolates were grown in the dark at 23 °C shaking at 200 rpm. Strains were grown on marine agar (MA) (Difco, Difco laboratories, Surray, KT8 2SE, UK), or SYP-SW agar (1% starch, 0.4% yeast extract, 0.2% peptone, 3.3% sea salt, 1.5% agar). Other bacterial isolates (indicator strains) were grown shaking at 200 rpm at 37 °C in LB (1% tryptone, 0.5% yeast extract, 0.5% salt). Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 Figure 6. No evidence for autoinduction by TDA or early induction by QS compounds. (a) Growth curve of Pseudovibrio strain W74 grown in the presence of C10-HSL and TDA, along with the relative controls DMSO and MeOH; (b) Brown pigment production between 24 and 36 h. Up to 24 h, cultures remained a light beige colour. No difference is seen in the growth or colour of W74, indicating that TDA is not auto-induced, nor is it induced by C10-HSL. Figure 6. No evidence for autoinduction by TDA or early induction by QS compounds. (a) Growth curve of Pseudovibrio strain W74 grown in the presence of C10-HSL and TDA, along with the relative controls DMSO and MeOH; (b) Brown pigment production between 24 and 36 h. Up to 24 h, cultures remained a light beige colour. No difference is seen in the growth or colour of W74, indicating that TDA is not auto-induced, nor is it induced by C10-HSL. (a) Growth curve of Pseudovibrio strain W74 grown in the presence of C10-HSL and TDA, along with the relative controls DMSO and MeOH; (b) Brown pigment production between 24 and 36 h. Up to 24 h, cultures remained a light beige colour. No difference is seen in the growth or colour of W74, indicating that TDA is not auto-induced, nor is it induced by C10-HSL. DMSO C10-HSL 100 nM MeOH TDA 1µM 18h 12h 24h 36h A B 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0 12 18 24 36 Absorbance measured at 600nm Time (hr) Growth Analysis DMSO C10-HSL 100 nM MeOH TDA 1 µM 3. Materials and Methods 3.1. Growth Conditions All Pseudovibrio strains were isolated from the sponges Axinella dissimilis Polymastia boletiformis and Haliclona simulans as previously described [16]. A selection of marine isolates from the UCC marine culture collection, previously isolated from the deep sea sponges of Hexactinellida and Poecillastra species, were used as indicator strains for TDA sensitivity assays. All marine sponge isolates were grown in the dark at 23 °C shaking at 200 rpm. Strains were grown on marine agar (MA) (Difco, Difco laboratories, Surray, KT8 2SE, UK), or SYP-SW agar (1% starch, 0.4% yeast extract, 0.2% peptone, 3.3% sea salt, 1.5% agar). Other bacterial isolates (indicator strains) were grown shaking at 200 rpm at 37 °C in LB (1% tryptone, 0.5% yeast extract, 0.5% salt). Mar. Drugs 2014, 12 DMSO C10-HSL 100 nM MeOH TDA 1µM 18h 12h 24h 36h A B 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0 12 18 24 36 Absorbance measured at 600nm Time (hr) Growth Analysis DMSO C10-HSL 100 nM MeOH TDA 1 µM Materials and Methods A 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0 12 18 24 36 Absorbance measured at 600nm Time (hr) Growth Analysis DMSO C10-HSL 100 nM MeOH TDA 1 µM A DMSO C10-HSL 100 nM MeOH TDA 1µM 18h 12h 24h 36h B B 3. Materials and Methods Mar. Drugs 2014, 12 All extracts—including a control of W74 grown in MB—showed TDA production at 36 h, suggesting that TDA production is not autoinduced or induced by 3-OH-C10-HSL. Research by Geng and co-workers [37] revealed that TDA produced by Silicibacter (Rugeria) and Pseudovibrio species induced the transcription of tdaC in a TDA-producing Ruegeria sp. Autoinduction was not observed in our marine isolates, further evidence that production of this secondary metabolite is not uniform among marine isolates. However, further analysis and functional genomics studies will be required to determine the factors underlying this divergence. 5970 3.3. Compound Extraction Compounds were extracted following a protocol adapted from Porsby et al., [24]. Briefly, Pseudovibrio strains were inoculated from an overnight culture into 10ml MB at an OD600 of 0.01 and incubated shaking at 150 rpm at 23 °C for >72 h (or until brown colour change occurred). The culture was then centrifuged (4472× g @ RT) and filtered through a 0.2 micron filter (514-0061 VWR, VWR International Ltd., Dublin, Ireland). Equal volume ethyl acetate (10 mL) plus 100 μL formic acid were added to the cell-free supernatant and incubated shaking at 23 °C at 200 rpm for 10 min. Samples were centrifuged at 4472× g for 10 min. Subsequently, the upper layer was removed and vacuum concentrated at 50 °C until dry. The dried compound was then resuspended in relevant mobile phase, i.e., 100 μL of MeOH for TLC and 200 μL of 85% acetonitrile/15% MilliQ water for UHPLC analysis. 3.4. Thin Layer Chromotography (TLC) Two microliters of extracts were spotted on a Silica gel TLC plate (60 F254, MERCK Millipore, Cork, Ireland). 2 μL of 10 μg/mL TDA (BVT-0152, BioViotica, Dransfeld, Germany), redissolved in MeOH was used as a control. The solvents used were dichloromethane and methanol at a ratio of 92.5:7.5, plus 0.15% acetic acid. When the front approached the top of the TLC plate, the plate was removed, dried and overlaid with 25 mL 0.5% LB agar containing bacterial indicator strains at an OD600 of 0.1, plus 5 mM 2,3,5-Triphenyltetrazolium Chloride (T8877, Sigma, St Louis, MO, USA) as a redox indicator of bacterial metabolism [42]. Plates were incubated for 24 h at 37 °C for pathogenic and laboratory indicator strains, and at 23 °C for marine isolates. Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 3.5. TDA Identification Pseudovibrio extracts were redissolved in 200 μL 85% acetonitrile/15% MilliQ water. TDA standard solutions were diluted in 85% acetonitrile/15% MilliQ from a 1 mM TDA (BioViotica, Dransfeld, Germany) solution in dimethyl sulfoxide. UHPLC-DAD-qTOFMS analysis was conducted on an Agilent 1290 UHPLC coupled to an Agilent 6550 qTOF (Santa Clara, CA, USA) equipped with a dual electrospray source [43]. Separation was performed at 40 °C on a 2.1 mm ID, 50 mm, 1.8 μm Agilent Eclipse Plus C18 column [44] using a water-acetonitrile gradient solvent system, with both water and acetonitrile containing 20 mM formic acid. Using a flow of 0.8 mL/min, the gradient was started at 15% acetonitrile and increased to 60% acetonitrile within 1.8 min, then to 100% in 0.2 min, keeping this for 0.8 min, returning to 15% acetonitrile in 0.2 min, and equilibrating for the next sample in 1.5 min (total runtime 4.5 min). TDA was determined in ESI+ mode and quantified from its [M + H]+ ion 212.9674 ± 0.005 with the same retention time as the authentic standard (1.05 min). The TDA standard had a concentration of 0.01 mg/mL. Quantification was done using regression based on the peaks area in the Agilent MassHunter Quant 6.0 software (Agilent Technologies, Santa Clare, CA, USA). 3.2. Antimicrobial Spot-Plate Overlay Assay Fifty microliters of Pseudovibrio strains grown overnight on Difco marine broth (MB, Difco laboratories, Surray, KT8 2SE, UK) were spotted at an OD600 of 0.2 onto MA or SYP-SW agar and incubated at 23 °C for 72 h. Bacterial indicator strains were grown overnight in LB and overlaid in 0.5% LB agar at an OD600 of 0.1. Zones of inhibition were measured after 24 h of incubation at 37 °C. 5971 Mar. Drugs 2014, 12 3.7. Pseudovibrio Induction Assay Pseudovibrio strain W74 was inoculated from an overnight culture into 3 × 200 mL MB in 1 L flasks, at OD600 nm of 0.01 in the presence of 100 nM 3-OH-C10-HSL (University of Nottingham, Nottingham, UK), or 1 μM TDA, to test for the induction and autoinduction of TDA, respectively. W74 was also grown in the presence of 100 μL of both DMSO and MeOH, as C10-HSL and TDA were suspended in these respectively. W74 was also grown in marine broth as a control. Cultures were incubated at 200 rpm at 23 °C, and the OD600 of the cultures was measured at 12 h, 18 h, 24 h and 36 h. The colour of the cultures was also observed and noted at the above time points, at which times TDA was also extracted from the samples and analysed via TLC overlayed with S. aureus NCD0 949 as indicator strain. Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 marine agar at an OD600 nm of 0.1. This was overlaid onto W74 spot-plates and incubated at 23 °C. Zones of inhibition were measured after 24 h. 3.8. RNA Isolation and cDNA Synthesis Pseudovibrio strain W74 was grown shaking in 20 mL MB in a 100 mL flask at 23 °C for 48 h. Growth was measured at 6, 12, 24, 30, 36 and 48 h and a growth curve was plotted. At the above time points, 500 μL culture was collected and stored in 1 mL RNA protect. RNA was isolated as per the RNeasy RNA Extraction Kit protocol (Qiagen GmbH, Hilden, Germany). Isolated RNA was subsequently treated with DNase (0.1 volume 10× Turbo DNase) and incubated at 37 °C for 30 min to remove any potentially contaminating DNA. DNase was then inactivated using 0.1 volume DNase inactivation reagent. A 16S targeted PCR using 63F and 1387R primers was performed on the isolated RNA to test whether the DNase treatment had been successful [45]. RNA was converted to cDNA using AMV Reverse Transcriptase (Promega, Madison, WI, USA), RNasin (100 U/μL) (Promega, Madison, WI, USA), random primers (0.5 μg/μL) (Promega, Madison, WI, USA) and 10 mM dNTPs (Promega, Madison, WI, USA). A 16S targeted PCR was also performed using 63F and 1387R primers to ensure cDNA integrity. 3.6. Antimicrobial Spot-Plate Overlay Assay for Marine Sponge Isolates The Pseudovibrio isolate W74 was spot-plated (5 μL) on MA at a starting OD600 of 0.2, and incubated at 23 °C for 72 h. Deep sea marine sponge isolates were grown overnight and inoculated into soft 0.5% 5972 4. Conclusions In this study, we have characterised the production and spectrum of activity of TDA from marine sponge bacterial isolates, while also revealing an apparent lack of auto-induction or of QS induced early induction of TDA production in Pseudovibrio. In contrast to previous studies, tolerance to TDA, which was prevalent among marine sponge isolates, was not associated with native TDA production in these strains. TDA was shown to be active against a broad spectrum of human and fish pathogens, including M. morganii and Pandoraeae sputonum, both multidrug resistant opportunistic pathogens commonly associated with nosocomial infections [10,50–53]. TDA-producing Pseudovibrio isolates also displayed antimicrobial activity against Salmonella enterica ssp. arizonae, a serious albeit uncommon multidrug-resistant human pathogen which can cause life-threatening infections, usually in immunocompromised hosts [54–56]. S. aureus, one of the top three pathogens of blood stream infections [53], also displayed sensitivity to TDA produced by Pseudovibrio isolates. As the search for new antibiotics continues, TDA produced by marine sponge isolates has potential as a platform molecule for clinical development. Indeed, synthetic modification of the TDA framework resulting in analogues with enhanced antimicrobial activity has recently been reported [57], evidence that chemical modification of the compound can be achieved. Furthermore, initial toxicity studies have revealed that TDA is non-toxic in two eukaryotic models [30]. However, more detailed understanding of the molecular mechanisms governing TDA production is needed to underpin the necessary development process. 3.10. Genetic Analysis of Pseudovibrio TDA Genes The 6 genes involved in the TDA pathway in Ruegeria sp. TM1040 [37] (Table 2) were used as a query to identify their homologs in the draft genomes of the three Pseudovibrio sp marine isolates W64, W74 and WM33 using NCBI BLAST+ 2.2.29 [49]. All-vs-all homology comparison of the 6 genes involved in the TDA pathway for the different species (Table 2) and the graphical representation was conducted by Circoletto [40]. Mar. Drugs 2014, 12 Mar. Drugs 2014, 12 gene gyrB. This gene was chosen on the basis that it is universally used for normalization of gene expression data in a broad spectrum of microbial species [47,48]. 3.9. RT-PCR Quantitative RT-PCR analysis of six genes (tdaA-F) involved in TDA production was performed on W74 cDNA using primers outlined in Supplementary Table S9. Specific RT-PCR primers and probes were designed using the Universal ProbeLibrary assay center [46] for each gene based on the W74 genome sequence. Ten serial dilutions of the gDNA standards from 108 to 104 copies were prepared and 2 blanks were used as a control. Quantitative real-time PCR analysis of expression was carried out using the FastStart TaqMAN probe master kit (12747422, Roche, Basel, Switzerland). cDNA samples were diluted 1/10, and 5 μL was added to the following mixture: 12.5 μL Probemaster, 0.2 μL probe, 0.5μL forward primer, 0.5 μL reverse primer (Supplementary Table S9), 6.3 μL H2O. 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Chen, Y.H.; Kuo, J.; Sung, P.J.; Chang, Y.C.; Lu, M.C.; Wong, T.Y.; Liu, J.K.; Weng, C.F.; Twan, W.H.; Kuo, F.W. Acknowledgments This research was supported in part by grants awarded by the Science Foundation of Ireland (SSPC2 12/RC/2275; 13-TIDA-B2625; 07/IN.1/B948; 12/TIDA/B2411; 12/TIDA/B2405; 09/RFP/BMT 2350); the Department of Agriculture, Fisheries and Food (DAFF11/F/009 MabS; FIRM/RSF/CoFoRD; FIRM 08/RDC/629); the Environmental Protection Agency (EPA 2008-PhD/S-2), the Irish Research Council for Science, Engineering and Technology (PD/2011/2414; RS/2010/2413), the European Commission (FP7-PEOPLE-2013-ITN, 607786; OCEAN2011-2, 287589; FP7-KBBE-2012-6, CP-TP 311975; FP7-KBBE-2012-6, CP-TP-312184; Marie Curie 256596); the Marine Institute (Beaufort award C2CRA 2007/082); Teagasc (Walsh Fellowship 2013) and the Health Research Board (HRA/2009/146). We thank Pat Higgins and David Woods for technical assistance. Mar. Drugs 2014, 12 5974 Mar. Drugs 2014, 12 Author Contributions F.O’G., M.J.M., F.J.R., C.A and A.D. conceived and designed the experiments; C.H., J.R, F.S. and J.-B.C. performed the experiments; K.F.N. and L.G. performed the UHPLC-TOFMS analysis of Pseudovibrio extracts; A.F.G. and F.O.G. analyzed the sequence data; C.H. C.A., F.J.R. and F.O’G. wrote the paper. Conflicts of Interest The authors declare no conflict of interest. The authors declare no conflict of interest. References Isolation of marine bacteria with antimicrobial activities from cultured and field-collected soft corals. World J. Microbiol. Biotechnol. 2012, 28, 3269–3279. 11. Rizzo, C.; Michaud, L.; Hormann, B.; Gerce, B.; Syldatk, C.; Hausmann, R.; de Domenico, E.; lo Giudice, A. Bacteria associated with sabellids (polychaeta: Annelida) as a novel source of surface active compounds. Mar. Pollut. Bull. 2013, 70, 125–133. Mar. Drugs 2014, 12 5975 12. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4312477544	https://www.nomos-elibrary.de/10.5771/9783748928690-301.pdf?download_full_pdf=1&page=1	German	null	3. Exkurs	Nomos Verlagsgesellschaft mbH & Co. KG eBooks	2,022	cc-by-sa	4,898	Der ARD-Zukunftsdialog Der ARD-Zukunftsdialog Der ARD-Zukunftsdialog Der ARD-Zukunftsdialog Matthias Trénel Einleitung Bereits seit vielen Jahren sieht sich der öffentlich-rechtliche Rundfunk einem steigenden Druck ausgesetzt, seine Finanzierung durch die Erhe bung von Rundfunkbeiträgen zu rechtfertigen. 2018 sorgte in der Schweiz die Volksabstimmung „No Billag“ für Aufsehen. Zwar hat sich die Be völkerung schlussendlich gegen die Abschaffung der Rundfunkabgaben entschieden, dennoch musste der öffentlich-rechtliche Rundfunk in der Folge ein großes Sparpaket verkraften. In Deutschland spitzte sich die Lage Ende 2020 zu, als das Bundesland Sachsen-Anhalt seine Zustimmung zur geplanten Erhöhung des Rundfunkbeitrags verweigerte und der erneuerte Medienstaatsvertrag in der Folge nicht in Kraft treten konnte. Als Reakti on darauf reichten die öffentlich-rechtlichen Programme Klage beim Bun desverfassungsgericht ein. Bereits seit vielen Jahren sieht sich der öffentlich-rechtliche Rundfunk einem steigenden Druck ausgesetzt, seine Finanzierung durch die Erhe bung von Rundfunkbeiträgen zu rechtfertigen. 2018 sorgte in der Schweiz die Volksabstimmung „No Billag“ für Aufsehen. Zwar hat sich die Be völkerung schlussendlich gegen die Abschaffung der Rundfunkabgaben entschieden, dennoch musste der öffentlich-rechtliche Rundfunk in der Folge ein großes Sparpaket verkraften. In Deutschland spitzte sich die Lage Ende 2020 zu, als das Bundesland Sachsen-Anhalt seine Zustimmung zur geplanten Erhöhung des Rundfunkbeitrags verweigerte und der erneuerte Medienstaatsvertrag in der Folge nicht in Kraft treten konnte. Als Reakti on darauf reichten die öffentlich-rechtlichen Programme Klage beim Bun desverfassungsgericht ein. g g Vor diesem Hintergrund wurde im Januar 2021 von den Intendant:in nen der Arbeitsgemeinschaft der öffentlich-rechtlichen Rundfunkanstalten der Bundesrepublik Deutschland (ARD) beschlossen, zwischen Mai und November 2021 den ARD-Zukunftsdialog durchzuführen. Das übergrei fende Ziel bestand darin, die Reformdiskussion über die Ausrichtung und Finanzierung des öffentlich-rechtlichen Rundfunks in einer aktiven Rolle mitzugestalten (Buhrow, 2021). Dies sollte nicht nur in den Rundfunkrä ten geschehen, in denen Politik und gesellschaftliche Gruppen vertreten sind, sondern auch in der Öffentlichkeit mit den Menschen selbst – und zwar unabhängig davon, ob von ihnen das Programmangebot des öffentlich-rechtlichen Rundfunks genutzt wird oder nicht. Ein weiteres konkreteres Ziel des Zukunftsdialogs war zudem, Erkenntnisse darüber zu gewinnen, welche Vorstellungen die Menschen zur Zukunft der ARD haben. Diese sollten im direkten Gespräch mit Mitarbeiter:innen der ARD ausgetauscht werden, um sie für Reformbestrebungen innerhalb der ARD nutzen zu können. 301 5 /9 83 – / – Matthias Trénel Allerdings schien die gewohnte Art und Weise, wie der öffentlich-recht liche Rundfunk Meinungen von Bürger:innen einholt, nämlich im Kon text von Medienforschung und Programmanalyse, wenig geeignet. 1 Für das Publikum bestand die Möglichkeit, Nachfragen zu stellen. Dies ändert jedoch nichts an der Einschätzung, dass der Mittelpunkt der Gespräche auf dem Podium lag. Einleitung Die Diskussion in solchen nicht-öffentlichen und kontrollierten Forschungs settings wird zwar gelegentlich „Publikumsdialog“ genannt, ist aber davon geprägt, dass Publikum mit Publikum vor den Forscher:innen diskutiert, welche sich in einer beobachtenden Rolle befinden. Die beteiligten Bür ger:innen erhalten für ihre Mitwirkung in den repräsentativen Fokusgrup pen – wie in der Markt- und Meinungsforschung üblich – eine Vergütung und haben in der Regel darüber hinaus keine Erwartungen, was mögli che Konsequenzen aus den Gesprächen betrifft. Auch die vom Zweiten Deutschen Fernsehen in den Jahren 2018 und 2019 als Podiumsdiskussion praktizierten „Bürgerdialoge“ eigneten sich kaum als Vorbild (ZDF, 2019). Sie waren gekennzeichnet durch eine klare Trennung zwischen Podium und Publikum: Im Wesentlichen diskutierte das ZDF mit Expert:innen vor Publikum1. Für den ARD-Zukunftsdialog wurde daher eine neue Form des Dialogs gesucht, eine die erstens auf einer quasi-repräsentativen Stichprobe von Bürger:innen fußt, die zweitens transparent in der Öffentlichkeit stattfindet und die drittens im Sinne einer verständigungsorientierten Öffentlichkeits arbeit (Burkart, 2012) einen wechselseitigen Diskurs von der ARD mit den Bürger:innen ermöglicht. Prozess Der ARD-Zukunftsdialog wurde als Prozess mit vier aufeinander aufbau enden Schritten konzipiert. Das „Herz“ des Prozesses war eine vierwöchige öffentliche Online-Beteiligung, die flankiert wurde durch zwei Großgrup penveranstaltungen mit Losbürger:innen – eine Auftaktkonferenz zur Vor strukturierung der Themen für die Online-Beteiligung und eine Abschluss konferenz zur Validierung der Ergebnisse von der Online-Beteiligung. Da ran schloss sich die Umsetzung der Erkenntnisse zur Weiterentwicklung der ARD an (siehe Tabelle 1). Die Abfolge von Großgruppenkonferenz, Online-Beteiligung und er neuter Großgruppenkonferenz ist ein bewährtes und robustes Dialogkon 302 / – Der ARD-Zukunftsdialog zept für die Bürgerbeteiligung in Veränderungsprozessen (Trénel, 2020). Dieses Modell „smarter Partizipation“ kombiniert die Vorteile großer Reichweite und Offenheit mit der Qualität kokreativ erarbeiteter Ergebnis se und dabei entstehendem Vertrauen. Tabelle 1: Ablauf des ARD-Zukunftsdialogs im Überblick 1. Auftakt- konferenz 2. Online-Be teiligung 3. Abschluss- konferenz 4. Umsetzung Zeit punkt 08.05.2021 31.05.-27.06.2 021 13.11.2021 ab 25.11.2021 Mitwir- kende Losbürger:in nen (n = 139) ARD-Vertre ter:innen (n = 35) Öffentlichkeit (n = 3.822) ARD The menpat:in. (n = 16) Losbürger:in nen (n = 91) ARD-Vertre ter:innen (n = 18) ARD-Sender und Kommis sionen Funkti on Future Search Crowdsour cing Reality Check Implementati on Aus gangs- fragen Wie nimmst du die ARD wahr? Was wünschst du dir für die Zu kunft? Und welche The men sind dir dabei wichtig? Welche Ideen und Wünsche hast du für die Zukunft der ARD? Wie bewertest und gewich test du die Zu kunftsthe men? Mit welchen Maßnahmen werden die ARD-Zu kunfts-the men in An griff genom men? Ergeb nis 9 Themen komplexe 3.761 Ideen in 7 Themenräu men Feedback zu 5 ARD-Zu kunftsthemen 13 ARD-Zu kunftsprojek te Tabelle 1: Ablauf des ARD-Zukunftsdialogs im Überblick Das Projektteam der ARD wurde bei der Konzeption und Realisierung des ARD-Zukunftsdialogs durch die Dialogdienstleister frischer wind (Gesamt konzeption, Großgruppenkonferenzen) und Zebralog (ebenfalls Gesamt konzeption, Losbürgerrekrutierung, Online-Beteiligung) unterstützt. Alle Prozessschritte wurden auf der Transparenzseite zum ARD-Zukunftsdialog dokumentiert (ARD, 2021a). Nachfolgend werden diese näher erläutert, Das Projektteam der ARD wurde bei der Konzeption und Realisierung des ARD-Zukunftsdialogs durch die Dialogdienstleister frischer wind (Gesamt konzeption, Großgruppenkonferenzen) und Zebralog (ebenfalls Gesamt konzeption, Losbürgerrekrutierung, Online-Beteiligung) unterstützt. Alle Prozessschritte wurden auf der Transparenzseite zum ARD-Zukunftsdialog dokumentiert (ARD, 2021a). Nachfolgend werden diese näher erläutert, 303 0.5771/978374 – - .5771/978374 – Matthias Trénel bevor die inhaltlichen Ergebnisse im anschließenden Kapitel dargestellt werden. bevor die inhaltlichen Ergebnisse im anschließenden Kapitel dargestellt werden. Auftaktkonferenz 1. Der Zukunftsdialog startete am 8. Mai 2021 mit einer Auftaktkonferenz, die aufgrund der COVID-19-Pandemie als Videokonferenz durchgeführt wurde. Daran nahmen 139 Bürger:innen teil, die aus dem gesamten Bundesge biet mithilfe eines telefonischen Losverfahrens ausgewählt wurden. Durch die Einladung nach dem Zufallsprinzip sollte eine proportionale Vertei lung aller Bevölkerungsgruppen erreicht werden, damit die Bürger:innen einen Querschnitt der Gesellschaft abbilden. Dies gelang im Großen und Ganzen mit folgenden Einschränkungen: Jüngere Altersgruppen waren relativ gering vertreten genauso wie Menschen aus den neuen Bundeslän dern. Zudem gab es einen leichten Überhang an Frauen. Zu den Teil nehmenden der Auftaktkonferenz gehörten weiterhin 35 Vertreter:innen der Landesrundfunkanstalten und Gemeinschaftseinrichtungen der ARD, darunter der damalige Vorsitzende der ARD und Intendant des Westdeut schen Rundfunks, Tom Buhrow. Die Funktion der Auftaktkonferenz bestand darin, ohne Vorgaben und Einengungen eine Themenstruktur für die kommenden Prozessschritte zu erarbeiten. Hierfür wurde die Methode „Future Search Conference“ ge wählt – also eine Methode, die vor allem dann eingesetzt wird, „wenn die Menschen sozusagen auf der grünen Wiese gemeinsam ihre Zukunft pla nen“ (Krummenacher et al., 2019, S. 20). Um eine rege Diskussion mit al len Teilnehmer:innen zu erreichen, wurden diese auf Kleingruppen von sieben bis acht Personen inklusive ein bis zwei Vertreter:innen der ARD verteilt. Im Wechsel zwischen Plenum und Kleingruppen wurden drei Runden durchlaufen: Zunächst ging es um die aktuellen Wahrnehmun gen der ARD. Dann wurde über Wünsche und Erwartungen für die Zu kunft gesprochen. Schließlich wurden Themen gesammelt, die die ARD in der Zukunft anpacken sollte. Jede Runde schloss mit einer übergreifenden Zusammenfassung aller Wortmeldungen durch das Projektteam der ARD und einem Voting durch die Bürger:innen zur Gewichtung der Vorschläge ab. Im Ergebnis wurden auf diese Weise neun Themenkomplexe erarbeitet (siehe Tabelle 2), die wiederum Ausgangspunkt für die darauffolgende Online-Beteiligung waren. 304 / – / – Der ARD-Zukunftsdialog Online-Beteiligung 2. Im nächsten Schritt wurde zwischen dem 31. Mai und dem 27. Juni 2021 eine öffentliche Online-Beteiligung durchgeführt (ARD, 2021b). Daran nahmen 3.822 Personen teil, die zusammen 14.601 Beiträge schrieben, davon 3.761 „Ideen“ und 10.840 Kommentare zu den „Ideen“. Insgesamt wurde die Online-Beteiligung von 15.626 Personen besucht (unique visitors). Die Intensität der Mitwirkung im zeitlichen Verlauf kor respondierte deutlich mit den öffentlichen Aufrufen zur Teilnahme im Online-, Fernseh- und Hörfunkangebot der ARD, zum Beispiel in der „ta gesschau“ am 31.05.2021 (ARD, 2021c). Auftaktkonferenz 1. Auf der Grundlage freiwilliger An gaben der Teilnehmenden ließ sich erkennen, dass sich jüngere Altersgrup pen und Menschen aus den neuen Bundesländern im Verhältnis zu ihrem Anteil in der deutschen Bevölkerung in stärkerem Umfang beteiligten als bei der Auftaktveranstaltung. Zu den Teilnehmenden gehörten zudem 16 Themenpat:innen der ARD, die sich aktiv in die Online-Diskussion einbrachten. Sie stammten aus verschiedenen Landesrundfunkanstalten und waren zum Teil Personen, die aus dem Programm der ARD einer größeren Öffentlichkeit bekannt sind (zum Beispiel Frank Bräutigam vom Südwestrundfunk, Hülya Deyneli vom Hessischen Rundfunk oder Roman Nuck vom Mitteldeutschen Rundfunk). Ziel der Online-Beteiligung war ein Crowdsourcing von Ideen und Wünschen zu den Themenkomplexen, die in der Auftaktkonferenz iden tifiziert wurden. Dafür wurden die Themenkomplexe vom ARD-Projekt team zur Vereinfachung auf sechs reduziert und um einen Komplex „Sonstiges“ ergänzt. Entsprechend wurde eine digitale Beteiligungsplatt form (Zebralog, 2021) mit sieben Themenräumen eingerichtet, in denen Ideen und Wünsche zur Zukunft der ARD als Textbeiträge gesammelt wurden. Die Online-Beteiligung war offen einsehbar für alle interessierten Bürger:innen. Um eigene Ideen beizutragen oder die Ideen anderer zu kommentieren, war eine Registrierung mit gültiger E-Mail-Adresse und einem Pseudonym obligatorisch. Außerdem musste jede Idee bei der Einreichung einem von ca. sechs vordefinierten Unterthemen je Themenraum zugeordnet werden. Diese „Tags“ wurden jeder Idee sichtbar angehängt und halfen dabei, sich über Filterfunktionen einen besseren Überblick über die abgegebenen Ideen zu verschaffen. Jeder Themenraum wurde von zwei bis drei Themenpat:innen der ARD betreut, um die Interaktion zwischen Publikum und ARD lebendig zu ge stalten. Über die Zeit der Online-Beteiligung wählten sie mehrmals in der Woche solche Ideen aus und hoben sie auf der Plattform hervor, die aus 305 0.5771/978374 – - Matthias Trénel Ihrer Sicht einen wichtigen Impuls für die Zukunftsdebatte der ARD lie ferten. Für die Auswahl waren vor allem Kriterien wie Kreativität, Kritik, Aktualität und thematische Vielfalt ausschlaggebend. Einige Themen pat:innen haben zudem an Live-Diskussionen teilgenommen, die am 9., 16. und 23. Juni zu bestimmten Themenschwerpunkten gestreamt wur den. Sie griffen darin Ideen aus der laufenden Online-Beteiligung auf und diskutierten sie weiter. Dabei wurden sie von Gebärdendolmetscher:innen begleitet. Die Bürger:innen brachten ihre Fragen und Meinungen derweil per Chat ein. Im Nachgang wurden die Live-Diskussionen auf der Platt form als Video zur Verfügung gestellt. g g g Insgesamt war die Online-Beteiligung von einer konstruktiven Atmo sphäre und konkreten Verbesserungsvorschlägen geprägt. Selbst bei Mei nungsunterschieden diskutierten die Teilnehmer:innen überwiegend sach lich miteinander, sodass polemische Äußerungen die Ausnahme blieben. Auftaktkonferenz 1. Einzelne Themen wie Gendern, Neutralität der ARD und Klimakrise wur den teils hitzig diskutiert. In diesen Fällen wirkte ein Team von Online- Moderator:innen auf der Grundlage öffentlich einsehbarer Dialogregeln darauf hin, den Austausch zu versachlichen (Gottwald, 2022). Tagsüber wurden alle Ideen und Kommentare unmittelbar bei ihrer Abgabe auf der Plattform veröffentlicht und nach kurzer Zeit von den Moderator:in nen geprüft. In der Nacht hingegen erschienen die abgegebenen Beiträge sicherheitshalber vorerst nicht, sondern wurden von den Moderator:innen erst zu Beginn des folgenden Tages freigeschaltet. g g g g Wie im folgenden Kapitel dargestellt, wurden die umfangreichen Er gebnisse der Online-Beteiligung systematisch ausgewertet (siehe Tabelle 3) und im September und Oktober 2021 in internen Workshops der Lan desrundfunkanstalten und ARD-Gemeinschaftseinrichtungen diskutiert. Schließlich wurden fünf Themenbündel von den ARD-Intendant:innen zu „ARD-Zukunftsthemen“ erklärt. 2 Zum Zeitpunkt des Verfassens von diesem Beitrag stammte die letzte Aktualisie rung vom 22. März 2022. Abschlusskonferenz 3. Die Abschlusskonferenz am 13. November 2021 rundete die aktive Phase des ARD-Zukunftsdialogs ab. Sie wurde – wie schon die Auftaktkonferenz – als Videokonferenz durchgeführt, um zur Eindämmung der COVID-19- Pandemie beizutragen. Somit wurde der ARD-Zukunftsdialog von Anfang bis Ende vollständig digital ohne jegliche persönliche Begegnung durchge führt. Von den Teilnehmenden der Auftaktkonferenz nahmen etwas weniger als die Hälfte erneut an der Abschlusskonferenz teil. Deshalb wurden 306 / – / – Der ARD-Zukunftsdialog Der ARD-Zukunftsdialog weitere Bürger:innen hinzugeladen. Einige hatten zuvor an der Online-Be teiligung mitgewirkt und andere wurden mithilfe desselben telefonischen Losverfahrens wie bei der Auftaktkonferenz neu gewonnen. Dabei lag der Fokus auf der Gruppe der unter 30-Jährigen, weil diese bereits bei der ersten Auftaktkonferenz unterrepräsentiert war und nun ein ausgewogenes Verhältnis erreicht werden sollte, was auch näherungsweise gelang. Aller dings waren Menschen aus den neuen Bundesländern weiterhin in gerin gerem Maße vertreten. Insgesamt diskutierten 91 Bürger:innen und 18 ARD-Vertreter.innen miteinander. Die Aufgabe der Abschlusskonferenz bestand darin, die Auswahl der deklarierten fünf ARD-Zukunftsthemen von einer quasi-repräsentativen Bürgerstichprobe zu validieren und einem „Reality Check“ zu unterzie hen. Schließlich wurden die fünf ARD-Zukunftsthemen mithilfe eines Votings gewichtet, wobei es zulässig war, auch mehr als einem Thema die Zustimmung zu geben. Das Ergebnis wird in Tabelle 4 dargestellt. Umsetzung 4. Die Ergebnisse der Abschlusskonferenz wurden von der ARD dafür ge nutzt, 13 Maßnahmen zu entwickeln, die sich zur Umsetzung der ARD- Zukunftsthemen eignen. Diese „ARD-Zukunftsprojekte“ wurden von den Intendant:innen der ARD verabschiedet und am 9. Dezember in einem Bericht zum Abschluss des ARD-Zukunftsdialogs bekannt gemacht (ARD, 2021d). In dem Bericht wird zudem dargelegt und begründet, welche Ide en und Wünsche die ARD nicht weiterverfolgen wird oder kann (siehe Ta belle 5). Gleichzeitig wurde eine „Transparenzseite“ (ARD, 2021a) einge richtet, in der seitdem in regelmäßigen Abständen über die Umsetzung der Zukunftsprojekte berichtet wird2. Auswertung und Ergebnisse Alle Prozessschritte wurden dokumentiert und ausgewertet, um eine Grundlage für die nachfolgenden Schritte zu erarbeiten. In den folgenden Tabellen werden die inhaltlichen Ergebnisse des ARD-Zukunftsdialogs im Überblick dargestellt – von der Definition der Themenkomplexe (Tabelle 307 https://doi.org/10.5771/978374 Open Access – - / – / – Matthias Trénel 2) über die Sammlung von Ideen und Wünschen (Tabelle 3) bis hin zur Gewichtung der Zukunftsthemen und zur Umsetzung der Zukunftspro jekte (Tabelle 4). 2) über die Sammlung von Ideen und Wünschen (Tabelle 3) bis hin zur Gewichtung der Zukunftsthemen und zur Umsetzung der Zukunftspro jekte (Tabelle 4). j Für die Auswertung der Online-Beteiligung wurden die 3.761 Ideen und 10.840 Kommentare in die Auswertungssoftware MaxQDA (Rädiker & Kuckartz, 2019) überführt. In dieser Umgebung erfolgte dann eine qualitative Inhaltsanalyse nach Mayring (2015), die zwei Etappen umfasste: Erstens wurden alle Beiträge (Ideen und Kommentare) in jedem The menraum nach ihrer Zuordnung zu den vorgegebenen und von den Teil j Für die Auswertung der Online-Beteiligung wurden die 3.761 Ideen und 10.840 Kommentare in die Auswertungssoftware MaxQDA (Rädiker & Kuckartz, 2019) überführt. In dieser Umgebung erfolgte dann eine qualitative Inhaltsanalyse nach Mayring (2015), die zwei Etappen umfasste: q y y g pp Erstens wurden alle Beiträge (Ideen und Kommentare) in jedem The menraum nach ihrer Zuordnung zu den vorgegebenen und von den Teil nehmenden selbst zugeordneten Unterthemen geordnet. Dann wurden alle Beiträge eines Unterthemas gelesen und auf induktive Weise Katego rien bzw. Themenschwerpunkte gebildet, um die Kernaussagen in den Beiträgen zusammenzufassen. Zweitens wurden alle Beiträge, die zu einem Unterthema gehörten, ein weiteres Mal gelesen. In dem Zuge wurde interpretiert, welche Text stellen einem der zuvor gebildeten Themenschwerpunkte zugeordnet wer den können. Für die Themenschwerpunkte mit den meisten zugeordne ten Ideen wurde eine Beschreibung erstellt, welche die zentrale Sinnein heit wiedergibt (Welche Anregung, Wunsch oder Forderung ist enthal ten?). Schließlich wurde für jeden Themenschwerpunkt ein repräsentatives Schlüsselzitat aus einem der zugeordneten Beiträge ausgewählt, um die Themenschwerpunkte durch den Originalton des Teilnehmers oder der Teilnehmerin zu veranschaulichen. Die auf diese Weise generierten Themenschwerpunkte sollen allerdings nicht als repräsentatives Meinungsbild verstanden werden. Der Vorteil einer Online-Beteiligung liegt vielmehr darin, anders als bei repräsentati ven Meinungsumfragen die Sichtweisen von Bürger:innen auf unverzerr te Weise zu erfassen, also unabhängig von vorgegebenen Fragen oder Antwortformaten. Zusätzlich lassen sich die emotionale Intensität sowie Überschneidungen und Konflikte zwischen Perspektiven erkennen. 308 Der ARD-Zukunftsdialog Tabelle 2: Ergebnisse des ARD-Zukunftsdialogs – Auftaktkonferenz ZUKUNFTSDIALOG DER ARD 1. Auswertung und Ergebnisse Auftaktkonferenz mit Losbürger:innen Wie nimmst du die ARD wahr? Was wünschst du dir für die Zukunft? Und welche Themen sind dir dabei wichtig? 9 Themenkomplexe: Wissen und Bil dung Journalismus mit positivem Fokus Meinungsvielfalt Digitales Programmangebote regionale und inter nationale Berichter stattung Diversität Angebote für Ju gendliche und jun ge Erwachsene Qualität und Kom munikation 309 https://doi.org/10.5771/9783748928690-301, am 24.10.2024, 06:07:59 Open Access – - https://www.nomos-elibrary.de/agb Matthias Trénel Tabelle 3: Ergebnisse des ARD-Zukunftsdialogs – Online-Beteiligung 2. Online-Beteiligung mit Öffentlichkeit Welche Ideen und Wünsche hast du für die Zukunft der ARD? 7 Themenräume zur Diskussion (mit Zahl der Beiträge/Kommentare): Wissen und Hin tergründe (302/726) Menschen und Meinungen (577/3.070) Mediathek und Audiothek (399/1.020) Programmideen (1.448/2.892) Region und Le bensgefühl (245/535) Generation Zu kunft (203/759) Das beschäftigt mich außerdem … (587/1.837) Bildung/Wissen/ Hintergr.: Bericht erstatt. für die Ge neration Zukunft; Sach-zusammen hänge über den klassischen „Schul stoff“ hinaus; Posi tive, lösungs-orien tierte Berichterstat tung Thematisch ausge wogenere Politfor mate Mehr konservative Stimmen Darstellung unter schiedlicher Sicht weisen Digitale Angebote ausbauen, Auffind barkeit verbessern Bessere Möglichkei ten für Personalisie rung bieten Ange bote möglichst Sport: Zu viel Sport -Bündelung in Spartenkanal; Zu viel Fußballbe richterstatt. in den Nachrichten; Mehr Platz für Breiten-/ Behind.-sp. Krimis: Krimis zu brutal und unrea listisch; Kritik an Inhalts- und Vielfalt Stadt/ Land: Mehr länd licher Blickwinkel; Mehr Sichtbarkeit des Regionalen in Mediathek/Audio thek; Mehr Angebo te für junge Men schen vom Land Gendern: Vorwurf von „Spracherzie hung“ bzw. „Sprachentstel lung“; Sprachliche Abbildung von Di versität; Wunsch nach Abstimmung in der Bevölkerung 310 Der ARD-Zukunftsdialog lange verfügbar machen Kommentar- funktion in Media thek/Audiothek Ausbau des Com munity Manage ments Produktions-quali tät der Krimis; Zu viele Produktionen und Wiederhol. Musik: Mehr Ab wechslung, weniger Wiederholungen; Mehr region. Künstler:innen; Mehr deutsch- und anderssprachige, weniger anglo-ame rikanische Musik Quiz: Mehr „nor male“ Bürger:in nen, weniger „B- Promis“; Mehr na tur-wissenschaftli che Themen; An spruchsvollere Ge staltung Diversität: Mehr Diversität bei Mo derator:innen/ Gästen; Unter schiedliche Lebens entwürfe abbilden; Diversere Besetzung von Talkshow-Run den Auftrag und Struktur: Abschaf fung von Mehrfach strukturen, Fusio nierung von Sen dern, Nutzung von Synergien; Wunsch nach „gerechterem“ Rundfunkbeitrag; Stärkere Mitbe stimmung der Bei tragszahler:innen Transparenzforma te einführen Zukunftsdialog fortführen lange verfügbar machen Kommentar- funktion in Media thek/Audiothek Ausbau des Com munity Manage ments Produktions-quali tät der Krimis; Zu viele Produktionen und Wiederhol. Musik: Mehr Ab wechslung, weniger Wiederholungen; Mehr region. Auswertung und Ergebnisse Künstler:innen; Mehr deutsch- und anderssprachige, weniger anglo-ame rikanische Musik Quiz: Mehr „nor male“ Bürger:in nen, weniger „B- Promis“; Mehr na tur-wissenschaftli che Themen; An spruchsvollere Ge staltung Diversität: Mehr Diversität bei Mo derator:innen/ Gästen; Unter schiedliche Lebens entwürfe abbilden; Diversere Besetzung von Talkshow-Run den Auftrag und Struktur: Abschaf fung von Mehrfach strukturen, Fusio nierung von Sen dern, Nutzung von Synergien; Wunsch nach „gerechterem“ Rundfunkbeitrag; Stärkere Mitbe stimmung der Bei tragszahler:innen Transparenzforma te einführen Zukunftsdialog fortführen Mehr Klimabe richterstatt.: Mehr Bewusstsein für Nach-haltig keitsthemen; Große Zusammenhänge aufzeigen; Vertiefte Hintergründe Serien: Weniger, dafür qualitativ hochwertigere Seri en; Mehr Themen von historischer, po litischer Bedeutung; Serien auch im Ori ginalton (Zweika nalton) Dokus: Mehr Do kus; Mehr inhaltli che Tiefe, längere Formate; „Bessere“ Sendezeiten lange verfügbar machen Kommentar- funktion in Media thek/Audiothek Ausbau des Com munity Manage ments Produktions-quali tät der Krimis; Zu viele Produktionen und Wiederhol. Musik: Mehr Ab wechslung, weniger Wiederholungen; Mehr region. Künstler:innen; Mehr deutsch- und anderssprachige, weniger anglo-ame rikanische Musik Quiz: Mehr „nor male“ Bürger:in nen, weniger „B- Promis“; Mehr na tur-wissenschaftli che Themen; An spruchsvollere Ge staltung Diversität: Mehr Diversität bei Mo derator:innen/ Gästen; Unter schiedliche Lebens entwürfe abbilden; Diversere Besetzung von Talkshow-Run den Auftrag und Struktur: Absc fung von Mehrf strukturen, Fusi nierung von Sen dern, Nutzung Synergien; Wun nach „gerechtere Rundfunkbeitra Stärkere Mitbe stimmung der B tragszahler:inne Transparenzfor te einführen Zukunftsdialog fortführen 311 Mehr Klimabe richterstatt.: Mehr Bewusstsein für Nach-haltig keitsthemen; Große Zusammenhänge aufzeigen; Vertiefte Hintergründe Serien: Weniger, dafür qualitativ hochwertigere Seri en; Mehr Themen von historischer, po litischer Bedeutung; Serien auch im Ori ginalton (Zweika nalton) Dokus: Mehr Do kus; Mehr inhaltli che Tiefe, längere Formate; „Bessere“ Sendezeiten 311 Matthias Trénel Tabelle 4: Ergebnisse des ARD-Zukunftsdialogs – Abschlusskonferenz und Umsetzung 3. Abschlusskonferenz mit Losbürger:innen 5 ARD-Zukunftsthemen (mit Gewichtung in Prozent, Mehrfachnennungen möglich): Nachhaltigkeit und Hin tergrund (49 %) Meinungen (45 %) Streaming (40 %) Dialog (39 %) Vielfalt der Gesellschaft (33 %) 4. Auswertung und Ergebnisse Umsetzung durch die ARD 13 Zukunftsprojekte – Was sich die ARD vorgenommen hat: Was ARD nicht wei ter-verfolgen wird oder kann: Die ARD tut mehr für das Wissen um die gro ßen und langfristigen Zusammenhänge: Erklärformate für komple xe Themen Ausbau des Doku-Bereichs Hochwertige Serien mit re levantem Hintergrund Die ARD bildet ein breiteres Meinungs- spektrum in ihren An geboten ab: Neues Konzept für Pro- und-Contra-Format Meinungen der Bürger:in nen stärker aufgreifen Die ARD bringt den Ausbau von ARD Me diathek und ARD Au diothek konsequent voran: Suche in Mediathek verbes sern Mehr Funktionen fürs ARD-Konto Die ARD verstärkt den Austausch mit ihren Nutzer:innen: Mehr Dialog und Commu nity Management Neues Format ARD-Check in Social Media Gründung Arbeitsgruppe Dialog Unterschiedliche Bevöl kerungsgruppen und Lebensentwürfe spielen in den Angeboten der ARD eine größere Rol le: Ländlichen Raum stärker abbilden Pilot für Austausch mit Menschen mit Migrations hintergrund Mehr Angebote in leichter Sprache Mehr Breitensport – weni ger Fußball Weniger Krimis und Ge walt Vielfältigere Musikauswahl und Konzerte Anspruchsvollere Quiz shows Gendern Auftrag und Struktur 312 Der ARD-Zukunftsdialog Der ARD-Zukunftsdialog Tabelle 5: Ergebnisse des ARD-Zukunftsdialogs – Begründung für Nicht-Umset zung von Ideen und Wünschen 4. Umsetzung durch die ARD (Fortsetzung) Was ARD nicht wei terverfolgen wird oder kann: Begründung der ARD: Mehr Brei tensport – weniger Fuß ball Fußball ist Lieblingssport der Deutschen Ein separater öffentlich-rechtlicher Sportkanal findet bis lang keine medienpolitische und finanzielle Unterstüt zung. Sportberichterstattung ist bereits vielfältig (sie umfasst mehr als 100 Sportarten), Breitensport ist Thema der Serie „No Sports“. Weniger Kri mis und Ge walt Krimis haben hohe Akzeptanz beim Publikum. Krimis sind ein Format, in dem Unterhaltung mit der Behandlung gesellschaftlich relevanter oder psychologisch schwierigerer Themen verknüpft werden kann. Alle Sender haben Kinder- und Jugendschutzbeauftragte. Krimis sind nur ein kleiner Teil des Programms. Vielfältigere Es ist nicht möglich, Angebote für jeden spezifischen Mu Tabelle 5: Ergebnisse des ARD-Zukunftsdialogs – Begründung für Nicht-Umset zung von Ideen und Wünschen zung von Ideen und Wünschen 4. Umsetzung durch die ARD (Fortsetzung) Was ARD nicht wei terverfolgen wird oder kann: Begründung der ARD: Mehr Brei tensport – weniger Fuß ball Fußball ist Lieblingssport der Deutschen Ein separater öffentlich-rechtlicher Sportkanal findet bis lang keine medienpolitische und finanzielle Unterstüt zung. Sportberichterstattung ist bereits vielfältig (sie umfasst mehr als 100 Sportarten), Breitensport ist Thema der Serie „No Sports“. Weniger Kri mis und Ge walt Krimis haben hohe Akzeptanz beim Publikum. Anspruchs vollere Quiz shows Auswertung und Ergebnisse Krimis sind ein Format, in dem Unterhaltung mit der Behandlung gesellschaftlich relevanter oder psychologisch schwierigerer Themen verknüpft werden kann. Alle Sender haben Kinder- und Jugendschutzbeauftragte. Krimis sind nur ein kleiner Teil des Programms. Vielfältigere Musikaus wahl und Konzerte Es ist nicht möglich, Angebote für jeden spezifischen Mu sikgeschmack zu schaffen. Jede Radiowelle hat bereits ein eigenes musikalisches Pro fil, um eine möglichst große Vielfalt anzubieten. Es gibt regelmäßige Umfragen unter Hörer:innen nach ihren musikalischen Vorlieben. Anspruchs vollere Quiz shows Die Rückmeldungen zu den Quizshows sind uneinheitlich. Gebraucht werden auch Quizformate, die von der ganzen Familie geschaut werden. Es gibt bereits eine Vielfalt an Quizformaten mit unter schiedlichen Anspruchsniveaus. Neue Quizformate werden in den dritten Programmen pi lotiert Es ist nicht möglich, Angebote für jeden spezifischen Mu sikgeschmack zu schaffen. g Jede Radiowelle hat bereits ein eigenes musikalisches Pro fil, um eine möglichst große Vielfalt anzubieten. g g Es gibt regelmäßige Umfragen unter Hörer:innen nach ihren musikalischen Vorlieben. Die Rückmeldungen zu den Quizshows sind uneinheitlich. Gebraucht werden auch Quizformate, die von der ganzen Familie geschaut werden. g Es gibt bereits eine Vielfalt an Quizformaten mit unter schiedlichen Anspruchsniveaus. Es gibt bereits eine Vielfalt an Quizformaten mit unter schiedlichen Anspruchsniveaus. Neue Quizformate werden in den dritten Programmen pi lotiert. Neue Quizformate werden in den dritten Programmen pi lotiert. 313 https://doi.org/10.5771/978374 Open Access – - / – Matthias Trénel Gendern Eine zentrale Vorgabe zum Gendern wird es auf absehbare Zeit nicht geben, weil darüber jede Landesrundfunkanstalt selbst entschiedet. Landesrundfunkanstalten haben unterschiedliche Einschät zungen zum Gendern. Aktuell gendert die Mehrheit der ARD-Programme im ge sprochenen Wort nicht. Auftrag und Struktur Auftrag, Struktur und Finanzierung des öffentlich-recht lichen Rundfunks werden durch die Medienpolitik be stimmt; die Sender haben nur bedingt Einfluss darauf. Hinweise zum Rundfunkbeitrag, zum Auftrag oder zur Anzahl der Sender werden gebündelt an die Medienpolitik weitergeleitet. Fazit Fazit Im Bericht zum ARD-Zukunftsdialog schreibt die ARD mutig: „Der ARD- Zukunftsdialog war erst der Anfang“ (ARD, 2021d, S. 38). Die Vieldeutig keit dieses Statements wirft gleichwohl neue Fragen auf: Wird damit in Aussicht gestellt, dass die Umsetzung der Zukunftsprojekte auf Dauer an gelegt ist? Oder steht der ARD-Zukunftsdialog allgemein für neue Metho den, die auch zukünftig zum Einsatz kommen sollen, um die Beziehungen zwischen der ARD und ihrem Publikum enger zu gestalten und ihre Inno vationsfähigkeit zu verbessern? Am 20. Auswertung und Ergebnisse Juli 2021 hat das Bundesverfassungsgericht der Beschwerde der ARD stattgegeben und die vom Land Sachsen-Anhalt blockierte Erhöhung des Rundfunkbeitrags wieder in Kraft gesetzt. Damit ist mitten in der Laufzeit des ARD-Zukunftsdialogs ein Anlass entfallen, der anfangs sicher lich einer der Trigger für den Beschluss war, das Partizipationsverfahren zu starten. Die im Raum stehende These, der ARD-Zukunftsdialog sei bloß Element einer Kampagne mit dem übergreifenden Ziel, das ARD-Image in der Öffentlichkeit zu verbessern, scheint allerdings nicht haltbar zu sein. Zumindest konnte nach dem Urteil des Bundesverfassungsgerichts, das aus Sicht der ARD erfolgreich ausfiel, nicht beobachtet werden, dass die Ernst haftigkeit in der Auseinandersetzung mit den inhaltlichen Dialogergebnis sen nachließ. Im Gegenteil: In der Anlage und Durchführung des Dialogs war bis zum Schluss durchgehend erkennbar, dass die Vertreter:innen der ARD das direkte Gespräch mit den Bürger:innen suchten und sich auch im Nachgang in internen Runden damit beschäftigten, die Vorschläge aus 314 / – Der ARD-Zukunftsdialog dem Zukunftsdialog mit den eigenen Vorhaben abzugleichen und darauf aufbauend Zukunftsprojekte zu definieren. Die laufenden politischen De batten über die Novelle des Medienstaatsvertrags und über Reformüberle gungen zum öffentlich-rechtlichen Rundfunk waren vermutlich ein güns tiges Umfeld für die Durchführung des ARD-Zukunftsdialogs. Ist der ARD-Zukunftsdialog nun eine gute Blaupause für zukünftige Formen des Publikumsdialogs? In vielerlei Hinsicht kann diese Frage bejaht werden: Es konnte demonstriert werden, wie über eine Kaskade von Prozess- und Auswertungsschritten substanzielle Ergebnisse entstan den. Der Wechsel zwischen quasi-repräsentativen Losbürger-Formaten und einem öffentlichen Format wie einer Online-Beteiligung erzeugte zudem hinreichend Legitimation und Aufmerksamkeit – in der Öffentlichkeit genauso wie innerhalb der ARD – damit die Ergebnisse als relevant für die Umsetzung erachtet wurden. Nicht zuletzt wurde auch die Erfahrung ge macht, dass viele Bürger:innen bereit sind, sich an einem solchen Dialog verfahren zur Weiterentwicklung der ARD ehrenamtlich, engagiert und konstruktiv zu beteiligen. g Gleichwohl kann der ARD-Zukunftsdialog dafür kritisiert werden, nicht genügend transparent gewesen zu sein. Die Auswahl der Fragestel lungen und Formate sowie die inhaltlichen Auswertungen waren allein Sa che der ARD und der von ihr beauftragten Dialogdienstleister. Somit gab es kein Instrument, mit dem das Bedürfnis nach Inszenierung, das vielen Organisationen innewohnt, eingehegt werden konnte. Um die Glaubwür digkeit ähnlicher Dialogangebote weiter zu stärken, könnte zukünftig eine „Spurgruppe“ oder eine andere Form von Begleitgremium, in dem die In teressen der Teilnehmenden Bürger:innen vertreten sind, in die Vor- und Nachbereitung der Prozessschritte einbezogen werden (Krummenacher et al., 2019, S. 37; Trénel, 2020, S. 81). Auswertung und Ergebnisse In diesem Zusammenhang sollte auch überlegt werden, welche Rolle die Aufsichtsgremien des öffentlich-rechtlichen Rundfunks, also die Rund funkräte und die Gremienvorsitzendenkonferenz (GVK), bei zukünftigen Formen der Publikums- und Bürgerbeteiligung der ARD spielen können. Die Möglichkeiten reichen von einer definierten Beobachterrolle über die Mitgliedschaft in einem Begleitgremium (wie beispielsweise einer Spur gruppe) bis hin zur Ausrichterrolle solcher Dialogangebote. Auf welche Weise auch immer – eine klare und stärkere institutionelle Aufhängung wird einen positiven Effekt darauf haben, den Stimmen des Publikums und der Bürger:innen nachhaltig Einfluss zu geben. 315 8928690-301, am 24.10.2024, 06:07:59 - https://www.nomos-elibrary.de/agb / – / – Matthias Trénel Literatur ARD (2021a, 17. Dezember). Transparenzseite zum ARD-Zukunftsdialog. Abgeru fen am 22. März, 2022, von https://ard.de/zukunftsdialog ARD (2021b, 28. Juni). Online-Beteiligung des ARD-Zukunftsdialogs. Abgerufen am 22. März, 2022, von https://ard-zukunftsdialog.de ARD (2021c, 31. Mai). ARD-Zukunftsdialog: Jetzt ist das Publikum gefragt. Abge rufen am 22. März, 2022, von https://www.tagesschau.de/inland/gesellschaft/ard -zukunftsdialog-101.html ARD (2021d, 9. Dezember). Bericht 2021 zum ARD-Zukunftsdialog. Abgerufen am 22. März, 2022, von https://daserste.de/ard/die-ard/spezial/ARD-Zukunftsdia log-Bericht-2021-100.pdf Buhrow, Tom (2021, 22. März). Wo die ARD im Jahr 2030 steht. Gastbeitrag in der Frankfurter Allgemeinen Zeitung. Abgerufen am 22. März, 2022, von https://w ww.faz.net/aktuell/feuilleton/medien/wdr-intendant-tom-buhrow-wo-die-ard-im -jahr-2030-steht-17258121.html?premium Burkart, Roland (2012). Verständigungsorientierte Öffentlichkeitsarbeit. In: Wal ter, Hömberg; Daniela, Hahn; & Timon B. Schaffer (Hrsg.), Kommunikation und Verständigung: Theorie – Empirie – Praxis, 17‒37. Wiesbaden, Deutsch land: Springer VS Verlag für Sozialwissenschaften. Gottwald, Anne. (2022, 26. Januar). ARD-Zukunftsdialog: Über die Moderation großer Online-Beteiligungsprozesse. Beitrag in der Kategorie „Praxis“ des Berlin Instituts für Partizipation – bipar. Abgerufen am 22. März, 2022, von https://ww w.bipar.de/ard-zukunftsdialog/ Krummenacher, Paul; Neff, Petra; Schjold, Inger; von Wurstemberger, Britta. (2019). Praxis der Großgruppenarbeit – Prozesse partizipativ gestalten. Stuttgart, Deutschland: Schäffer-Poeschel Verlag. Mayring, Philipp (2015). Qualitative Inhaltsanalyse. Weinheim/Basel: Beltz Verlag. Rädiker, Stefan; & Kuckartz, Udo (2019). Analyse qualitativer Daten mit MAXQ DA: Text, Audio und Video. Wiesbaden, Deutschland: Springer VS Verlag für Sozialwissenschaften. Trénel, Matthias. (2020). Gelebte digitale Partizipation. Bürgerbeteiligung in Ver änderungsprozessen am Beispiel der Stadt Zürich. Zeitschrift für Organisations entwicklung, 20 (2), 80‒86. ZDF (2019, 11. Dezember). Das ZDF im Dialog mit den Bürgern. Nachrichten in eigener Sache der heute-Redaktion. Abgerufen am 22. März, 2022, von https://w ww.zdf.de/nachrichten/in-eigener-sache/zdf-buerdialoge-gesammelt-100.html Zebralog (2021, 28. Juni). Die Dialogzentrale: Plattform für digitale Beteiligung. Abgerufen am 22. März, 2022, von https://www.zebralog.de/dialogzentrale 316
https://openalex.org/W4366428416	https://www.scienceopen.com/document_file/1dab4668-c8c8-405b-bbef-41afb129e7b2/ScienceOpen/Zoonoses20230003.pdf	English	null	Next-Generation Vaccines against COVID-19 Variants: Beyond the Spike Protein	Zoonoses	2,023	cc-by	7,849	REVIEW ARTICLE REVIEW ARTICLE Next-Generation Vaccines against COVID-19 Variants: Beyond the Spike Protein Srinivasa Reddy Bonam1 and Haitao Hu1,2,3,* Received: January 5 2023 Revised: March 4 2023 Accepted: March 26 2023 Published Online: April 19 2023 Abstract Corresponding author: E-mail: haihu@UTMB.edu (HH) Vaccines are among the most effective medical countermeasures against infectious diseases. The emergence of the Coronavirus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spurred scientific strategies to fight against the disease. Since 2020, in response to the pandemic, many vaccines based on different platforms have been under development, among which mRNA, adenoviral vectors, and subunit vaccines have been clinically approved for use in humans. These first-generation COVID-19 vaccines largely target the viral spike (S) protein and are aimed at eliciting potent neutralizing antibodies. With the emergence of SARS-CoV-2 variants, particularly the highly transmissible Omicron strains, S-based vaccine strategies have faced a continuing challenge of strong immune escape by variants. The coronavirus nucleocapsid (N) protein is a viral protein that induces strong T-cell immunity and is more conserved than S protein across different SARS-CoV-2 variants. Inclusion of N protein in the development of COVID-19 vaccines has been reported. Here, we briefly review and discuss COVID-19, current S-protein-based vaccine strategies, the immunobiology of N protein in SARS-CoV-2 host immunity, and next- generation vaccine strategies involving N protein to combat current and emerging SARS-CoV-2 variants. 1Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA 2Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555, USA 3Sealy Institute for Vaccine Sciences, University of Texas Medical Branch, Galveston, TX 77555, USA Received: January 5 2023 Revised: March 4 2023 Accepted: March 26 2023 Published Online: April 19 2023 Received: January 5 2023 Revised: March 4 2023 Accepted: March 26 2023 Published Online: April 19 2023 Key words: COVID-19, SARS-CoV-2 variants, vaccine, mRNA, nucleocapsid Bonam et al. Zoonoses (2023) 3:18 DOI 10.15212/ZOONOSES-2023-0003 Bonam et al. Zoonoses (2023) 3:18 DOI 10.15212/ZOONOSES-2023-0003 COVID-19 PATHOPHYSIOLOGY COVID-19 pathophysiology has been extensively dis- cussed elsewhere [11–13]. Detailed research on host-vi- rus interactions has revealed that SARS-CoV-2 enters hosts through different routes, including nasal and oral routes, and binds the functional receptor angiotensin- converting enzyme 2 (ACE2) [14]. ACE2 is highly expressed on alveolar epithelial cells, enterocytes, endothelial cells, and the oral mucosa. The spike (S) protein of SARS-CoV-2 enters cells via ACE2, and SARS-CoV-2 RNA subsequently is replicated and prop- agated by the host machinery. However, the possibili- ties of other receptors supporting viral entry (neuropilin 1, B0AT1 [neutral amino acid transporter]) should not be ruled out [15]. Because of its rapid replication, the virus damages infected organs, particularly the lungs. During the acute phase, immune cells, particularly den- dritic cells and macrophages, secrete signaling mole- cules, which recruit other immune cells to the infected environment [11]. This process is not transient and leads to the hyperactivation of immune cells, which copiously secrete cytokines and chemokines that damage infected organs, in a response termed a “cytokine storm” [16]. Furthermore, the involvement of diverse pathways has been proposed, including cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) mediated pathways [17, 18], programmed cell death mediated by stimula- tion extracellular neutrophils traps (NETosis) [19], and other pathways [20]. In addition, the S protein-ACE2 interaction results in down-regulation of ACE2 on lung epithelial cells; this response is believed to be a primary cause of lung injury [21]. ACE2 simultaneously upreg- ulates ACE1 via negative feedback mechanisms [22], thus leading to excessive angiotensin-II. Angiotensin II in turn binds angiotensin II receptor type 1 (AGTR1A) receptors and elicits excessive vascular pulmonary activ- ity consistent with the lung pathology observed in pul- monary destruction [22, 23]. Irrespective of the signal- ing pathways, COVID-19 progresses through processes including lymphopenia, cytokine storms, accumulation of macrophages and neutrophils in the lungs, immune dysregulation, acute respiratory distress syndrome. INTRODUCTION East Respiratory Syndrome Coronavirus [MERS-CoV] and SARS-CoV), SARS- CoV-2 has a higher replication rate and attack rate in communities and induces high fatality rates in hospitalized patients [5]. Nevertheless, the infection and recov- ery patterns scarcely depend on demo- graphic factors [6], and the underlying mechanisms are not fully understood to date. COVID-19 symptoms are simi- lar to those of influenza, including fever, frontal headache, retro-orbital or tempo- ral headache, gastrointestinal symptoms, ground-glass opacity, pleural effusion [7], loss of taste or smell, and neurological symptoms. Laboratory findings including East Respiratory Syndrome Coronavirus [MERS-CoV] and SARS-CoV), SARS- CoV-2 has a higher replication rate and attack rate in communities and induces high fatality rates in hospitalized patients [5]. Nevertheless, the infection and recov- ery patterns scarcely depend on demo- graphic factors [6], and the underlying mechanisms are not fully understood to date. COVID-19 symptoms are simi- lar to those of influenza, including fever, frontal headache, retro-orbital or tempo- ral headache, gastrointestinal symptoms, ground-glass opacity, pleural effusion [7], loss of taste or smell, and neurological symptoms. Laboratory findings including In early 2020, the causative agent of Coronavirus Disease 2019 (COVID-19) was identified as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) [1, 2]. Although the precise origin of the virus is unknown, it shows high similar- ity with several bat coronaviruses. After the World Health Organization issued global warnings, SARS-CoV-2 crossed interna- tional borders and led to a pandemic [3]. As of December 2022, SARS-CoV-2 has caused 650 million infections and 6.6 mil- lion deaths globally [4]. Compared with other beta-coronaviruses (e.g., Middle © 2023 The Authors. Creative Commons Attribution 4.0 International License 2 Bonam et al. EMERGENCE OF SARS-COV-2 VARIANTS lymphopenia, hypersensitive C-reactive protein, erythrocyte sedimentation rate, creatine kinase, transaminases (aspartate aminotransferase and alanine aminotransferase), thrombo- cytopenia are more common biomarkers for diagnosis in patients with COVID-19 [8], and thus may serve as biomark- ers [8]. Similarly, lung radiographic findings indicate ground grass opacity, predominantly peripheral distribution, and interlobular septal thickening in patients with COVID-19. Patients with severe COVID-19 and respiratory distress are transferred to intensive care for mechanical ventilation. Of note, SARS-CoV-2 infection severity is associated with comorbidities, such as cardiovascular diseases, diabetes, and obesity, thus leading to high mortality [9, 10]. The human immune system is tightly regulated during infection and exhibits long-lasting antigen/pathogen- specific memory over time [24–26]. To evade the host immune system, continually evolving viruses may either hijack antiviral pathways or produce diverse mutations increasing genetic diversity. These mutations can lead to significant changes in viral protein structure, including antigenic drift, folding/masking of critical antigenic res- idues exposed to the host, and production of new protein sequences [27, 28]. Multiple new SARS-CoV-2 variants have arisen worldwide, including the Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Epsilon (B.1.427 and B.1.429), Eta (B.1.525), Iota (B.1.526), Kappa (B.1.617.1), Mu (B.1.621, B.1.621.1), Zeta (P.2), Theta (P.3), and Omicron (B.1.1.529, BA.1, BA.1.1, BA.2, BA.3, BA.4, and BA.5) variants (Fig 1) [29–32]. Changes in viral S protein alter the viral receptor binding pattern in the host, thus enhancing the infection poten- tial, evading conventional detection methods, producing diverse disease outcomes, and decreasing susceptibility to the immune response raised against earlier circulating SARS-CoV-2 strains or vaccines [33]. The emergence of these variants has posed continuing challenges to host immunity induced by first-generation vaccines or prior infection. FIRST-GENERATION COVID-19 VACCINES Despite causing unusual economic and livelihood loss globally, the COVID-19 pandemic has also driven remarkable scientific and technological progress in the past several years. A complete SARS-CoV-2 genome was quickly determined [34], and structural elucidation was performed by mapping the host receptor interactions [35]. Substantial success was attained in vaccine devel- opment against SARS-CoV-2. More than 90 vaccine candidates are being developed worldwide through var- ious strategies, including live inactivated vaccines, viral vector-based vaccines, recombinant subunit vaccines, and nucleic acid (DNA or RNA) vaccines [36, 37]. Among them, two mRNA-based vaccines (Pfizer-BioNTech and Moderna) (Fig 2) and an adenovirus 26-based vaccine (Johnson & Johnson) were approved by the United States Food and Drug Administration for clinical use under emergency authorization. Since then, the two mRNA vaccines have received full approval. Subsequently, additional vaccines have been approved for COVID-19, including Novavax’s adjuvanted vaccine (SARS-CoV-2 S protein with Matrix-M adjuvant). Millions of doses of these vaccines have been distributed worldwide and are being used for immunizing both infected and non-infected populations [38]. These first-generation COVID-19 vaccines have been extensively reviewed elsewhere [37, 39–41]. Despite these incredible achievements, new problems have been emerged that challenge the long-term control Next-Generation Vaccines Against COVID-19 Variants: Beyond the Spike Protein 3 FIGURE 1 \| Emerging SARS-CoV-2 variants. Emergence of SARS-CoV-2 variants of concern and variants of interest (https://www.who.int/ activities/tracking-SARS-CoV-2-variants). FIGURE 1 \| Emerging SARS-CoV-2 variants. Emergence of SARS-CoV-2 variants of concern and variants of interest (https://www.who.int/ activities/tracking-SARS-CoV-2-variants). SARS-COV-2 NUCLEOCAPSID (N) PROTEIN BIOLOGY of the pandemic; these problems include emerging viral variants (Fig 1) with greater transmissibility and immune escape ability, and waning immunity over time in vacci- nated individuals [32, 42]. N is an important structural protein of SARS-CoV-2 and is abundantly expressed in infected cells [53]. Hundreds of copies of N are present in the viral core, which encloses the genomic RNA. Like other viral struc- tural proteins, N protein plays a crucial role in the CoV life cycle, including mRNA transcription, cytoskeletal organization, RNA replication, packaging, and immune regulation [54]. Liquid-liquid phase separation of N protein regulates viral transcription and assembly [55]. Initial studies elucidating N protein structure by nuclear magnetic resonance have indicated that its N-terminal domain (NTD) has an overall right-handed fold struc- ture composed of a β-sheet core with an extended cen- tral loop [56]. The protein is composed of two alpha helices and five beta sheets, containing a total of 419 amino acids. It is functionally divided into two domains: the NTD and C-terminal domain (CTD) (Fig 3). The NTD, which contains an RNA-binding domain, acts as a linker between the RNA and matrix, thus enabling Most first-generation COVID-19 vaccines approved for clinical use or under clinical development tar- get the viral S protein, its subunits (S1 and S2) or its receptor binding domain (RBD) (Fig 3A), and are aimed at eliciting a protective neutralizing antibody response. Variants with S proteins bearing structural changes—particularly those of the Delta and Omicron strains with increased transmissibility and immune escape—have emerged and become predominant [43–50]. For example, multiple S variants have been shown to have diminished sensitivity to neutralizing antibodies induced by the first-generation S-targeting vaccines [45–47]. Clinical research has also indicated that S-targeting COVID-19 vaccines show dimin- ished efficacy against the Delta [51] and Omicron [52] strains. Thus, novel vaccine strategies targeting con- served regions of the virus to induce broader protection against COVID-19 variants are needed. Bonam et al. 4 FIGURE 2 \| Mode of action of mRNA vaccines. mRNA encapsulation by lipid nanoparticles (LNPs) aids in targeted delivery. After the mRNA enters the cell via endocytosis, the ionizable lipids in the LNPs facilitate mRNA release into the cytosol. Host ribosomes convert the mRNA into the corresponding protein, which in turn is degraded into peptides by the proteasome. SARS-COV-2 NUCLEOCAPSID (N) PROTEIN BIOLOGY The presentation of the peptides by the major histocompatibility complex to corresponding T lymphocytes stimulates both cellular and humoral immune responses. FIGURE 2 \| Mode of action of mRNA vaccines. mRNA encapsulation by lipid nanoparticles (LNPs) aids in targeted delivery. After the mRNA enters the cell via endocytosis, the ionizable lipids in the LNPs facilitate mRNA release into the cytosol. Host ribosomes convert the mRNA into the corresponding protein, which in turn is degraded into peptides by the proteasome. The presentation of the peptides by the major histocompatibility complex to corresponding T lymphocytes stimulates both cellular and humoral immune responses. capabilities. More detailed information can be found in comprehensive reviews published elsewhere [61, 62]. virion formation. N binds the positive single stranded RNA to the ribonucleoprotein core and has a “beads-on- a-string” appearance in micrographs. The CTD assists in RNA binding and contains a dimerization sequence, which facilitates RNA synthesis via interaction with the replication-transcription complexes [57, 58]. The CTD dimerization sequence also facilitates viral genome incorporation into virions. The intrinsically disordered region (IDR), between the domains, physically links the positive viral RNA genome and matrix protein [55]. Interestingly, the presence of the IDR favors N protein proteolysis, which is a key strategy for viral prolifera- tion [59]. The proteolytic products, particularly N1−209, interact with immunophilin (i.e., Cyclophilin A) in host cells and promote the SARS-CoV-2 replication cycle [59]. HOST IMMUNITY TO SARS-COV-2 N PROTEIN In addition, the NTD and CTD are flanked by N-arm and C-tail IDRs, respectively. FIGURE 3 \| Structure of SARS-CoV-2 and its nucleocapsid protein. (A) SARS-CoV-2 and its four structural proteins: S, envelope, membrane, and N proteins. Physical interaction of N with the viral RNA genome. (B) Structural organization of N protein. The N-terminal domain (NTD) and C-terminal domain (CTD) are connected by an intrinsically disordered region (IDR) known as the central linker region (LKR). In addition, the NTD and CTD are flanked by N-arm and C-tail IDRs, respectively. FIGURE 3 \| Structure of SARS-CoV-2 and its nucleocapsid protein. (A) SARS-CoV-2 and its four structural proteins: S, envelope, membrane, and N proteins. Physical interaction of N with the viral RNA genome. (B) Structural organization of N protein. The N-terminal domain (NTD) and C-terminal domain (CTD) are connected by an intrinsically disordered region (IDR) known as the central linker region (LKR). In addition, the NTD and CTD are flanked by N-arm and C-tail IDRs, respectively. SARS-CoV-1 and SARS-CoV-2 N protein has indi- cated that N-specific memory T cells from SARS-CoV-1 infected individuals are long-lasting and cross-reactive with SARS-CoV-2 N protein. The N-specific memory T cells remained detectable in SARS-CoV-1 infected indi- viduals 17 years after the SARS outbreak in 2003; more- over, these T cells showed strong cross-reactivity to N protein of SARS-CoV-2 [66]. primed with coronavirus [70]. SARS-CoV-2 infection induces strong CD4+ and CD8+ T cell responses, particu- larly in recovered patients with severe rather than mild disease [10, 71]. Importantly, in recovered patients with mild disease, a high N-specific CD8+ T cell response has been observed [71]. More detailed information on cellular immunity induced by SARS-CoV-2 infection or vaccina- tion has been reviewed elsewhere [72]. Similarly, CD8+ T cells specific to an immunodomi- nant SARS-CoV-2 N epitope cross-react with selective seasonal coronaviruses [69]. Screening of SARS-CoV-2 peptide pools has revealed that the N protein induces an immunodominant response in recovered patients with HLA-B7+ COVID-19 that is also detectable in SARS- CoV-2 unexposed donors [70]. A single N-encoded epitope that is highly conserved across circulating corona- viruses drives this immunodominant response. Notably, CD8+ T cell responses against the N protein epitope (N105–113) restricted by B07:02 demonstrate strong anti- viral activity and correlate with protection against severe disease [70]. HOST IMMUNITY TO SARS-COV-2 N PROTEIN Indeed, the most immunodominant CD8+ T cell response known to date is the N105 peptide pre- sented by HLA-B*07:02, which arises from a high fre- quency of T cells within the naive T cell repertoire that recognize N105, rather than from human cells previously HOST IMMUNITY TO SARS-COV-2 N PROTEIN Most patients with COVID-19 mount a specific immune response against the full length and fragments of the N protein and, to a lesser extent, against a fragment con- taining amino acids 300–685 of S protein [63]. A total of 82% of convalescent patients have detectable N-specific IgG 12 months after SARS-CoV-2 infection, in addition to S-specific antibodies [64]. In terms of T cell responses, data from convalescent patients have indicated that the S, M, and N proteins each account for 27%, 21%, and 11% of the total CD4+ response, respectively [65]. Higher fre- quencies of multi-cytokine production (poly-functional) by M- and N-specific CD8+ T cells are associated with mild COVID-19 [66]. Moreover, N-specific T cell and B cell (in terms of antibody secretion) responses have been reported in different cohorts of COVID-19 infected patients [65, 67, 68]. N induces robust both CD4+ and CD8+ T cell responses. CD4+ and CD8+ T cells have been found to recognize multiple regions of N protein in all individuals who have recovered from COVID-19 [66]. Another study comparing the T cell response to Because of its abundant expression after SARS-CoV-2 infection, N protein has been used as a diagnostic marker for several antigenic tests, including IgG binding assays [60]. Conventionally, the coronavirus N protein has also been shown to modulate the host intracellular machin- ery and play regulatory roles during the viral life cycle. Therefore, the characteristics of domains of N-protein have been targets for antiviral development via inhibiting or blocking the RNA-binding activity or oligomerization Next-Generation Vaccines Against COVID-19 Variants: Beyond the Spike Protein 5 FIGURE 3 \| Structure of SARS-CoV-2 and its nucleocapsid protein. (A) SARS-CoV-2 and its four structural proteins: S, envelope, membrane, and N proteins. Physical interaction of N with the viral RNA genome. (B) Structural organization of N protein. The N-terminal domain (NTD) and C-terminal domain (CTD) are connected by an intrinsically disordered region (IDR) known as the central linker region (LKR). In addition, the NTD and CTD are flanked by N-arm and C-tail IDRs, respectively. FIGURE 3 \| Structure of SARS-CoV-2 and its nucleocapsid protein. (A) SARS-CoV-2 and its four structural proteins: S, envelope, membrane, and N proteins. Physical interaction of N with the viral RNA genome. (B) Structural organization of N protein. The N-terminal domain (NTD) and C-terminal domain (CTD) are connected by an intrinsically disordered region (IDR) known as the central linker region (LKR). NUCLEOCAPSID-BASED SARS-COV-2 VACCINES Beyond the S protein, the N protein is a well-recognized dominant target of antibody and T cell responses in SARS-CoV-2-infected individuals and therefore has been suggested as a potential immunogen to augment vaccine-mediated protective immunity [73]. Despite its use as a diagnostic marker, N-specific IgG in the first week of SARS-CoV-2 infection is associated with rapid symptom resolution [74]. Although current S-protein- targeting vaccines confer strong protection against ances- tral SARS-CoV-2, the emerging variants have greater immune evasion of vaccine- and/or infection-elicited S-protein-specific neutralization than prior variants [75]. Therefore, vaccines expressing N protein have potential 6 Bonam et al. non-human primates (NHPs) [86]. The efficacy of this vaccine against major SARS-CoV-2 variants remains to be determined. The vaccine has been evaluated in a phase I clinical trial, which has indicated that the vaccine is well tolerated and elicits S- and N-specific immune responses in healthy participants [87]. Similarly, another independ- ent study has investigated the effect of a recombinant MVA vaccine expressing S and N proteins (MVA/SdFCS-N) in NHPs against the SARS-CoV-2 Delta variant [88]. Among the different routes of administration, MVA/ SdFCS-N administration via an intramuscular route has been found to better protection against the Delta variant than other routes [88]. value in vaccine development. Indeed, efforts a decade ago were aimed at investigating N protein as a potential vac- cine immunogen against coronaviruses. The COVID-19 pandemic has led to new light being shed on these inves- tigations [61, 73, 76–78]. Several N-based vaccine immu- nogens have been developed via various technologies. Adenovirus type-5 (Ad5) vector expressing N protein of ancestral SARS-CoV-2 has been investigated as a vaccine candidate [79, 80]. The vaccine is immunogenic and elic- its both antibody and T-cell (CD4+ and CD8+) responses in mice [79]. Ad5-N alone has been found to induce very modest protection against SARS-CoV-2 in a K18-hACE2 transgenic mouse model (expressing human angioten- sin-converting enzyme 2 [hACE2] receptor). However, combining Ad5-N with Ad5 expressing S (Ad5-S) has been observed to have synergistic effects and to induce stronger protection in the mouse brain (but not the lungs) than Ad5-S alone [79]. Another independent study testing Ad5-N vaccine has observed vaccine-induced protective immunity in Syrian hamsters and K18-hACE2 mice, as evidenced by decreased animal weight loss and viral loads [81]. NUCLEOCAPSID-BASED SARS-COV-2 VACCINES Another study has focused on the development of tri- valent COVID-19 vaccines by using adenoviral vectors of human and chimpanzee origin expressing the S1, N, and RNA-dependent RNA polymerase of SARS-CoV-2 [89] and also has compared intramuscular and intranasal immu- nization strategies. Of note, a single intranasal mucosal immunization led to strong, systemic neutralizing anti- bodies. In addition, intranasal vaccination increased tissue resident memory CD8+ T cells in the respiratory mucosa as well as locally trained macrophages. The intranasal strategy conferred protection against the parent strain of SARS-CoV-2 as well as other variants of concern (B.1.1.7 and B.1.351) [89]. N protein-based subunit vaccines have also been devel- oped and tested. SARS-CoV-2 N protein either alone or in combination with well-known adjuvants (e.g., Freund’s adjuvant, alum, or QS-21) has been shown to be immu- nogenic in animal models [82, 83]. A synthetic peptide vaccine comprising N epitopes (HLA class I bound cyto- toxic T lymphocyte peptide) along with adjuvants (Toll- like receptor [TLR] 4 agonist (MPLA) and TLR9 agonist [CpG oligonucleotide]) have shown moderate protection against SRAS-CoV-2 in Rhesus macaques [84]. Using the human Ad5 vector, ImmunityBio has devel- oped an approach targeting both SARS-CoV-2 S and N proteins. The S and Enhanced T-cell Stimulation Domain (N-ETSD) of the N protein are used together in the vac- cine, and both have been found to be immunogenic in mice. Subcutaneous priming and intranasal or subcuta- neous boosting with this dual antigen vaccine induces greater Th1-biased T-cell and humoral responses than Ad5-S alone [90]. In a phase I trial, a single dose (1 × 1011 viral particle) vaccination in healthy adults has been found to elicit a strong T cell response [91]. This plat- form supports the feasibility of needleless immunizations in general. However, viral challenge studies are needed to evaluate the vaccine efficacy in animal models. Our group has generated a nucleoside-modified, lipid-nanoparticle (LNP)-formulated mRNA vaccine that encodes the full-length N protein of SARS-CoV-2 (Wuhan-Hu-1 strain) (mRNA-N) [60]. Immunogenicity analyses in mice have indicated that mRNA-N is highly immunogenic and induces strong N-specific binding antibody and T cell (both CD4+ and CD8+) responses. As expected, no neutralizing antibody response is elicited by mRNA-N [60]. Similarly to N-expressing vaccines based on other platforms [79, 80], the mRNA-N vaccine has been found to induce modest protection against mouse- adapted SARS-CoV-2 and the Delta strain in mice and hamsters [60]. NUCLEOCAPSID-BASED SARS-COV-2 VACCINES Vesicular stomatitis virus is a commonly used vector for mucosal delivery. Intranasal administration of a vesicular stomatitis virus-based vaccine expressing S and N pro- teins has been found to elicit a mucosal immune response and protective effects in a hamster viral challenge model, whereas the same vaccine administered intramuscularly is only marginally protective [92]. MULTIVALENT SARS-COV-2 VACCINES EXPRESSING MULTIPLE VIRAL PROTEINS In addition to viral vectors, multivalent vaccines based on DNA or proteins have been reported. Mice immunized with a DNA vaccine encoding S/RBD and N protein have been found to develop broad neutralizing antibodies and N-specific T cell responses [93]. The DNA vaccine has been found to protect mice against lethal SARS-CoV-2 infection [93]. Similarly, a phase I clinical trial of a DNA vaccine (GX-19N™) encoding the RBD and N protein has demonstrated its safety and immunogenicity [94], although the efficacy of this vaccine in animal models is To elicit both neutralizing antibodies and broadly protec- tive T cell responses, several multivalent SARS-CoV-2 vaccines expressing both S and N proteins have been developed, mainly through use of viral vectors. A syn- thetic modified vaccinia Ankara (MVA) vaccine express- ing SARS-CoV-2 S and N protein (COH04S1) has been shown to be immunogenic [85] and to confer protec- tion against a challenge with ancestral SARS-CoV-2 in Next-Generation Vaccines Against COVID-19 Variants: Beyond the Spike Protein 7 unclear. Hong and colleagues have immunized macaques with multivalent protein subunit vaccine comprising the RBD fused with tetanus toxoid epitope P2 (RBD-P2) and N protein, and shown that addition of N protein induces slightly faster SARS-CoV-2 clearance than that induced by RBD-P2 alone in NHPs [95]. continually mutate, the design and selection of VOC- specific sequences will be become less optimal. Given the more conserved sequence of N than S protein across dif- ferent variants, as well as the cross-reactive, long-lasting N-protein-specific T cell immunity [96, 97], our study and those from other groups have clearly demonstrated the benefit and utility of including N immunogen in the next-generation COVID-19 vaccines for VOCs. Our study has indicated that, despite the use of ancestral sequences, the inclusion of mRNA-N along with mRNA-S for vaccination elicits a robust protection against both the Delta and Omicron strains, which is not achieved by mRNA-S alone. Given the demonstrated safety profile of the mRNA-LNP platform in large human populations, the bivalent mRNA-S+N vaccine should be advanced to clinical testing. Although the above multivalent vaccine approaches have been found to elicit various levels of protection against SARS-CoV-2 or its variants in different animal models, a direct comparison of their efficacy in protecting against SARS-CoV-2 VOCs, with respect to the efficacy of current clinically approved first-generation COVID-19 vaccines, particularly the mRNA-S vaccines, is critically lacking. ACKNOWLEDGEMENTS H.H. is supported by NIH grants AI157852 and AI147903. The figures were created in BioRender.com under a paid subscription. H.H. is supported by NIH grants AI157852 and AI147903. The figures were created in BioRender.com under a paid subscription. H. is supported by NIH grants AI157852 and AI147903. The References 1. Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al. A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med. 2020;382:727-733. MULTIVALENT SARS-COV-2 VACCINES EXPRESSING MULTIPLE VIRAL PROTEINS Their efficacy against the currently predominant Omicron variants also remains to be determined through comparison with the clinically approved S-protein- targeting vaccines. Several important questions remain to be explored. First, the durability of vaccine-elicited protective immu- nity is critical [98, 99]. Whether the dual mRNA-S+N vaccine induces more durable protective immunity than mRNA-S alone is unclear and is currently under inves- tigation. Second, because a large human population has been vaccinated with first-generation vaccines or natu- rally infected with the virus, further testing the inclusion of N immunogen as a booster strategy will be likely to reveal additional insights into the utility of this bivalent vaccine approach against SARS-CoV-2 VOCs. Third, further validating the safety and efficacy of the vaccine approach in larger animal models will be helpful before clinical testing. Finally, because COVID-19 VOCs can infect individuals through vaccine breakthrough and antibody escape, screening for broadly neutralizing or pan-neutralizing antibodies against COVID-19 VOCs is also important for the development of a pan-COVID-19 vaccine, as well as the prevention and treatment of VOC infections in the future. g g As discussed earlier, the mRNA vaccine expressing the full-length N protein (mRNA-N) generated by our group confers modest protection against a mouse adap- tive SARS-CoV-2 and Delta strain. To enable comparison with first-generation S-protein-targeting vaccines, we also generated an mRNA-LNP vaccinee expressing the prefusion-stabilized SARS-CoV-2 S protein (mRNA-S), whose immunogenicity and protective efficacy against multiple SARS-CoV-2 VOCs was evaluated either alone or in combination with mRNA-N (mRNA-S+N) in rodent models [60]. Our data showed that mRNA S+N vaccination induced markedly stronger protection than mRNA-S alone against multiple SARS-CoV-2 strains, including a mouse-adapted strain, Delta, and Omicron, in both the lower and upper respiratory tracts. The dif- ference between mRNA-S alone and mRNA-S+N was most profound in the protection against Omicron: whereas mRNA-S alone had a substantially diminished efficacy against Omicron because of strong immune escape, mRNA-S+N vaccination conferred substantial protection against Omicron (four of five animals had no detectable viral RNA and titers in the lungs) [60], thus indicating induction of cross-protective immunity against VOCs by mRNA-S+N. In vivo CD8+ T cell depletion supports that CD8+ T cells play a critical role in protection against VOCs after mRNA-S+N vaccine immunization [60]. Collectively, our findings suggest that the bivalent mRNA-S+N is a potential pan-COVID-19 vaccine for emerging SARS-CoV-2 variants. CONFLICTS OF INTEREST No conflicts of interest are declared by the authors. No conflicts of interest are declared by the authors. SUMMARY AND FUTURE DIRECTIONS 2. Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;579:270-273. As new variants of SARS-CoV-2 continue to arise, pan- COVID-19 vaccines that induce broad protection against emerging strains must urgently be developed. Current strategies to develop vaccines against COVID-19 VOCs, include utilization of VOC-specific S immunogens. Although a VOC-targeted S protein booster has been clinically approved in the two mRNA vaccinees, this strategy is likely to be challenging, because as S proteins As new variants of SARS-CoV-2 continue to arise, pan- COVID-19 vaccines that induce broad protection against emerging strains must urgently be developed. Current strategies to develop vaccines against COVID-19 VOCs, include utilization of VOC-specific S immunogens. Although a VOC-targeted S protein booster has been clinically approved in the two mRNA vaccinees, this strategy is likely to be challenging, because as S proteins 3. World Health Organization. 2020. 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Multiple roles of SARS-CoV-2 N protein facilitated by proteoform-specific interactions with RNA, host proteins, and convalescent antibodies. JACS Au. 2021;1(8):1147-1157. 77. Dutta NK, Mazumdar K, Lee BH, Baek MW, Kim DJ, Na YR, et al. Search for potential target site of nucleocapsid gene for the design of an epitope-based SARS DNA vaccine. Immunol Lett. 2008;118(1):65-71. 60. SUMMARY AND FUTURE DIRECTIONS Kakodkar P, Kaka N, Baig MN. A comprehensive literature review on the clinical presentation, and management of the pandemic coronavirus disease 2019 (COVID-19). Cureus. 2020;12:e7560. 9 Next-Generation Vaccines Against COVID-19 Variants: Beyond the Spike Protein SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed individuals. Cell. 2020;181(7):1489-1501.e1415. 47. Garcia-Beltran WF, Lam EC, St Denis K, Nitido AD, Garcia ZH, Hauser BM, et al. Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity. Cell. 2021;184(9):2372-2383.e2379. 66. Le Bert N, Tan AT, Kunasegaran K, Tham CYL, Hafezi M, Chia A, et al. SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls. Nature. 2020;584(7821):457-462. 48. Cao Y, Wang J, Jian F, Xiao T, Song W, Yisimayi A, et al. Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies. Nature. 2022;602(7898):657-663. 67. Ni L, Ye F, Cheng ML, Feng Y, Deng YQ, Zhao H, et al. Detection of SARS-CoV-2-specific humoral and cellular immunity in COVID- 19 convalescent individuals. Immunity. 2020;52(6):971-977.e973. 49. Planas D, Saunders N, Maes P, Guivel-Benhassine F, Planchais C, Buchrieser J, et al. Considerable escape of SARS-CoV-2 Omicron to antibody neutralization. Nature. 2022;602(7898):671-675. 68. Woo PCY, Lau SK, Wong BH, Tsoi HW, Fung AM, Chan KH, et al. Detection of specific antibodies to severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein for serodiagnosis of SARS coronavirus pneumonia. J Clin Microbiol. 2004;42(5):2306-2309. 50. Liu L, Iketani S, Guo Y, Chan JF, Wang M, Liu L, et al. Striking antibody evasion manifested by the Omicron variant of SARS- CoV-2. Nature. 2022;602(7898):676-681. 51. Tang P, Hasan MR, Chemaitelly H, Yassine HM, Benslimane FM, Al Khatib HA, et al. BNT162b2 and mRNA-1273 COVID-19 vaccine effectiveness against the SARS-CoV-2 Delta variant in Qatar. Nat Med. 2021;27(12):2136-2143. 69. Lineburg KE, Grant EJ, Swaminathan S, Chatzileontiadou DSM, Szeto C, Sloane H, et al. CD8(+) T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope cross-react with selective seasonal coronaviruses. Immunity. 2021;54(5):1055-1065.e1055. 52. Andrews N, Stowe J, Kirsebom F, Toffa S, Rickeard T, Gallagher E, et al. Covid-19 vaccine effectiveness against the omicron (B.1.1.529) variant. N Engl J Med. 2022;386(16):1532-1546. 70. Nguyen THO, Rowntree LC, Petersen J, Chua BY, Hensen L, Kedzierski L, et al. CD8(+) T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naive precursor frequency and TCR promiscuity. Immunity. 2021;54(5):1066-1082.e1065. 53. He Y, Zhou Y, Wu H, Kou Z, Liu S, Jiang S. SUMMARY AND FUTURE DIRECTIONS Hajnik RL, Plante JA, Liang Y, Alameh MG, Tang J, Bonam SR, et al. Dual spike and nucleocapsid mRNA vaccination confer protection against SARS-CoV-2 Omicron and Delta variants in preclinical models. Sci Transl Med. 2022;14(662):eabq1945. 78. Wu W, Cheng Y, Zhou H, Sun C, Zhang S. The SARS-CoV-2 nucleocapsid protein: its role in the viral life cycle, structure and functions, and use as a potential target in the development of vaccines and diagnostics. Virol J. 2023;20(1):6. 61. Chang Ck, Hou MH, Chang CF, Hsiao C-D, Huang T-h. The SARS coronavirus nucleocapsid protein – Forms and functions. Antiviral Res. 2014;103:39-50. 79. Dangi T, Class J, Palacio N, Richner JM, Penaloza MacMaster P. Combining spike- and nucleocapsid-based vaccines improves distal control of SARS-CoV-2. Cell Rep. 2021;36(10):109664. 62. Matsuo T. Viewing SARS-CoV-2 nucleocapsid protein in terms of molecular flexibility. Biology. 2021;10(6):454. 80. Dangi T, Sanchez S, Class J, Richner M, Visvabharathy L, Chung YR, et al. Improved control of SARS-CoV-2 by treatment with a nucleocapsid-specific monoclonal antibody. J Clin Invest. 2022;132(23):e162282. 63. Smits VAJ, Hernández-Carralero E, Paz-Cabrera MC, Cabrera E, Hernández-Reyes Y, Hernández-Fernaud JR, et al. The Nucleocapsid protein triggers the main humoral immune response in COVID-19 patients. Biochem Biophys Res Commun 2021;543:45-49. 81. Matchett WE, Joag V, Stolley JM, Shepherd FK, Quarnstrom CF, Mickelson CK, et al. Cutting edge: nucleocapsid vaccine elicits spike-independent SARS-CoV-2 protective immunity. J Immunol. 2021;207(2):376-379. 64. Guo L, Wang G, Wang Y, Zhang Q, Ren L, Gu X, et al. SARS- CoV-2-specific antibody and T-cell responses 1 year after infection in people recovered from COVID-19: a longitudinal cohort study. Lancet Microbe. 2022;3(5):e348-e356. 82. Silva EKVB, Bomfim CG, Barbosa AP, Noda P, Noronha IL, Fernandes BHV, et al. Immunization with SARS-CoV-2 nucleocapsid protein triggers a pulmonary immune response in rats. PLoS One. 2022;17(5):e0268434. 65. Grifoni A, Weiskopf D, Ramirez SI, Mateus J, Dan JM, Moderbacher CR, et al. Targets of T cell responses to 10 Bonam et al. ten-fold increases in mean T-cell responses in phase 1 subjects that are sustained against spike variants. medRxiv. 2021;2021.2004.2005.21254940. ten-fold increases in mean T-cell responses in phase 1 subjects that are sustained against spike variants. medRxiv. 2021;2021.2004.2005.21254940. 83. Feng W, Xiang Y, Wu L, Chen Z, Li Q, Chen J, et al. Nucleocapsid protein of SARS-CoV-2 is a potential target for developing new generation of vaccine. J Clin Lab Anal. 2022;36(6):e24479. 92. O’Donnell KL, Gourdine T, Fletcher P, Clancy CS, Marzi A. SUMMARY AND FUTURE DIRECTIONS Protection from COVID-19 with a VSV-based vaccine expressing the spike and nucleocapsid proteins. Front Immunol. 2022;13:1025500. 84. Harris PE, Brasel T, Massey C, Herst CV, Burkholz S, Lloyd P, et al. A synthetic peptide CTL vaccine targeting nucleocapsid confers protection from SARS-CoV-2 challenge in rhesus macaques. Vaccines (Basel). 2021;9(5):520. 93. Appelberg S, Ahlén G, Yan J, Nikouyan N, Weber S, Larsson O, et al. A universal SARS-CoV DNA vaccine inducing highly cross- reactive neutralizing antibodies and T cells. EMBO Mol Med. 2022;14(10):e15821. 85. Chiuppesi F, Salazar MD, Contreras H, Nguyen VH, Martinez J, Park Y, et al. Development of a multi-antigenic SARS-CoV-2 vaccine candidate using a synthetic poxvirus platform. Nat Commun. 2020;11(1):6121. 94. Ahn JY, Lee J, Suh YS, Song YG, Choi YJ, Lee KH, et al. Safety and immunogenicity of two recombinant DNA COVID-19 vaccines containing the coding regions of the spike or spike and nucleocapsid proteins: an interim analysis of two open- label, non-randomised, phase 1 trials in healthy adults. Lancet Microbe. 2022;3(3):e173-e183. 86. Chiuppesi F, Nguyen VH, Park Y, Contreras H, Karpinski V, Faircloth K, et al. Synthetic multiantigen MVA vaccine COH04S1 protects against SARS-CoV-2 in Syrian hamsters and non- human primates. NPJ Vaccines. 2022;7(1):7. 87. Chiuppesi F, Zaia JA, Frankel PH, Stan R, Drake J, Williams B, et al. Safety and immunogenicity of a synthetic multiantigen modified vaccinia virus Ankara-based COVID-19 vaccine (COH04S1): an open-label and randomised, phase 1 trial. Lancet Microbe. 2022;3(4):e252-e264. 95. Hong SH, Oh H, Park YW, Kwak HW, Oh EY, Park HJ, et al. Immunization with RBD-P2 and N protects against SARS-CoV-2 in nonhuman primates. Sci Adv. 2021;7(22):eabg7156. 96. Zhao J, Zhao J, Mangalam AK, Channappanavar R, Fett C, Meyerholz DK, et al. Airway memory CD4(+) T cells mediate protective immunity against emerging respiratory coronaviruses. Immunity. 2016;44(6):1379-1391. 88. Routhu NK, Gangadhara S, Lai L, Davis-Gardner ME, Floyd K, Shiferaw A, et al. A modified vaccinia Ankara vaccine expressing spike and nucleocapsid protects rhesus macaques against SARS- CoV-2 Delta infection. Sci Immunol. 2022;7(72):eabo0226. 97. McKinstry KK, Strutt TM, Kuang Y, Brown DM, Sell S, Dutton RW, et al. Memory CD4+ T cells protect against influenza through multiple synergizing mechanisms. J Clin Invest. 2012;122(8):2847-2856. 89. Afkhami S, D’Agostino MR, Zhang A, Stacey HD, Marzok A, Kang A, et al. Respiratory mucosal delivery of next- generation COVID-19 vaccine provides robust protection against both ancestral and variant strains of SARS-CoV-2. Cell. 2022;185(5):896-915.e19. 98. SUMMARY AND FUTURE DIRECTIONS Qu P, Faraone JN, Evans JP, Zheng YM, Yu L, Ma Q, et al. Durability of booster mRNA vaccine against SARS-CoV-2 BA.2.12.1, BA.4, and BA.5 subvariants. N Engl J Med. 2022;387(14):1329-1331. 90. Rice A, Verma M, Shin A, Zakin L, Sieling P, Tanaka S, et al. Intranasal plus subcutaneous prime vaccination with a dual antigen COVID-19 vaccine elicits T-cell and antibody responses in mice. Sci Rep. 2021;11(1):14917. 99. Townsend JP, Hassler HB, Sah P, Galvani AP, Dornburg A. The durability of natural infection and vaccine-induced immunity against future infection by SARS-CoV-2. Proc Natl Acad Sci U S A. 2022;119(31):e2204336119. 91. Sieling P, King T, Wong R, Nguyen A, Wnuk K, Gabitzsch E, et al. Prime hAd5 spike + nucleocapsid vaccination induces Srinivasa Reddy Bonam is a senior postdoctoral fellow at the Department of Microbiology and Immunology at the University of Texas Medical Branch in Galveston, Texas, and is working with Dr. Haitao Hu on novel vaccine development for emerging infectious diseases. He completed his PhD (2018) from the Indian Institute of Chemical Technology (IICT), India, under the supervision of Dr. H. M. Sampath Kumar. In his PhD, his research was focused on novel immunomodulators and vaccine adjuvants. During his PhD, he was awarded the Raman Charpak Fellowship (RCF) for 6 months to work under the guidance of Prof. Sylviane Muller at the Institut de biologie moléculaire et cellulaire (IBMC), University of Strasbourg, France (2017), where he worked on P140 (Lupuzor®), a 21-mer peptide acting against systemic lupus erythematosus (SLE). From 2018 to early 2020, he worked as a postdoctoral researcher with Prof. Sylviane Muller. Later, he joined with Prof. Jagadeesh Bayry at INSERM, Centre de Recherche des Cordeliers, Paris, France, to continue his postdoctoral research on various aspects of immunology, including regulatory T cells (Tregs), basophils, “next-generation flu vaccines for the world (INDIGO)”, and COVID-19 (“COVIMUNE: Étude de la immunitaire interactive dans COVID19”). He has published 60 research and review papers and 5 book chapters, and has a couple of patents to his credit. Haitao Hu is an Associate Professor of Microbiology and Immunology at the University of Texas Medical Branch in Galveston, Texas. He received his PhD training at Drew Weissman Laboratory at the University of Pennsylvania. His research focuses on host mechanisms regulating viral infection, antiviral immunity, and vaccine development, including mRNA vaccines. SUMMARY AND FUTURE DIRECTIONS A current research effort of his laboratory is developing broadly protective vaccines against emerging SARS-CoV-2 variants.
https://openalex.org/W4248846820	https://www.qeios.com/read/4RHXQB/pdf	English	null	Recurrent Oligodendroglioma	Definitions	2,020	cc-by	58	Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Recurrent Oligodendroglioma National Cancer Institute National Cancer Institute Qeios ID: 4RHXQB · https://doi.org/10.32388/4RHXQB Source National Cancer Institute. Recurrent Oligodendroglioma. NCI Thesaurus. Code C160737. National Cancer Institute. Recurrent Oligodendroglioma. NCI Thesaurus. Code C160737 The reemergence of oligodendroglioma after a period of remission. 1/1
https://openalex.org/W4382934270	https://www.biorxiv.org/content/biorxiv/early/2023/07/03/2023.06.30.547281.full.pdf	English	null	Local Orchestration of Global Functional Patterns Supporting Loss and Restoration of Consciousness in the Primate Brain	bioRxiv (Cold Spring Harbor Laboratory)	2,023	cc-by	17,659	. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint these authors share first authorship these authors share senior authorship Correspondence: al857@cam.ac.uk (AIL); rodrigocofre@gmail.com (RC). these authors share first authorship *these authors share senior authorship Correspondence: al857@cam.ac.uk (AIL); rodrigocofre@gmail.com (RC). these authors share first authorship *these authors share senior authorship p Correspondence: al857@cam.ac.uk (AIL); rodrigocofre@gmail.com (RC). Correspondence: al857@cam.ac.uk (AIL); rodrigocofre@gmail.com (R Local Orchestration of Global Functional Patterns Supporting Loss and Restoration of Consciousness in the Primate Brain Andrea I. Luppi1,2,, Lynn Uhrig3,4,, Jordy Tasserie3,5,, Camilo M. Signorelli3,6,7, Emmanuel Stamatakis1, Alain Destexhe8, Bechir Jarraya3,9, Rodrigo Cofre8** 1Division of Anaesthesia and Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK. 1Division of Anaesthesia and Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK. 2 g g 2Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada. 3Cognitive Neuroimaging Unit, CEA, INSERM, Université Paris-Saclay, NeuroSpin Center, 91191 Gif/Yvette, France 2Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada. 3Cognitive Neuroimaging Unit, CEA, INSERM, Université Paris-Saclay, NeuroSpin Center, 91191 Gif/Yvette, France , 4Department of Anesthesiology and Critical Care, Necker Hospital, AP-HP, Université de Paris Cité, Paris, France. partment of Anesthesiology and Critical Care, Necker Hospital, AP-HP, Université de s Cité, Paris, France. Center for Brain Circuit Therapeutics Department of Neurology Brigham & Women’s 5Center for Brain Circuit Therapeutics Department of Neurology Brigham & Women’s Hospital Harvard Medical School Boston MA USA 5Center for Brain Circuit Therapeutics Department of Neurology Brigham & Women’s H i l H d M di l S h l B MA USA p 6Physiology of Cognition, GIGA-CRC In Vivo Imaging, University of Liège, Liège, Belgium 7Department of Computer Science, University of Oxford, Oxford, 7 Parks Rd, Oxford OX1 3QG 6Physiology of Cognition, GIGA-CRC In Vivo Imaging, University of Liège, Liège, Belgium 7Department of Computer Science, University of Oxford, Oxford, 7 Parks Rd, Oxford OX1 3QG nstitute of Neuroscience (NeuroPSI), Paris-Saclay University, Centre National de la echerche Scientifique (CNRS), Gif-sur-Yvette, France. 8Institute of Neuroscience (NeuroPSI), Paris-Saclay University, Centre National de la Recherche Scientifique (CNRS), Gif-sur-Yvette, France. 9 q ( ) 9Department of Neurology, Hopital Foch, 92150, Suresnes, France. 9Department of Neurology, Hopital Foch, 92150, Suresnes, France. Introduction Despite significant progress in recent years, understanding how the activity and connectivity of the brain support and causally determine different states of consciousness remains a major challenge for modern neuroscience (Dehaene, Lau and Kouider, 2017; Kringelbach and Deco, 2020). The traditional approach to the neuroscience of consciousness has been to look for specific and localised brain regions that support consciousness (Crick and Koch, 1998; Seth and Bayne, 2022). However, there is now increasingly compelling evidence for a role of distributed, large-scale patterns of cortical organisation supporting cognitive function, dysfunction, (Margulies et al., 2016; Huntenburg, Bazin and Margulies, 2018; Fulcher et al., 2019; Paquola et al., 2019; Bethlehem et al., 2020; Cross et al., 2021) and consciousness (Dehaene and Naccache, 2001; Dehaene and Changeux, 2011; Atasoy et al., 2017; Atasoy, Deco, et al., 2018). Recent work has demonstrated that multiple pathological and pharmacological perturbations of consciousness induce consistent reorganisation of the brain’s functional architecture along functional and anatomical axes of cortical organisation (Huang, Mashour and Hudetz, 2023; Li et al., 2023; Luppi, Hansen, et al., 2023). This distributed approach goes beyond viewing the brain in terms of brain regions and fixed networks, emphasising the role of dynamics and functional organisation. For instance, when consciousness is lost, the functional interactions between brain regions (“functional connectivity”) become increasingly similar to the pattern of direct anatomical connections between regions (“structural connectivity”) (Barttfeld et al., 2015; Ma, Hamilton and Zhang, 2017; Uhrig et al., 2018; Demertzi et al., 2019; Gutierrez-Barragan et al., 2022; Tasserie et al., 2022). More recently, evidence has shown that the organisation of distributed brain function is perturbed in a scale-specific manner under several pharmacological and pathological perturbations of consciousness (Huang, Mashour and Hudetz, 2023; Luppi, Vohryzek, et al., 2023) - with anaesthesia and disorders of consciousness increasing the dependence of brain function on structural connectivity across scales. Here, we seek to integrate the localised and distributed approaches to characterise the functional architecture of the macaque brain. To this end, we leverage functional MRI (fMRI) data from non-human primates in the awake state, under loss of consciousness induced by three different anaesthetics (sevoflurane, propofol, ketamine) and restoration of consciousness by deep brain stimulation (DBS). Specifically, we consider the brain’s hierarchical organisation across scales, by studying its principal gradient of functional connectivity, and its complex relationship with the other functional eigenmodes of the brain. Abstract CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint Keywords Anaesthesia; fMRI; Deep Brain Stimulation; Thalamus; Harmonic modes; Gradients; Macaque; Consciousness. Abstract A central challenge of neuroscience is to elucidate how the orchestration of brain function is modulated by different states of consciousness. Here, we investigate the link between distributed structural and functional brain organisation in functional MRI signals of non-human primates, through bi-directional causal manipulations of consciousness. During varying levels of propofol, sevoflurane, or ketamine anaesthesia, and subsequent restoration of responsiveness by deep brain stimulation of the central thalamus, we investigate how loss of consciousness impacts distributed patterns of structure-function organisation across scales. Combining the specificity of electrical stimulation with global fMRI coverage of the entire cortex, we report that distributed brain activity under anaesthesia is increasingly constrained by brain structure across scales, coinciding with anaesthetic-induced collapse of multiple dimensions of hierarchical cortical organisation. Crucially, we show that these distributed signatures of anaesthetic-induced loss of consciousness are observed across different anaesthetics, and they are reversed by electrical stimulation of the central thalamus, coinciding with recovery of behavioural markers of consciousness during propofol anaesthesia. No such effects were observed upon stimulation of a control anatomical site, ventral lateral thalamus, demonstrating specificity. Through causal manipulations of consciousness that integrate pharmacology and electrical intracranial stimulation of the thalamus, our results identify global signatures of consciousness that are under local causal control by specific nuclei of the thalamus. Overall, the present work broadens our understanding of the link between brain network organisation and distributed function in supporting consciousness, and the interplay between local and global functional architecture. A central challenge of neuroscience is to elucidate how the orchestration of brain function is modulated by different states of consciousness. Here, we investigate the link between distributed structural and functional brain organisation in functional MRI signals of non-human primates, through bi-directional causal manipulations of consciousness. During varying levels of propofol, sevoflurane, or ketamine anaesthesia, and subsequent restoration of responsiveness by deep brain stimulation of the central thalamus, we investigate how loss of consciousness impacts distributed patterns of structure-function organisation across scales. C bi i th ifi it f l t i l ti l ti ith l b l fMRI f th ti 1 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . Introduction We then integrate brain structure and function, by decomposing brain activity into distributed patterns of structure-function coupling, termed structural eigenmodes. Through this decomposition, we quantify the extent to which brain activity is constrained by the underlying network of structural connectivity across multiple spatial scales, from large-scale to localised. 2 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint Comparing the brain-wide effects of different anaesthetics enables us to disentangle which aspects of the brain’s functional organisation support consciousness, being consistently targeted by anaesthetics, despite their distinct molecular mechanisms. Crucially, we also aim to obtain more stringent evidence for the causal relevance of distributed signature of consciousness, by determining whether the reorganisation of distributed brain function consistently induced by different anaesthetics can be reversed by targeted stimulation of different subregions of the thalamus, a brain structure that has been repeatedly associated with supporting consciousness (Tononi, 2004; Schiff et al., 2007; Staunton, 2008; Mhuircheartaigh et al., 2010; Lioudyno et al., 2013; Ní Mhuircheartaigh et al., 2013; Vijayan et al., 2013; Akeju et al., 2014; Lewis et al., 2015; Flores et al., 2017; Hemmings et al., 2019; Kelz et al., 2019; Zucca et al., 2019; Mashour et al., 2020; Redinbaugh et al., 2020, 2022; Afrasiabi et al., 2021; Bastos et al., 2021; Setzer et al., 2022; Gammel et al., 2023; Kantonen et al., 2023). We do so, using a complementary but independently acquired dataset of fMRI in macaques recorded in the awake state and during anaesthesia with and without concurrent deep brain stimulation of the centro-medial thalamus (CT) or ventro-lateral thalamus (VT), previously shown to induce recovery of responsiveness according to the location and intensity of the electrical input (Tasserie et al., 2022). Results Here, we studied distributed functional patterns in the macaque cerebral cortex. Specifically, we measure how these patterns are reorganised under different anaesthetic agents and by direct stimulation of different thalamic nuclei. Introduction This dual causal manipulation - pharmacology and electrical stimulation - provides us with a unique opportunity to study functional changes that are observed during loss of consciousness and reappear upon recovery of consciousness, despite continuous anaesthetic infusion. By combining the broad spatial coverage of fMRI with stimulation of specific nuclei of the thalamus, we can consider both local and global effects. Through this approach, we show that distributed cortical signatures of consciousness are under local control specifically by the central thalamus, thereby integrating the distributed and localised perspectives. Anaesthetic-Induced Collapse of the Principal Functional Gradient Our first approach to study distributed brain function and its alteration under anaesthesia is through the lens of the principal gradient of functional connectivity. Functional gradients, as the eigenmodes of the brain’s functional connectivity, provide a low-dimensional representation of the similarity between different regions’ patterns of functional connectivity. Along each dimension (represented by an eigenmode), regions whose connectivity with the rest of the brain is more similar will exhibit more similar values - whose spatial variation delineates the gradient. In particular, the principal functional gradient (eigenmode associated with the principal eigenvalue) captures the direction of maximal spatial variation in functional organisation: its range can be interpreted as reflecting the distance between the two extremes of the cortical processing hierarchy, reflecting the depth of information processing. This principal gradient can also be reshaped by pharmacological intervention (Girn et al., 2022; 3 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint Timmermann et al., 2023), rendering this approach a promising tool for the present study. Here, we hypothesise that this depth should be reduced in the unconscious brain, when processing of information is manifestly impaired. Indeed, such an observation was very recently reported in humans, with different gradients of functional connectivity collapsing as a result of different pharmacological and pathological perturbations, including anaesthesia (Huang, Mashour and Hudetz, 2023). Here, we delineate gradients using the common nonlinear dimensionality reduction technique known as diffusion map embedding (see Methods). We observed anaesthetic-induced collapse of the principal gradient of macaque functional connectivity, which is restored by centro-median thalamic stimulation at high amplitude. The range of the principal gradient of macaque functional connectivity is significantly reduced by anaesthesia, regardless of the specific agent used (Figure 1 and Figure S1), and is significantly increased back to awake levels by low amplitude stimulation of the centro-median thalamus (Figure 2 and Figure S1). Anaesthetic-Induced Collapse of the Principal Functional Gradient In general, the second gradient appears less sensitive to perturbations, both in terms of reflecting the effect of anaesthetics, and in terms of reflecting restoration due to CT stimulation (Figure S2). Surprisingly, a weaker effect was observed when stimulation at the same location at high amplitude, although both induce significant restoration of the principal functional gradient, compared with stimulation of the control site in the ventral-lateral thalamus. 4 4 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint Figure 1. Anaesthetic-induced collapse of the principal gradient of macaque functional connectivity. (A) Scatter plots show the first two principal gradients of macaque functional connectivity (obtained from diffusion graph embedding: see Methods) for the group-averaged FC matrix of the awake condition, and each anaesthetised condition. The gradients are also plotted on the cortical surface of the macaques, with colour representing the position of each region along each gradient. (B) The range of the principal gradient of macaque functional connectivity across wakefulness and different anaesthetic conditions. Box plots indicate the median and interquartile range of the distribution. * p < 0.05; p < 0.01; * p < 0.001 (FDR-corrected), compared against Awake condition; see Figure S1 for comparisons between all pairs; see Figure S2 for gradient 2 results). Figure 1. Anaesthetic-induced collapse of the principal gradient of macaque functional connectivity. (A) Scatter plots show the first two principal gradients of macaque functional connectivity (obtained from diffusion graph embedding: see Methods) for the group-averaged FC matrix of the awake condition, and each anaesthetised condition. Anaesthetic-Induced Collapse of the Principal Functional Gradient The gradients are also plotted on the cortical surface of the macaques, with colour representing the position of each region along each gradient. (B) The range of the principal gradient of macaque functional connectivity across wakefulness and different anaesthetic conditions. Box plots indicate the median and interquartile range of the distribution. * p < 0.05; p < 0.01; * p < 0.001 (FDR-corrected), compared against Awake condition; see Figure S1 for comparisons between all pairs; see Figure S2 for gradient 2 results). 5 5 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint Figure 2. Anaesthetic-induced collapse of the principal gradient of macaque functional connectivity is restored by thalamic stimulation. (A) Scatter plots showing the first two principal gradients of macaque functional connectivity (obtained from diffusion graph embedding) for the group-averaged FC matrix of the awake condition, and of the anaesthetized state, with and without DBS thalamic stimulation. The gradients are also plotted on the cortical surface of the macaques, with colour representing the position of each region along each gradient. (B) Effect of thalamic deep-brain stimulation on the range of the principal gradient of functional connectivity in macaques during anaesthesia. Box plots indicate the median and interquartile range of the distribution. * p < 0.05; p < 0.01; * p < 0.001 (FDR-corrected), compared against Awake condition; * p < 0.05; p < 0.01; * p < 0.001 (FDR-corrected), compared against CT high condition; see Figure S1 for comparisons between all pairs; see Figure S2 for gradient 2 results). Figure 2. Anaesthetic-induced collapse of the principal gradient of macaque functional connectivity is restored by thalamic stimulation. (A) Scatter plots showing the first two principal gradients of macaque functional connectivity (obtained from diffusion graph embedding) for the group-averaged FC matrix of the awake condition, and of the anaesthetized state, with and without DBS thalamic stimulation. Anaesthetic-Induced Collapse of the Principal Functional Gradient The gradients are also plotted on the cortical surface of the macaques, with colour representing the position of each region along each gradient. (B) Effect of thalamic deep-brain stimulation on the range of the principal gradient of functional connectivity in macaques during anaesthesia. Box plots indicate the median and interquartile range of the distribution. * p < 0.05; p < 0.01; * p < 0.001 (FDR-corrected), compared against Awake condition; * p < 0.05; p < 0.01; * p < 0.001 (FDR-corrected), compared against CT high condition; see Figure S1 for comparisons between all pairs; see Figure 2. Anaesthetic-induced collapse of the principal gradient of macaque functional connectivity is restored by thalamic stimulation. (A) Scatter plots showing the first two principal gradients of macaque functional connectivity (obtained from diffusion graph embedding) for the group-averaged FC matrix of the awake condition, and of the anaesthetized state, with and without DBS thalamic stimulation. The gradients are also plotted on the cortical surface of the macaques, with colour representing the position of each region along each gradient. (B) Effect of thalamic deep-brain stimulation on the range of the principal gradient of functional connectivity in macaques during anaesthesia. Box plots indicate the median and interquartile range of the distribution. * p < 0.05; p < 0.01; * p < 0.001 (FDR-corrected), compared against Awake condition; * p < 0.05; p < 0.01; * p < 0.001 (FDR-corrected), compared against CT high condition; see Figure S1 for comparisons between all pairs; see Figure S2 for gradient 2 results). 6 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint Hierarchical Integration Across Scales is Compromised under Anaesthesia * p < 0.05; p < 0.01; * p < 0.001 (FDR-corrected), compared against Awake condition; * p < 0.05; p < 0.01; * p < 0.001 (FDR-corrected), compared against CT high condition; see Figure S3 for comparisons between all pairs, and Figure S4 for hierarchical segregation). Hierarchical Integration Across Scales is Compromised under Anaesthesia It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint We observe that the hierarchical brain integration of macaque eigenmodes is reshaped by loss and recovery of consciousness. Eigenmode-based hierarchical integration is significantly reduced in the macaque brain under anaesthesia, whether induced by sevoflurane, propofol, or ketamine (Figure 4 and Figure S3). In contrast, hierarchical segregation is nearly insensitive to the effects of anaesthesia (Figure S4). The reduction in eigenmode-based hierarchical integration induced by anaesthesia is also specifically restored by high amplitude electrical stimulation delivered in the centro-median nucleus of the thalamus, bringing it back to the level of the awake state (Figure 4). These results reveal novel signatures of consciousness restored by deep brain stimulation of the central thalamus under anaesthesia. Figure 4. Hierarchical brain integration from macaque eigenmodes is reshaped by loss and recovery of consciousness. (A) Eigenmode-based hierarchical integration is significantly reduced in the macaque brain under anaesthesia, whether induced by sevoflurane, propofol, or ketamine. (B) The reduction in eigenmode-based hierarchical integration induced by anaesthesia is restored to awake levels by electrical stimulation of the centro- median thalamus with high (5V) current. Box plots indicate the median and interquartile range of the distribution. * p < 0.05; p < 0.01; * p < 0.001 (FDR-corrected), compared against Awake condition; * p < 0.05; p < 0.01; * p < 0.001 (FDR-corrected), compared against CT high condition; see Figure S3 for comparisons between all pairs, and Figure S4 for hierarchical segregation). Figure 4. Hierarchical brain integration from macaque eigenmodes is reshaped by loss and rec Figure 4. Hierarchical brain integration from macaque eigenmodes is reshaped by loss and recovery of consciousness. (A) Eigenmode-based hierarchical integration is significantly reduced in the macaque brain under anaesthesia, whether induced by sevoflurane, propofol, or ketamine. (B) The reduction in eigenmode-based hierarchical integration induced by anaesthesia is restored to awake levels by electrical stimulation of the centro- median thalamus with high (5V) current. Box plots indicate the median and interquartile range of the distribution. Hierarchical Integration Across Scales is Compromised under Anaesthesia Whereas the range of the principal functional gradient reflects the putative depth of the information-processing hierarchy, hierarchical organisation can also manifest in terms of nested relationships between the system’s parts at different scales: lower elements in the hierarchy are recursively combined to form the higher elements (Hilgetag and Goulas, 2020). This perspective makes it possible to consider the interplay of global integration and local segregation of brain signals - which is a central feature of prominent scientific accounts of consciousness. Classical graph-theoretic measures such as small-worldness and modularity quantify integration and segregation at a single scale, making them inadequate to capture these properties across multiple hierarchical modules (Newman, 2006; Rubinov and Sporns, 2010; Wang et al., 2021). Instead, we can obtain insight into the hierarchical relationships between different scales of distributed activation in the brain by going beyond the principal gradient alone, and instead considering all functional eigenmodes (Wang et al., 2021). This is achieved by characterising the similarities and differences of regions’ allegiance across eigenmode scales, resulting in a nested, modular structure that identifies a hierarchical sub- division of the functional connectome into nested modules (see Figure 3). Hierarchical integration and segregation can then be quantified by the relative prevalence of the different eigenmodes, indicated by their associated eigenvalues (Wang et al., 2021). Figure 3. Hierarchical integration quantified from brain functional eigenmodes. Top: Each eigenmode (except the first) divides cortical regions into two groups, at progressively finer scales. Combining the different groupings identifies a hierarchical sub-division of the functional connectome into nested modules. Bottom: The relative weight of each eigenmode is given by its associated eigenvalue. The first eigenmode corresponds to global integration, and segregation is then reflected by the contribution of the other eigenmodes. Figure 3. Hierarchical integration quantified from brain functional eigenmodes. Top: Each eigenmode (except the first) divides cortical regions into two groups, at progressively finer scales. Combining the different groupings identifies a hierarchical sub-division of the functional connectome into nested modules. Bottom: The relative weight of each eigenmode is given by its associated eigenvalue. The first eigenmode corresponds to global integration, and segregation is then reflected by the contribution of the other eigenmodes. 7 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Harmonic Mode Decomposition Reveals Increased Structural Constraints on Brain Activity Functional brain activity and connectivity unfold over the network of physical white matter pathways between brain regions: the structural connectome. Therefore, for our final investigation we go beyond function alone, by explicitly integrating brain activity and structural 8 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint connectivity. To this end, inspired by the work of Atasoy and colleagues (2016) in humans, we leverage the mathematical framework of "harmonic mode decomposition" (Atasoy, Donnelly and Pearson, 2016), which decomposes brain activity in terms of contributions from multi- scale patterns of coactivation using the network organisation of the structural connectome (Figure 5). connectivity. To this end, inspired by the work of Atasoy and colleagues (2016) in humans, we leverage the mathematical framework of "harmonic mode decomposition" (Atasoy, Donnelly and Pearson, 2016), which decomposes brain activity in terms of contributions from multi- scale patterns of coactivation using the network organisation of the structural connectome (Figure 5). Each harmonic mode is a cortex-spanning activation pattern (eigenmode of the structural connectome) characterised by a specific granularity (spatial frequency, Figure 6): large-scale, coarse-grained patterns (e.g., left and right or front and back of the brain) are strongly constrained by the organisation of the structural connectome, such that strongly interconnected regions are predicted to exhibit similar activation (Luppi, Vohryzek, et al., 2023). In contrast, high-frequency harmonic modes are relatively unconstrained by the underlying network structure, such that regions may exhibit different activity even if they are strongly connected in the structural network (Figure 5). Therefore, decomposing functional MRI signals in terms of contributions from different connectome harmonics provides a quantification of the extent to which brain activity is constrained by the network structure of the connectome. In other words, harmonic mode decomposition is analogous to the well- known Fourier decomposition, but operating in the spatial domain (Luppi, Vohryzek, et al., 2023). Figure 5. Harmonic Mode Decomposition Reveals Increased Structural Constraints on Brain Activity Structural eigenmode decomposition generalises the Fourier transform to the network structure of the brain. (A) In traditional Fourier analysis, a signal in the time domain (represented in terms of successive time points) is decomposed into temporal harmonics of different frequencies and thereby rendered in terms of a new set of basis functions. (B) High-frequency temporal harmonics correspond to rapidly varying signals, such that Figure 5. Structural eigenmode decomposition generalises the Fourier transform to the network structure of the brain. (A) In traditional Fourier analysis, a signal in the time domain (represented in terms of successive time points) is decomposed into temporal harmonics of different frequencies and thereby rendered in terms of a new set of basis functions. (B) High-frequency temporal harmonics correspond to rapidly varying signals, such that 9 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint data points may have very different values even if they are close in time. In contrast, low-frequency temporal harmonics correspond to signals that change slowly over time, such that temporally contiguous have similar values, reflecting a greater time dependence of the signal. (C) Harmonic decomposition of the connectome involves decomposing a signal in the spatial domain (represented in terms of fMRI activation at discrete spatial locations over the cortex) into harmonic modes of the structural connectome, resulting in a new set of basis functions in terms of whole-brain distributed patterns of activity propagation distributed throughout the brain at different spatial scales (granularity) , from global patterns of smooth variation along geometrical axes (left–right and anterior– posterior being the most prominent) to increasingly localised patterns. Note that here, frequency is not about time, but about spatial scale. (D) Low-frequency (coarse-grained) connectome harmonics indicate that the spatial organisation of the functional signal closely matches the underlying organisation of the structural connectome: Nodes that are strongly connected exhibit similar functional signals (indicated by colour). High-frequency (fine- grained) patterns indicate divergence between the spatial organisation of the functional signal and the underlying network structure, where nodes may exhibit different functional signals even if they are closely connected in the structural network (Luppi, Vohryzek, et al., 2023). Harmonic Mode Decomposition Reveals Increased Structural Constraints on Brain Activity The use of connectome-specific harmonic decomposition of cortical activity allows us to quantify the contribution of structural organisation to brain activity, across different spatial scales: from large-scale to localised. This approach therefore goes beyond previous investigations that simply assessed the similarity of structural and functional connectivity at a single scale (Barttfeld et al., 2015; Demertzi et al., 2019; Gutierrez-Barragan et al., 2021). Specifically, for each time-point, the magnitude of contribution of each harmonic to the BOLD activation across the brain (weighted by its associated eigenvalue) is termed the energy (see Methods). Through this formalism, we tested whether the different anaesthetics induced changes in the contribution of structural connectivity to functional activity, and whether such changes (if any) would be reversed upon restoration of responsiveness by thalamic DBS. 10 10 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint Figure 6. Harmonic modes of the macaque structural connectome. The first four non-uniform harmonic modes of the macaque structural connectome and the last four are shown on the surface of the macaque brain. Note that the first, low-frequency harmonics reveal large-scale patterns, while the last, high-frequency harmonics correspond to localised patterns. The total number of harmonics, and therefore the maximum resolution, corresponds to the number of brain regions in the connectome (here 82). Please note that in the original formulation of Atasoy and colleagues (2016), “connectome harmonics” are specifically defined as the harmonic modes of a high-resolution human structural connectome, obtained from combining long-range white matter tracts and local connectivity within the grey matter. Here we use instead the harmonic modes obtained from a parcellated macaque connectome. To avoid confusion, we refer to the eigenmodes obtained in this way as “harmonic modes”. Figure 6. Harmonic modes of the macaque structural connectome. Harmonic Mode Decomposition Reveals Increased Structural Constraints on Brain Activity It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint amplitude (5V) stimulation of the centro-median thalamic nucleus brought the overall harmonic energy significantly back closer to awake levels, whereas the same stimulation delivered at low amplitude (3V) induced a significant but smaller effect. When the same electrical input (both low and high amplitude) was delivered to the ventro-lateral part of the thalamus, the overall harmony energy did not significantly differ from the anaesthesia condition (Figure 7). Figure 7. Energy of harmonic modes in the macaque is reshaped by loss and recovery of responsiveness. (A) Energy of connectome harmonics is significantly increased in the macaque brain under deep (but not light) anaesthesia, whether induced by sevoflurane, propofol, or the dissociative anaesthetic ketamine. (B) Effect of thalamic deep-brain stimulation on the energy of connectome harmonics during anaesthesia. Box plots indicate the median and interquartile range of the distribution. * p < 0.05; p < 0.01; * p < 0.001 (FDR-corrected), compared against Awake condition; * p < 0.05; p < 0.01; * p < 0.001 (FDR-corrected), compared against CT high condition; see Figure S5 for comparisons between all pairs. Figure 7. Energy of harmonic modes in the macaque is reshaped by loss and recovery of responsiveness. (A) Energy of connectome harmonics is significantly increased in the macaque brain under deep (but not light) anaesthesia, whether induced by sevoflurane, propofol, or the dissociative anaesthetic ketamine. (B) Effect of thalamic deep-brain stimulation on the energy of connectome harmonics during anaesthesia. Box plots indicate the median and interquartile range of the distribution. * p < 0.05; p < 0.01; * p < 0.001 (FDR-corrected), compared against Awake condition; * p < 0.05; p < 0.01; * p < 0.001 (FDR-corrected), compared against CT high condition; see Figure S5 for comparisons between all pairs. Harmonic Mode Decomposition Reveals Increased Structural Constraints on Brain Activity The first four non-uniform harmonic modes of the macaque structural connectome and the last four are shown on the surface of the macaque brain. Note that the first, low-frequency harmonics reveal large-scale patterns, while the last, high-frequency harmonics correspond to localised patterns. The total number of harmonics, and therefore the maximum resolution, corresponds to the number of brain regions in the connectome (here 82). Please note that in the original formulation of Atasoy and colleagues (2016), “connectome harmonics” are specifically defined as the harmonic modes of a high-resolution human structural connectome, obtained from combining long-range white matter tracts and local connectivity within the grey matter. Here we use instead the harmonic modes obtained from a parcellated macaque connectome. To avoid confusion, we refer to the eigenmodes obtained in this way as “harmonic modes”. Figure 6. Harmonic modes of the macaque structural connectome. The first four non-uniform harmonic modes of the macaque structural connectome and the last four are shown on the surface of the macaque brain. Note that the first, low-frequency harmonics reveal large-scale patterns, while the last, high-frequency harmonics correspond to localised patterns. The total number of harmonics, and therefore the maximum resolution, corresponds to the number of brain regions in the connectome (here 82). Please note that in the original formulation of Atasoy and colleagues (2016), “connectome harmonics” are specifically defined as the harmonic modes of a high-resolution human structural connectome, obtained from combining long-range white matter tracts and local connectivity within the grey matter. Here we use instead the harmonic modes obtained from a parcellated macaque connectome. To avoid confusion, we refer to the eigenmodes obtained in this way as “harmonic modes”. Our results indicate that the energy of connectome harmonics in the macaque is reshaped by loss and recovery of consciousness. In particular, the energy of connectome harmonics is significantly increased in the macaque brain under deep (but not light) anaesthesia, whether induced by sevoflurane, propofol, or ketamine (Figure 7 and Figure S5). Remarkably, this pattern is replicated in an independent dataset of propofol anaesthesia, and it is reversed under the effects of thalamic stimulation with both amplitude and site dependence: high 11 . CC-BY 4.0 International license made available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Multi-dimensional Representation of Loss and Recovery of Consciousness Finally, we bring together our three complementary analyses to generate a multi-dimensional profile that might better characterise perturbations of consciousness. We show this by using the magnitude of effect size for each condition of perturbed consciousness compared with wakefulness (Figure 8). For the multi-anaesthetic dataset, we find that hierarchical integration displays the largest effect sizes across all anaesthetics; additionally, we clearly see the greater effect of deep anaesthesia, with all three drugs (propofol, sevoflurane, and ketamine) pushing the effect sizes further away from zero (Figure 8A). Interestingly, hierarchical integration was 12 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint also strongly affected by high amplitude electrical stimulation of the centro-median thalamus, essentially reducing the effect of anaesthesia back to a near-zero effect size, when compared with wakefulness (Figure 8B). In general, this experimental condition was the most effective in counteracting the effects of propofol on the distributed functional signatures (indicated by the smallest effect sizes when compared against wakefulness), except for the range of the principal FC gradient, which was more restored by low- rather than high-amplitude stimulation of the centro-median thalamic nucleus (Figure 8B). Overall, this multi-dimensional representation in terms of structural and functional eigenmode reorganisation provides a compact description, identifying relevant axes along which perturbations of consciousness can manifest. also strongly affected by high amplitude electrical stimulation of the centro-median thalamus, essentially reducing the effect of anaesthesia back to a near-zero effect size, when compared with wakefulness (Figure 8B). In general, this experimental condition was the most effective in counteracting the effects of propofol on the distributed functional signatures (indicated by the smallest effect sizes when compared against wakefulness), except for the range of the principal FC gradient, which was more restored by low- rather than high-amplitude stimulation of the centro-median thalamic nucleus (Figure 8B). Multi-dimensional Representation of Loss and Recovery of Consciousness Overall, this multi-dimensional representation in terms of structural and functional eigenmode reorganisation provides a compact description, identifying relevant axes along which perturbations of consciousness can manifest. Figure 8. Representing perturbations of consciousness in terms of structural and functional eigenmode reorganisation. (A): Radial plot showing the magnitude (absolute value) of the effect size obtained when comparing each state of perturbed consciousness against wakefulness for the multi-anaesthesia data set. (B) Radial plot shows the magnitude (absolute value) of the effect size obtained when comparing each state of perturbed consciousness against wakefulness, for the DBS dataset. Figure 8. Representing perturbations of consciousness in terms of structural and functional eigenmode reorganisation. (A): Radial plot showing the magnitude (absolute value) of the effect size obtained when comparing each state of perturbed consciousness against wakefulness for the multi-anaesthesia data set. (B) Radial plot shows the magnitude (absolute value) of the effect size obtained when comparing each state of perturbed consciousness against wakefulness, for the DBS dataset. Discussion Here, we capitalised on two unique fMRI datasets of macaque brains during anaesthesia (propofol, ketamine, sevoflurane) with versus without simultaneous intracranial electrical stimulation of different thalamic nuclei. We investigated anaesthetic-induced reorganisation of macaque brain functional architecture, its relationship to brain structure across brain scales, and its restoration by causal intervention (electrical thalamic stimulation). Our goals were twofold. First, we sought to identify consistent signatures of consciousness in terms of distributed brain patterns, across different ways of consciousness loss, by considering anaesthetics with distinct molecular mechanisms. Second, we aimed to determine whether the reorganisation of distributed brain function consistently induced by different anaesthetics can be reversed by targeted stimulation of different subregions of the thalamus. 13 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint Thanks to the broad spatial coverage provided by fMRI, we were able to characterise distributed brain function in terms of its relationship to the underlying structural organisation and in terms of how it supports different facets of hierarchical organisation. We showed that large-scale patterns are reliably altered when consciousness is suppressed, regardless of the anaesthetic agent, and that they are restored upon DBS-induced awakening, even in the presence of continued anaesthetic infusion. Taken together, these observations indicate that such changes are neither specific to a particular anaesthetic agent nor exclusively related to the mere presence of the anaesthetic in the bloodstream: rather, they are associated with behavioural markers of wakefulness, with the stimulation results being both location- and amplitude-dependent (Tasserie et al., 2022). We examined how anaesthetic-induced unconsciousness reshapes distributed functional organisation of the primate brain, by studying a dataset of functional MRI recordings from macaque monkeys anaesthetised with propofol, sevoflurane, or ketamine (see the Methods section and (Uhrig et al., 2018) for details). Discussion CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint millimetres of cortex. Therefore, even the most fine-grained eigenmodes from the macaque 82-node connectome correspond to dividing the cortex in fewer than 100 patches - a number that lies firmly in the low-frequency (structurally-constrained) range for the human 18,000- eigenmode decomposition. In other words, when only a resolution of fewer than 100 harmonic eigenmodes is considered, the present results coincide with those reported by Luppi et al (2023): unconsciousness manifests as an increased contribution of structural constraints to cortical functional activation. Future work using higher-resolution anatomical connectivity may enable us to obtain finer-grained insights about the respective roles of low-frequency versus high-frequency structural eigenmodes, such as by using the “structural decoupling index” to quantify their balance (Preti and Van De Ville, 2019). millimetres of cortex. Therefore, even the most fine-grained eigenmodes from the macaque 82-node connectome correspond to dividing the cortex in fewer than 100 patches - a number that lies firmly in the low-frequency (structurally-constrained) range for the human 18,000- eigenmode decomposition. In other words, when only a resolution of fewer than 100 harmonic eigenmodes is considered, the present results coincide with those reported by Luppi et al (2023): unconsciousness manifests as an increased contribution of structural constraints to cortical functional activation. Future work using higher-resolution anatomical connectivity may enable us to obtain finer-grained insights about the respective roles of low-frequency versus high-frequency structural eigenmodes, such as by using the “structural decoupling index” to quantify their balance (Preti and Van De Ville, 2019). Decomposition in terms of harmonic modes of the structural connectome characterises distributed brain function in terms of the relationship between functional activation and distributed patterns derived from brain anatomical network organisation, showing how this relationship changes when consciousness is lost. Discussion Previous work has consistently shown increased correspondence between structural and functional connectivity under anaesthesia (Barttfeld et al., 2015; Uhrig et al., 2018; Demertzi et al., 2019; Gutierrez-Barragan et al., 2022; Tasserie et al., 2022). However, existing investigations of structure-function correspondence during anaesthesia have typically relied on correlation and analogous distance metrics, which operate at a single spatial scale, whereas it is well established that brain structure and function both exhibit multi-scale, network organisation. This motivates the use of a multi-scale approach, provided by the framework of structural eigenmode decomposition, which simultaneously considers all available scales of organisation: from a single region to the entire cortex). Our results showed that increased multi-scale coupling between brain activity and structural eigenmodes, as recently observed in the human brain during pharmacological and pathological loss of consciousness (Luppi et al., 2023), can be generalised to the macaque brain - thereby supporting previous results that operated at a single scale (and which were obtained in terms of functional connectivity rather than activity). We also showed that the signatures that we identified generalise across anaesthetic agents, including ketamine. Unlike sevoflurane and propofol, ketamine does not act primarily as an agonist of GABA-A receptors, but rather as an antagonist of NMDA receptors. Note that this result is not in contrast with the ketamine-induced decreased structural coupling observed in humans by Luppi and colleagues (2023), because that study employed a sub-anaesthetic dose of ketamine (i.e., having psychedelic-like effects), whereas the dose of ketamine used in the present study induced general anaesthesia. Thus, showing that previously identified signatures of unconsciousness can be generalised to this additional pharmacological perturbation, improve the robustness of consciousness-specificity of these signatures, while also suggesting that ketamine can have opposite effects on structure-function relationships in the brain, depending on its dosage and the corresponding changes in subjective experience. In the human brain, considering highly fine-grained harmonic modes of the human connectome in the order of 18,000 surface vertices (Luppi et al., 2023), harmonic mode decomposition has revealed that anaesthesia and disorders of consciousness induce a shift in the relative contribution of different harmonics, increasing the contribution of low-frequency (structurally-constrained) harmonics at the expense of high-frequency (liberal) ones. We note that the resolution of harmonic modes is fundamentally limited by the resolution of the underlying structural connectome: in the present case, 82 regions, each spanning several 14 . Discussion It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint Crucially, the restoration of such distributed cortical patterns can be triggered by selective stimulation of a specific subcortical region, as demonstrated by our spatially specific causal intervention: the centro-median thalamic nucleus - in contrast to the much weaker effects elicited by control site stimulation of the ventral lateral nucleus of the thalamus. Therefore, both wakefulness and its associated large-scale cortical functional patterns are under causal control by a very selective locus. Thus, our unique combination of broad recording coverage (from fMRI) and specific causal intervention (from DBS) enables us to show that global functional patterns are orchestrated locally, showing how the locationist and distributed approaches to the neural correlates of consciousness are not antithetical, but rather complement each other. In this sense, our results help reconcile the traditional locationist approach to the neural correlates of consciousness with recent advances in understanding brain function in terms of distributed patterns (Suzuki and Larkum, 2020). Our findings regarding thalamic control of distributed cortical functions are consistent with recent views on the thalamus as a controller of brain dynamics (Liu et al., 2015; Shine, 2019, 2021), and more broadly on its role in supporting consciousness (Lioudyno et al., 2013; Vijayan et al., 2013; Akeju et al., 2014; Flores et al., 2017), including connectivity with cortical regions displaying consciousness-specific signatures (Luppi et al., 2019) and the effects of selective thalamic stimulation on consciousness and arousal in animals (Alkire et al., 2007, 2009; Lewis et al., 2015; Bastos et al., 2021; Tasserie et al., 2022) and human patients (Tononi, 2004; Schiff et al., 2007; Staunton, 2008; Mashour et al., 2020). Discussion However, the harmonic modes themselves do not change, being based on structural connectivity, which is relatively stable at short timescales and unaffected by anaesthesia. Therefore, we complemented our investigation of distributed structure-function relationships with an additional investigation of distributed patterns arising from functional connectivity, termed functional gradients. This alternative perspective identified diminished hierarchical character of brain function under unconsciousness: both in terms of the processing distance (contraction of the principal functional gradient), and in terms of diminished hierarchical integration across nested functional modules, when considering all functional eigenmodes, rather than just the principal one. We also note that both approaches that considered all eigenmodes (hierarchical integration and harmonic mode decomposition) exhibited greater effect sizes than consideration of the principal gradient (principal functional eigenmode) alone. Indeed, the measure of gradient range also exhibited a deviation from the behavioural pattern, being more influenced by low- than high-amplitude stimulation of the central thalamus - although we note that both low- and high-amplitude CT stimulation were nevertheless clearly superior to stimulation of the control VT site. As previously reported, low-amplitude stimulation of the central thalamus has weaker effect on behaviour, than high-amplitude stimulation (Tasserie et al., 2022). Thus, gradient range is influenced by CT stimulation even before this stimulation is sufficient to induce restoration of responsiveness, whereas high-amplitude stimulation is more closely aligned with behaviour. In contrast, the structural eigenmode decomposition and hierarchical integration, which consider all eigenmodes (structural and functional, respectively), both displayed the largest effect for high-amplitude stimulation of the central thalamus, coinciding with the fullest restoration of consciousness. These observations reinforce the value of considering distributed brain function across multiple scales: the full effects of high-amplitude stimulation appear to be spread across multiple functional eigenmodes, such that only considering the first one provides an incomplete picture with weaker link to behaviour. On the other hand, although here we did not focus on the specific topography of functional gradients, but rather on their extent and relationships, we expect that future work will also benefit from such an approach. Overall, these convergent results from distributed function extend previous observations from macaques and humans under anaesthesia and disorders of consciousness (Signorelli et al., 2021; Luppi et al., 2022). 15 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Discussion ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint signatures may hold additional promise for unveiling the rich interplays that support consciousness. Another limitation is that our analysis was exclusively cortical (except for the thalamic stimulation), yet other subcortical structures such as the basal ganglia and brainstem (Moruzzi and Magoun, 1949; Brown, Lydic and Schiff, 2010; Mhuircheartaigh et al., 2010; Braun et al., 2011; Hudetz, 2012; Hemmings et al., 2019; Guang et al., 2021; Spindler et al., 2021) are known to play an important role in anaesthetic-induced and pathological loss of responsiveness (Bastos et al., 2021; Tasserie et al., 2022). Future work including subcortical regions may therefore provide additional insights. Future directions can also combine methods from eigenmode analysis and combinatorial topology to construct efficient representations of high-order information-theoretic signals (Medina-Mardones et al., 2021), which are a hallmark of many complex systems including the brain in its different states of consciousness. Additionally, here we relied on a symmetrised version of the macaque connectome, which is required for eigendecomposition in order to ensure real eigenmodes. However, since a directed macaque connectome is available, we expect that generalisation of harmonic mode analysis to account for directed connections, may provide a more refined picture. Pertaining to alternative avenues of eigenmode decomposition, recent work suggested that structural eigenmodes obtained from the geometry of the human brain may outperform those obtained from the human connectome, in terms of explaining variance in the functional signals (Pang et al., 2023). Thus, future work may investigate whether cortical geometry provides insights into the reorganisation of consciousness, beyond those provided by the connectome. In the absence of subjective reports from animals, behavioural markers alone play a more prominent role, although it is known from research in humans that on occasion behaviour and subjective experience can be dissociated - including with ketamine. More broadly, our analysis is based on fMRI acquired in resting state, i.e. in the absence of a stimulus or a task. Discussion Indeed, recent studies also reported that electrical stimulation of specific central-lateral thalamic nuclei (but not control sites) can restore arousal during anaesthetic-induced loss of responsiveness in macaques (Tononi, 2004; Schiff et al., 2007; Staunton, 2008; Mhuircheartaigh et al., 2010; Lioudyno et al., 2013; Ní Mhuircheartaigh et al., 2013; Vijayan et al., 2013; Akeju et al., 2014; Flores et al., 2017; Hemmings et al., 2019; Kelz et al., 2019; Mashour et al., 2020; Redinbaugh et al., 2020, 2022; Afrasiabi et al., 2021; Bastos et al., 2021; Gammel et al., 2023; Kantonen et al., 2023), counteracting the loss of high-frequency (gamma-band) activity and communication between the thalamus and deep cortical layers, which is induced by anaesthesia. However, these studies were limited by the use of electrodes for recording, given the authors’ interest in different frequency bands of neural activity - consequently limiting spatial coverage to 2-8 sites. In contrast, the present work combined thalamic stimulation with global coverage of the entire cortex, thanks to the use of functional MRI. This unique set-up enabled us to study both local and distributed neural contributions to consciousness. Our evidence that selective thalamic DBS can restore not only arousal, but also distributed neural signatures of consciousness that are disrupted across anaesthetics, lends further support for the translational potential of DBS as an avenue for treating the challenging condition of patients suffering from disorders of consciousness (Cohadon and Richer, 1993; Schiff et al., 2007; Lemaire et al., 2018; Edlow et al., 2021) - although with the clear caveat that such patients typically exhibit widespread cortical and subcortical damage, unlike the non-human primates in the present study. An important limitation of this work is that our distributed approach to brain function does not provide insights about the role of specific brain regions: instead, we focused on different whole-brain patterns. A substantial body of previous work has focused on specific regions, and our goal here was to provide an alternative perspective. Additionally, thanks to the DBS dataset we were still able to identify localised contributions to consciousness. Nevertheless, future analytic approaches capable of simultaneously identifying both local and distributed 16 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. Discussion Though beyond the scope of the present work, analysis of distributed patterns pertaining to the dysfunctional spread of naturalistic and synthetic stimuli, which was also observed in the same anaesthetised animals, will provide a more comprehensive understanding of how such patterns underpin information-processing and its perturbation by anaesthetics. Conversely, a strength of this work is the replication of our results not only across multiple anaesthetics, but also in the independent DBS-fMRI dataset, showing their robustness to acquisition differences such as the presence or absence of MION contrast agent. Likewise, the DBS data enabled us to assess the respective roles of both location (central versus ventral-lateral thalamus) and stimulation strength. Overall, we showed that distributed patterns of functional activity and connectivity of the primate brain provide rich characterisations of consciousness, its suppression by different anaesthetics, and its restoration by thalamic stimulation. The resulting insights align both with the well-known increase in structure-function coupling observed across a variety of pharmacological and pathological states of unconsciousness across species, and also with prominent theories of consciousness that postulate a central role for the integration of information, which we show here to be reliably disrupted in the unconscious primate brain. We have also provided two convergent lines of evidence for a reduced hierarchical character of cortical functions in the unconscious primate brain. 17 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint Animals For the anaesthesia dataset, the details of the acquisitions have been previously reported in (Barttfeld et al., 2015; Uhrig et al., 2018; Signorelli et al., 2021). Five rhesus macaques were included for analyses (Macaca mulatta, one male, monkey J, and four females, monkey A, K, Ki, and R, 5-8 kg, 8-12 yr of age), in a total of six different arousal conditions: awake state, ketamine, light propofol, deep propofol, light sevoflurane, and deep sevoflurane anaesthesia. Three monkeys were used for each condition: awake state (monkeys A, K, and J), ketamine (monkeys K, R and Ki), propofol (monkeys K, R, and J), sevoflurane (monkeys Ki, R, and J). Each monkey had fMRI resting-state acquisitions on different days and several monkeys were scanned in more than one experimental condition. All procedures are in agreement with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (Directive 2010/63/EU) and the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. Animal studies were approved by the institutional Ethical Committee (Commissariat à l'Energie atomique et aux Énergies alternatives; Fontenay aux Roses, France; protocols CETEA \#10-003 and 12-086). For the DBS dataset, details were provided in (Tasserie et al., 2022). Five male rhesus macaques (Macaca mulatta, 9 to 17 years and 7.5 to 9.1 kg) were included, three for the awake (non-DBS) experiments (monkeys B, J, and Y) and two for the DBS experiments (monkeys N and T). All procedures are in agreement with 2010/63/UE, 86-406, 12-086 and 16-040. Anaesthesia Protocol During sevoflurane anesthesia, monkeys received first an intramuscular injection of ketamine (20 mg/kg; Virbac) for induction, followed by sevoflurane anesthesia (light sevoflurane, sevoflurane inspiratory/expiratory, 2.2/2.1 volume percent; deep sevoflurane, sevoflurane inspiratory/expiratory, 4.4/4.0 volume percent; Abbott, France). Only 80 minutes after the induction, the scanning sessions started for the sevoflurane acquisitions to get a washout of the initial ketamine injection (Schroeder et al., 2016). To avoid artefacts related to potential movements throughout magnetic resonance imaging acquisition, a muscle-blocking agent was coadministered (cisatracurium, 0.15 mg/kg bolus intravenously, followed by continuous intravenous infusion at a rate of 0.18 mg · kg–1 · h–1; GlaxoSmithKline, France) during the ketamine and light propofol sessions. maintain anesthesia. Atropine (0.02 mg/kg intramuscularly; Aguettant, France) was injected 10 min before induction, to reduce salivary and bronchial secretions. For propofol anesthesia, monkeys were trained to be injected an intravenous propofol bolus (5 to 7.5 mg/kg; Fresenius Kabi, France), followed by a target-controlled infusion (Alaris PK Syringe pump, CareFusion, USA) of propofol (light propofol sedation, 3.7 to 4.0 µg/ml; deep propofol anesthesia, 5.6 to 7.2 µg/ml) based on the "Paedfusor" pharmacokinetic model (Absalom and Kenny, 2005). During sevoflurane anesthesia, monkeys received first an intramuscular injection of ketamine (20 mg/kg; Virbac) for induction, followed by sevoflurane anesthesia (light sevoflurane, sevoflurane inspiratory/expiratory, 2.2/2.1 volume percent; deep sevoflurane, sevoflurane inspiratory/expiratory, 4.4/4.0 volume percent; Abbott, France). Only 80 minutes after the induction, the scanning sessions started for the sevoflurane acquisitions to get a washout of the initial ketamine injection (Schroeder et al., 2016). To avoid artefacts related to potential movements throughout magnetic resonance imaging acquisition, a muscle-blocking agent was coadministered (cisatracurium, 0.15 mg/kg bolus intravenously, followed by continuous intravenous infusion at a rate of 0.18 mg · kg–1 · h–1; GlaxoSmithKline, France) during the ketamine and light propofol sessions. For the DBS dataset, anaesthesia was induced with an intramuscular injection of ketamine (10 mg/kg; Virbac, France) and dexmedetomidine (20 μg/kg; Ovion Pharma, USA) and then the same method as described above for deep propofol sedation was used (Monkey T: TCI, 4.6 to 4.8 µg/ml; monkey N: TCI, 4.0 to 4.2 µg/ml). Two monkeys were implanted with a clinical DBS electrode (Medtronic, Minneapolis, MN, USA, lead model 3389).The DBS protocol is thoroughly described in Tasserie et al, 2022. Anaesthesia Protocol The right centro-median thalamus was targeted by stereotactic surgery using a neuro-navigation system (BrainSight, Rogue, Canada), guided by the rhesus macaque atlases (paxinos et al., Academic press 2008 and Saleem Logothetis Academic Press 2012) and a preoperative and intraoperative anatomical MRI. Anaesthesia Protocol The anaesthesia protocol is thoroughly described in previous studies (Barttfeld et al., 2015; Uhrig et al., 2018). Monkeys received anesthesia either with ketamine (Uhrig et al., 2018), propofol (Barttfeld et al., 2015; Uhrig et al., 2018) or sevoflurane (Barttfeld et al., 2015; Uhrig et al., 2018), with two different levels of anesthesia for propofol and sevoflurane anesthesia (light and deep). The anesthesia levels were defined according to the monkey sedation scale, based on spontaneous movements and the response to external stimuli (presentation, shaking or prodding, toe pinch), and corneal reflex (Uhrig, 2016). For each scanning session, the clinical score was determined at the beginning and end of the scanning session, together with continuous electroencephalography monitoring (Uhrig et al., 2016). Monkeys were intubated and ventilated as previously described (Barttfeld et al., 2015; Uhrig et al., 2018). Heart rate, noninvasive blood pressure, oxygen saturation, respiratory rate, end-tidal carbon dioxide, and cutaneous temperature were monitored (Maglife, Schiller, France) and recorded online (Schiller). During ketamine, deep propofol, and deep sevoflurane anesthesia, monkeys stopped responding to all stimuli, reaching a state of general anesthesia. For ketamine anesthesia, ketamine was injected intramuscular (20 mg/kg; Virbac, France) for induction of anesthesia, followed by a continuous intravenous infusion of ketamine (15 to 16 mg · kg–1 · h–1) to 18 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint maintain anesthesia. Atropine (0.02 mg/kg intramuscularly; Aguettant, France) was injected 10 min before induction, to reduce salivary and bronchial secretions. For propofol anesthesia, monkeys were trained to be injected an intravenous propofol bolus (5 to 7.5 mg/kg; Fresenius Kabi, France), followed by a target-controlled infusion (Alaris PK Syringe pump, CareFusion, USA) of propofol (light propofol sedation, 3.7 to 4.0 µg/ml; deep propofol anesthesia, 5.6 to 7.2 µg/ml) based on the "Paedfusor" pharmacokinetic model (Absalom and Kenny, 2005). Functional Magnetic Resonance Imaging Data Acquisition For the awake condition, monkeys were implanted with a magnetic resonance compatible head post and trained to sit in the sphinx position in a primate chair (Uhrig, Dehaene and Jarraya, 2014)). For the awake scanning sessions, monkeys sat inside the dark magnetic resonance imaging scanner without any task and the eye position was monitored at 120 Hz (Iscan Inc., USA). The eye-tracking was performed to make sure that the monkeys were awake during the whole scanning session and not sleeping. The eye movements were not regressed out from rfMRI data. For the anesthesia sessions, animals were positioned in a sphinx position, mechanically ventilated, and their physiologic parameters were monitored. No eye-tracking was performed in anesthetic conditions. For the anesthesia dataset, before each scanning session, a contrast agent, monocrystalline iron oxide nanoparticle (Feraheme, AMAG Pharmaceuticals, USA; 10 mg/kg, intravenous), was injected into the monkey’s saphenous vein (Vanduffel et al., 2001). Monkeys were scanned at rest on a 3-Tesla horizontal scanner (Siemens Tim Trio, Germany) with a single transmit-receive surface coil customized to monkeys. Each functional scan consisted of gradient-echo planar whole-brain images (repetition time = 2,400 ms; echo time = 20 ms; 1.5-mm3 voxel size; 500 brain volumes per run). 19 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint For the DBS dataset, monkeys were scanned at rest on a 3-Tesla horizontal scanner (Siemens, Prisma Fit, Erlanger Germany) with a customized eight-channel phased- array surface coil (KU Leuven, Belgium). The parameters of the functional MRI sequences were: echo planar imaging (EPI), TR = 1250 ms, echo time (TE) = 14.20 ms, 1.25-mm isotropic voxel size and 500 brain volumes per run. Data preprocessing and time series extraction For the anaesthesia dataset, a total of 157 functional magnetic imaging runs were acquired (Uhrig et al., 2018): Awake, 31 runs (monkey A, 4 runs; monkey J, 18 runs; monkey K, 9 runs), Ketamine, 25 runs (monkey K, 8 runs; monkey Ki, 7 runs; monkey R, 10 runs), Light Propofol, 25 runs (monkey J, 2 runs; monkey K, 10 runs; monkey R, 12 runs), Deep Propofol, 31 runs (monkey J, 9 runs; monkey K, 10 runs; monkey R, 12 runs), Light Sevoflurane, 25 runs (monkey J, 5 runs; monkey Ki, 10 runs; monkey R, 10 runs), Deep Sevoflurane anaesthesia, 20 runs (monkey J, 2 runs; monkey Ki, 8 runs; monkey R, 11 runs). For details, check the supplementary tables for (Barttfeld et al., 2015; Uhrig et al., 2018; Signorelli et al., 2021) (http://links.ww.com/ALN/B756). Functional images were reoriented, realigned, and rigidly coregistered to the anatomical template of the monkey Montreal Neurologic Institute (Montreal, Canada) space with the use of Python programming language and Oxford Centre Functional Magnetic Resonance Imaging of the Brain Software Library software (United Kingdom, http://www.fmrib.ox.ac.uk/fsl/; accessed February 4, 2018) (Uhrig, Dehaene and Jarraya, 2014)). From the images, the global signal was regressed out to remove any confounding effect due to physiologic changes (e.g., respiratory or cardiac changes). For the DBS dataset, a total of 199 Resting State functional MRI runs were acquired: Awake 47 runs (monkey B: 18 runs; monkey J: 13 runs; monkey Y: 16 runs), anaesthesia (DBS-off) 38 runs (monkey N: 16 runs,; monkey T: 22 runs), low amplitude centro-median thalamic DBS 36 runs (monkey N: 18 runs; monkey T: 18 runs), low amplitude ventro-lateral thalamic DBS 20 runs (monkey T), high amplitude centro-median thalamic DBS 38 runs (monkey N: 17 runs; monkey T: 21 runs), and high amplitude ventro-lateral thalamic DBS 20 runs (monkey T: 20 runs). Images were preprocessed using Pypreclin (Python preclinical pipeline) (Tasserie et al., Neuroimage 2020). Functional images were corrected for slice timing and B0 inhomogeneities, reoriented, realigned, resampled (1.0 mm isotropic), masked, coregistered to the MNI macaque brain template (Frey et al., 2011), and smoothed (3.0-mm Gaussian kernel). Anatomical images were corrected for B1 inhomogeneities, normalised to the anatomical MNI macaque brain template, and masked. Images were preprocessed using Pypreclin (Python preclinical pipeline) (Tasserie et al., Neuroimage 2020). Data preprocessing and time series extraction Functional images were corrected for slice timing and B0 inhomogeneities, reoriented, realigned, resampled (1.0 mm isotropic), masked, coregistered to the MNI macaque brain template (Frey et al., 2011), and smoothed (3.0-mm Gaussian kernel). Anatomical images were corrected for B1 inhomogeneities, normalised to the anatomical MNI macaque brain template, and masked. Data were parcellated according to the Regional Map parcellation (Kötter and Wanke, 2005). This parcellation comprises 82 cortical ROIs (41 per hemisphere; Supplementary Table 4). Voxel time series were filtered with low-pass (0.05-Hz cutoff) and high-pass (0.0025-Hz cutoff) 20 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint filters and a zero-phase fast-Fourier notch filter (0.03 Hz) to remove an artifactual pure frequency present in all the data (Barttfeld et al., 2015; Uhrig et al., 2018). filters and a zero-phase fast-Fourier notch filter (0.03 Hz) to remove an artifactual pure frequency present in all the data (Barttfeld et al., 2015; Uhrig et al., 2018). Furthermore, an extra cleaning procedure was performed to ensure the quality of the data after time-series extraction (Signorelli et al., 2021). The procedure was based on a visual inspection of the time series for all the nodes, the Fourier transform of each signal, the functional connectivity for each subject, and the dynamical connectivity computed with phase correlation. Trials were kept when the row signal did not present signs of artifactual activity, functional connectivity was coherent with the average and dynamical connectivity presented consistent patterns across time. Anatomical Parcellation and Structural Connectivity Anatomical (structural) connectivity data were derived from the recent macaque connectome of (Shen et al., 2019), which combines diffusion MRI tractrography with axonal tract-tracing studies, representing the most complete representation of the macaque connectome available to date. Structural (i.e., anatomical) connectivity data are expressed as a matrix in which the 82 cortical regions of interest are displayed in x-axis and y-axis. Each cell of the matrix represents the strength of the anatomical connection between any pair of cortical areas. Data preprocessing and time series extraction Finally, for the anaesthesia data set a total of 119 runs are analysed in subsequent sections: awake state 24 runs, ketamine anaesthesia 22 runs, light propofol anaesthesia 21 runs, deep propofol anaesthesia 23 runs, light sevoflurane anaesthesia 18 runs, deep sevoflurane anaesthesia 11 runs. For the DBS data set, a total of 156 runs are analysed in subsequent sections: awake state 36 runs, Off condition 28 runs, On 3V 31 runs, On 3V control 18 runs, On 5V 25 runs, On 5V control 18 runs. Structure-Function Coupling via Harmonic Mode Decomposition Functional spatiotemporal patterns of neural activity derived from fMRI can be decomposed in terms of anatomically-based distributed building blocks: eigenvectors of the graph Laplacian of the structural connectome (Atasoy et al., 2017; Atasoy, Deco, et al., 2018; Atasoy, Vohryzek, et al., 2018). Here we are inspired by the original Connectome Harmonic Decomposition formulated by of Atasoy and colleagues (Atasoy, Donnelly and Pearson, 2016), who used the harmonic modes of a high-resolution human structural connectome, obtained from combining long-range white matter tracts and local connectivity within the grey matter. Since here we use instead the harmonic modes obtained from a parcellated macaque connectome of diffusion MRI and tract-tracing, we refer to the eigenmodes obtained in this way as “harmonic modes”, and reserve the term “connectome harmonics” for those developed by Atasoy and colleagues. 21 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint Following the method developed by Atasoy and colleagues (Atasoy, Donnelly and Pearson, 2016), we compute the symmetric graph Laplacian on the matrix that represents the macaque structural connectome. To ensure symmetry of the corresponding connectivity matrix, and real eigenvalues, we averaged entries above and below the diagonal to obtain an undirected connectome. Thereafter, we estimate the connectome Laplacian (discrete counterpart of the Laplace operator applied to the network of the macaque structural brain connectivity): where is the diagonal "degree matrix" of the graph i.e. . We then calculate the harmonic modes by solving the following eigenvalue equation: where is the corresponding eigenvalue of the eigenvector . In other words, and are the eigenvalues and eigenvectors of the Laplacian of the primate structural connectivity matrix (macaque connectome). Therefore, if is the harmonic pattern of the spatial frequency, then the corresponding eigenvalue is a term relating to the intrinsic energy of that particular harmonic mode (see figure 1). Decomposition of fMRI data At each timepoint (corresponding to one TR), the spatial pattern of cortical activity over brain regions at time , denoted as , was decomposed as a linear combination of the set of harmonic modes : with the contribution of each harmonic mode at time being estimated as the projection (dot product) of the fMRI data onto : with the contribution of each harmonic mode at time being estimated as the projection (dot product) of the fMRI data onto : Structure-Function Coupling via Harmonic Mode Decomposition With an increasing harmonic number ,we obtain more complex and fine-grained spatial patterns (see Figures 5 and 6). Energy of harmonic modes Once the fMRI cortical activation pattern at time has been decomposed into a linear combination of harmonic modes, the magnitude of each harmonic’s contribution to the cortical 22 . CC-BY 4.0 International license made available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint activity of each harmonic , (regardless of sign) at any given timepoint , denoted . ,is called its "power", for analogy with the Fourier transform, is computed as the amplitude of its contribution: activity of each harmonic , (regardless of sign) at any given timepoint , denoted . ,is called its "power", for analogy with the Fourier transform, is computed as the amplitude of its contribution: The normalized frequency-specific contribution of each harmonic at timepoint , termed "energy", is estimated by combining the magnitude strength of activation (power) of a particular harmonic mode with its own intrinsic energy given by the associated eigenvalue : Functional Gradient Mapping ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint provide a balance between local and global contributions to the embedding space estimation (Coifman et al., 2005). The high-dimensional similarity matrix is treated as a graph, with "connections" (entries of the similarity matrix) reflecting the similarity between the regional patterns of FC. The technique estimates a low-dimensional set of embedding components (gradients); in this low- dimensional space, proximity reflects similarity of the patterns of FC: regions with similar FC patterns (which are strongly connected in the network) are placed close to each other, and regions with low similarity are placed far apart. In this way, each gradient represents one dimension of covariance in the inter-regional similarity between FC patterns, with a small number of gradients capturing most of the dimensions of inter-regional similarity, which can then be visualised by a low-dimensional scatter plot (Figure 4) (Coifman et al., 2005; Margulies et al., 2016). In the embedding space, each gradient can be understood to be "anchored" at regions that have the strongest values for that gradient, suggesting that this particular embedding dimension captures their similarity profiles of FC well. In contrast, regions that are close to the origin (i.e. have a low absolute value for a particular gradient) mean that they are only minimally similar to the "anchor points" of that gradient, which overall does not strongly capture their FC similarity profile well overall (Coifman et al., 2005; Margulies et al., 2016). Therefore, the more different the extremes of a gradient differ along the axis of the gradient, the more the differentiation between regions is being captured by that gradient. To quantify this formally, we calculated the difference between the maximum and minimum values of each scan along the first gradient (Girn et al., 2022) (which mathematically captures most of the variation in FC profiles within each scan) and compared these differences across conditions. Note that the dimension of greatest variability (first gradient) is assessed in a data-driven manner and need not be identical across different scans. Functional Gradient Mapping Macaque cortical functional gradients were calculated using the BrainSpace toolbox https://github.com/ MICA-MNI /BrainSpace (Vos de Wael et al., 2020)as implemented in MATLAB, with default parameters for kernel, similarity metric, and sparsity (see below). We calculate the functional connectivity matrix (FC) as the Pearson correlation between each pair of regional fMRI signals per scan per condition. Following previous work, each matrix was z-transformed and thresholded row-wise to achieve 90% sparsity, retaining only the strongest connections in each row (Girn et al., 2022). The cosine similarity matrix was calculated on the thresholded z-matrix to generate a similarity matrix reflecting the similarity in whole-brain connectivity patterns between vertices. While the FC matrix reflects how similar each pair of regions are in terms of their temporal cofluctuations, this similarity matrix reflects how similar two regions are in terms of their patterns of FC. The similarity matrix is required as input to the diffusion map embedding algorithm that we have used here in agreement with previous work on functional gradients and how they are reshaped by pharmacological interventions (Girn et al., 2022). Diffusion Map Embedding is a nonlinear manifold learning technique (Coifman et al., 2005; Margulies et al., 2016), that exploits the properties of the graph Laplacian to model the diffusion process, and is therefore related to harmonic mode decomposition - though performed on functional data rather than using a common structural connectome, to reveal its contributions to the functional activations (indeed, there is a deep mathematical analogy exists between diffusion graph embedding on the functional connectome and the recent extension of CHD called "functional harmonics" (Glomb et al., 2021; Lioi et al., 2021). The relative influence of density of sampling points on the manifold is controlled by an additional parameter in the range of 0 to 1, which for diffusion map embedding is set to 0.5 to 23 . CC-BY 4.0 International license made available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. Hierarchical Integration The overall level of segregation across the hierarchy is quantified as , the sum of contributions from all eigenmodes except the first (i.e., all eigenmodes that involve a partitioning of the cortex), each weighted by the corresponding number of modules (further corrected for heterogeneous modular sizes, since a partition into two modules of size 1 and N-1 is clearly not as segregated as a partition into two equally-sized modules). Since the first eigenmode encompasses the entire cortex into one global module, the corresponding eigenvalue quantifies the overall contribution of global integration, . The overall level of segregation across the hierarchy is quantified as , the sum of contributions from all eigenmodes except the first (i.e., all eigenmodes that involve a partitioning of the cortex), each weighted by the corresponding number of modules (further corrected for heterogeneous modular sizes, since a partition into two modules of size 1 and N-1 is clearly not as segregated as a partition into two equally-sized modules). Hierarchical Integration A third perspective on distributed brain function that can be obtained from studying the brain's eigenmodes emerges from its hierarchical organisation, and how the latter supports the balance between integration and segregation across scales - which is a central feature of many prominent scientific theories of consciousness. Classical graph-theoretic measures such as small-worldness and modularity quantify integration and segregation at a single scale and are not suitable for capturing these properties across multiple hierarchical modules (Newman, 2006; Rubinov and Sporns, 2010). However, a recently introduced formalism based on eigenmodes of functional connectivity can provide such quantification (Wang et al., 2021). The functional connectivity (FC) is a symmetric matrix, which can be decomposed as , where is an orthogonal matrix whose columns are eigenvectors (eigenmodes) of FC, and is a diagonal matrix whose entries are the eigenvalues of FC. Each eigenmode of functional connectivity identifies a distinct pattern of regions that are jointly activated (same 24 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint sign) or alternate (opposite sign). Therefore, hierarchical modules can be identified based on the concordance or discordance of signs between regions across eigenmodes, progressively partitioning the FC into a larger number of modules and submodules, up to the level where each module coincides with a single region, indicative of completely segregated activity. The first eigenmode has the same sign throughout the entire cortex, reflecting global integration. At the next level, two partitions can be detected based on their different signs in the second eigenmode, and each in turn is subdivided at the following level of the hierarchy (i.e., from the third eigenmode ) on the basis of regional signs. Hierarchical Integration Thus, segregated modules at one level of the hierarchy can become integrated by being part of the same superordinate module (note that this hierarchical modularity based on eigenmodes is not equivalent to the clustering or modularity maximisation methods (Newman, 2006; Rubinov and Sporns, 2010). During this nested partitioning process, we obtain the module number and the modular size at each level. sign) or alternate (opposite sign). Therefore, hierarchical modules can be identified based on the concordance or discordance of signs between regions across eigenmodes, progressively partitioning the FC into a larger number of modules and submodules, up to the level where each module coincides with a single region, indicative of completely segregated activity. The first eigenmode has the same sign throughout the entire cortex, reflecting global integration. At the next level, two partitions can be detected based on their different signs in the second eigenmode, and each in turn is subdivided at the following level of the hierarchy (i.e., from the third eigenmode ) on the basis of regional signs. Thus, segregated modules at one level of the hierarchy can become integrated by being part of the same superordinate module (note that this hierarchical modularity based on eigenmodes is not equivalent to the clustering or modularity maximisation methods (Newman, 2006; Rubinov and Sporns, 2010). During this nested partitioning process, we obtain the module number and the modular size at each level. Each level \textit{i} of the hierarchy is characterised by two quantities: the number of modules into which the cortex is divided, and the covariance explained by the corresponding eigenmode (given by its squared eigenvalue ). However, the number of modules alone may not properly describe the picture of nested segregation and integration because the size of modules may be heterogeneous. The correction factor was calculated as which reflects the deviation from the optimized modular size in the ith level. Thus, the correction effect is stronger for a larger deviation of modular size from homogeneity (Wang et al., 2021). Since the first eigenmode encompasses the entire cortex into one global module, the corresponding eigenvalue quantifies the overall contribution of global integration, . Conflicts of Interest The authors have no conflicts of interest to declare. The authors have no conflicts of interest to declare. Acknowledgments AIL was supported by a Gates Cambridge Scholarship (OPP 1144), a Travel Grant of the Boehringer Ingelheim Fonds, and the visitor program of the European Institute for Theoretical Neuroscience (EITN). JT was supported by the Fondation pour la Recherche Médicale (FRM grant number ECO20160736100). CMS was supported by FNRS, project MIS/VA - F.4512.21 and grant Embodied-Time - 40011405, Belgium. EAS is supported by the Stephen Erskine Fellowship of Queens’ College, Cambridge. AD and RC are supported by the CNRS and the European Community (Human Brain Project, H2020-945539). BJ was supported by the Fondation Bettencourt Schueller, Fondation de France, Human Brain Project (Corticity project), Institut National de la Santé et de la Recherche Médicale, UVSQ, Commissariat à l’Energie Atomique, Collège de France. 25 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint Author contributions A.I.L., R.C., A.D., conceived the analysis; J.T., L.U., and B.J. designed the experiments; J.T., L.U., and B.J. collected the data; A.I.L. and C.M.S. analysed the data; A.I.L., C.M.S., and R.C. wrote the manuscript with feedback from all co-authors.. B.J., A.D., E.A.S., supervised the project. All authors approved the manuscript References CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 3, 2023. ; https://doi.org/10.1101/2023.06.30.547281 doi: bioRxiv preprint Brown, E.N., Lydic, R. and Schiff, N.D. (2010) ‘General anesthesia, sleep, and coma’, The New England journal of medicine, 363(27), pp. 2638–2650. 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https://openalex.org/W2140289152	https://www.frontiersin.org/articles/10.3389/fcvm.2015.00005/pdf	English	null	MRI in Chronic Aortic Dissection: A Systematic Review and Future Directions	Frontiers in cardiovascular medicine	2,015	cc-by	7,835	INTRODUCTION with clinical compromise or who are inadequately managed with medical therapy alone (2, 3). Aortic dissection is a catastrophic complication of aortic wall disease associated with high mortality and morbidity. The under- lying process of the aortic wall disruption is most commonly secondary to atherosclerotic disease (especially with older age) or a knownconnectivetissuedisease[suchasMarfansyndrome(MFS); thoracic aortic aneurysm and dissection syndrome (TAAD); or bicuspid aortic valve (BAV)]. Using data from the International Registry of Acute Aortic Dissection (IRAD), aortic dissection is more common in men,with mean age of 63 years and an incidence of up to 0.8% at autopsy (1–4). According to the Stanford classi- ﬁcation, type A aortic dissections involve the proximal/ascending aorta (and may extend distally) while type B aortic dissections involve the descending thoracic aorta without any proximal exten- sion (5, 6). The aims of surgical intervention in type A dissection are the prevention of aortic rupture, severe aortic valve regur- gitation, coronary artery dissection, and cardiac tamponade (2). Compared with type B dissection, type A confers a higher risk of neurological complications, including stroke. For type B, surgical or endovascular intervention is typically reserved only for those It is well recognized that persisting or chronic aortic dissec- tion (>2 weeks after initial intimal injury) is a risk factor for further aortic dilatation and dissection extension (7–9). In these cases, intervention may be required to prevent progressive aortic dilatation (1). Re-operation rates in type A dissection for areas of aneurysmal dilatation or persisting dissection range from 10 to 20% in the ﬁrst 10 years following initial surgery (10). A key issue regarding the management of chronic aortic dis- sections is a progressive increase in the size of the false lumen (FL). The reported incidence of partial or complete distal aortic FL patency in type A dissection patients is signiﬁcant, ranging between 31 and 89% (9). For type B dissection patients who have been medically managed, a persisting FL in the chronic phase cor- relates with an increased risk of aortic enlargement (11). Hence, regardless of the initial management in aortic dissection, care- ful follow-up with appropriate imaging is mandated (12). Several international guidelines recommend the close follow-up of these patients; however multiple imaging modalities are currently used without a clear consensus of the gold standard (2–5). This“gap”in the evidence is speciﬁcally noted in the current European Society of Cardiology guidelines on aortic disease management (4). Abbreviations:2D,two-dimensional;3D,three-dimensional;4D,four-dimensional; BAV, bicuspid aortic valve; CFD, computational ﬂuid dynamics; CT, computed tomography; CTA, computed tomography angiography; ECG, electrocardiography; FL,false lumen;GRE,gradient-echo;IRAD,international registry of acute aortic dis- section; IVUS, intravascular ultrasound; MFS, Marfan syndrome; MRA, magnetic resonance angiography; MRI, magnetic resonance imaging; PC, phase-contrast; PET, positron emission tomography; SE, spin-echo; T, Tesla; TAAD, thoracic aor- tic aneurysm and dissection syndrome; TL, true lumen; TEE, trans-esophageal echocardiography. REVIEWS IN MEDICINE published: 19 February 2015 doi: 10.3389/fcvm.2015.00005 REVIEWS IN MEDICINE published: 19 February 2015 doi: 10.3389/fcvm.2015.00005 REVIEWS IN MEDICINE Andrew G. Sherrah1,2, Stuart M. Grieve1,3,4,5, Richmond W. Jeremy 1,2, Paul G. Bannon1,2, Michael P. Vallely 1,2,6 and Rajesh Puranik 1,7* Andrew G. Sherrah1,2, Stuart M. Grieve1,3,4,5, Richmond W. Jeremy 1,2, Paul G. Bannon1,2, Michael P. Vallely 1,2,6 and Rajesh Puranik 1,7* 1 Sydney Medical School, University of Sydney, Sydney, NSW, Australia 2 The Baird Institute, Royal Prince Alfred Hospital, Sydney, NSW, Australia 3 Department of Radiology, Royal Prince Alfred Hospital, Sydney, NSW, Australia 4 Charles Perkins Centre, University of Sydney, Sydney, NSW, Australia 5 Heart Research Institute, University of Sydney, Sydney, NSW, Australia 6 Australian School of Advanced Medicine, Macquarie University, Sydney, NSW, Australia 7 Cardiovascular Magnetic Resonance Sydney, Sydney, NSW, Australia Correspondence: Keywords: chronic aortic dissection, aortic type B dissection, aortic typeA dissection, magnetic resonance imaging, follow-up INTRODUCTION Edited by: The acute event of thoracic aortic dissection carries with it high mortality and morbidity. Despite optimal initial surgical or medical management strategies, the risk of further com- plications in the long-term, including aneurysmal dilatation and false lumen (FL) expansion, are not insigniﬁcant. Adequate follow-up of such conditions requires dedicated imaging where relevant prognostic indicators are accurately assessed. We perform a systematic review of the literature and report the current evidence for the use of magnetic resonance imaging (MRI) in assessment of chronic aortic dissection. We then make a comparison with traditional imaging modalities including computed tomography and echocardiography. We discuss new ways in which MRI may extend existing aortic assessment, including identiﬁcation of blood-ﬂow dynamics within the TL and FL using phase-contrast imaging. Edited by: Sebastian Kelle, German Heart Institute Berlin, Germany Reviewed by: Peter Bernhardt, University of Ulm, Germany Daniel R. Messroghli, Deutsches Herzzentrum Berlin, Germany Correspondence: Rajesh Puranik, RPAH Medical Centre, Suite 401, Newtown, NSW 2042, Australia e-mail: raj.puranik@cmrs.org.au INTRODUCTION The most commonly used modalities are computed tomog- raphy (CT) and echocardiography, however magnetic resonance imaging (MRI) has recently emerged as a comprehensive, non- ionizing imaging tool well suited to serial measurements in this group of patients (Figure 1) (13). In the acute setting of aortic dissection, the most suitable imaging modality has been February 2015 \| Volume 2 \| Article 5 \| 1 www.frontiersin.org MRI in chronic aortic dissection Sherrah et al. FIGURE 1 \| MRI follow-up of a 63-year-old male with chronic descending thoracic aortic dissection. The patient had undergone surgical replacement of the ascending aorta for type A aortic dissection 4 years earlier. (A) Sagittal gadolinium-contrast-enhanced MRA (magnetic resonance angiography) view; (B) axial black blood view of the proximal descending thoracic aorta; (C) axial true FISP (steady state-free precession) cine view; and (D) axial phase-contrast view, showing ﬂow patterns in the true and false lumens of the descending aorta. The true lumen is indicated by the white arrow (Courtesy: Cardiovascular Magnetic Resonance, Sydney, Australia). descending thoracic aorta; (C) axial true FISP (steady state-free precession) cine view; and (D) axial phase-contrast view, showing ﬂow patterns in the true and false lumens of the descending aorta. The true lumen is indicated by the white arrow (Courtesy: Cardiovascular Magnetic Resonance, Sydney, Australia). FIGURE 1 \| MRI follow-up of a 63-year-old male with chronic descending thoracic aortic dissection. The patient had undergone surgical replacement of the ascending aorta for type A aortic dissection 4 years earlier. (A) Sagittal gadolinium-contrast-enhanced MRA (magnetic resonance angiography) view; (B) axial black blood view of the proximal descending thoracic aorta; (C) axial true FISP (steady state-free precession) cine view; and (D) axial phase-contrast view, showing ﬂow patterns in the true and false lumens of the descending aorta. The true lumen is indicated by the white arrow (Courtesy: Cardiovascular Magnetic Resonance, Sydney, Australia). FIGURE 1 \| MRI follow-up of a 63-year-old male with chronic descending thoracic aortic dissection. The patient had undergone surgical replacement of the ascending aorta for type A aortic dissection 4 years earlier. (A) Sagittal gadolinium-contrast-enhanced MRA (magnetic resonance angiography) view; (B) axial black blood view of the proximal MRI AND CHRONIC AORTIC DISSECTION extensively examined, given the multiple critical clinical factors that affect time to diagnosis and appropriate management (2, 14, 15). Here, the utility of imaging modalities is limited by availabil- ity,time to acquire diagnostic imaging,cost,degree of invasiveness, the need for intravenous contrast, radiation exposure, and ease of intra-operative access. The availability of computed tomography angiography (CTA), together with the accurate visualization of the aortic root using trans-esophageal echocardiography (TEE), form the standard work-up in the majority of centers and offers a good combination of whole aortic coverage, characterization of dissection severity, and timely imaging (6). In the chronic setting, options for follow-up imaging are less constrained by the need for rapid image acquisition. We performed a systematic review of the current literature using pre-existing guidelines (16), to ascertain whether in patients with chronic aortic dissection, there is beneﬁt from MRI-based follow- up when compared to other imaging modalities. The Cochrane Central Register of Controlled Trials, MEDLINE, and EMBASE were searched using the terms [(chronic dissection) and (aort) and (MRI OR magnetic resonance imag)]. Exclusion criteria were existing topic reviews, studies without a direct comparison of MRI and another imaging modality, and studies not in English. Case reports and conference abstracts were included due to a predicted paucity of available prospective trials. Twelve studies were included for this review from the literature selection process (summarized in Figure 2). No prospective stud- ies were identiﬁed where patients with chronic dissection were randomized to follow-up with MRI or follow-up with another imagingmodality.TheincludedstudiescomparedMRIandatleast oneotherimagingmodality,namelyCT(17–21),TEE(22–26),tra- ditional aortography (21, 27), or intravascular ultrasound (IVUS) This review aims to compare the clinical utility of alternative imaging modalities for the follow-up of chronic aortic dissec- tion by means of systematic review of the current literature, with emphasis upon the advantages and disadvantages of MRI in this setting,and discuss the future role of MRI in assessment of chronic aortic dissection. February 2015 \| Volume 2 \| Article 5 \| 2 Frontiers in Cardiovascular Medicine \| Cardiovascular Imaging MRI in chronic aortic dissection Sherrah et al. FIGURE 2 \| Illustrated summary of study collection and inclusion. URE 2 \| Illustrated summary of study collection and inclusion (26, 28), in the follow-up of individual patients with heterogeneity in both MRI techniques utilized and reported radiological ﬁndings (Table 1). MRI AND CHRONIC AORTIC DISSECTION Additionally, the majority of included studies (10 of 12) were published in the year 2000 and earlier, when MRI technol- ogy was vastly different to today. Pertinent aspects of the included studies, related to the era of MRI use, are discussed below. nature of early (from greater than 10 years ago) MRI studies utilizinglow-ﬁeldstrengths(0.5–1.0 T)toassesschronicaorticdis- section, results are favorable. Compared with CTA, the assessment of FL partial thrombosis was shown to be comparable (20, 30). Using spin-echo (SE) “black blood” techniques, the near absence of signal from blood ﬂowing at a normal velocity affords a nat- ural contrast that highlights ﬂowing versus static blood in the aorta, in comparison to CTA which requires exogenous contrast to accurately deﬁne ﬂuid compartments (20). Clinically useful assessment of FL patency was also demonstrated in early stud- ies using phase-contrast (PC) gradient-echo (GRE) sequences to differentiate thrombus from slow ﬂow (31). THE EARLY APPLICATION OF MRI IN CHRONIC AORTIC DISSECTION IN 1980–2000 In addition to adequate assessment of FL patency, degree of thrombosis, and aortic diameter, MRI is capable of quantifying potentially prognostic hemodynamic parameters such as complex ﬂow patterns, localized wall shear stress, valvular function, and pulse wave velocity. MRI technology is rapidly advancing, and recent improvements in gradient technology,radiofrequency coils, parallel imaging, pulse sequence design, and post-processing have greatly improved image quality. Despite the relatively rudimentary CURRENT MRI TECHNOLOGY While these early studies highlighted the potential of MRI in imaging aortic dissection, current MRI scanners possess far superior technology and the ability to assess additional dynamic February 2015 \| Volume 2 \| Article 5 \| 3 www.frontiersin.org www.frontiersin.org MRI in chronic aortic dissection Sherrah et al. Table 1 \| Patient and MRI data from the included studies. Reference MRI patients, n MRI tesla MRI technique Comparison imaging modality Pathology Clough et al. (17) 12 3.0 Breath-hold or respiratory-gated, ECG-triggered 3D SSFP CT Medically managed type B dissection Bijnens et al. (18) 44 – Not described CT Descending aortic dissection Di Cesare et al. (22) 29 1.5 T1 spin-echo, cardiac-gated, and MRA TEE Descending aortic dissection following surgery for type A dissection Maspes et al. (27) 2 1.5 Breath-hold 3D MRA Aortography Chronic aortic dissection Cecconi et al. (23) 42 1.5 T1 spin-echo with ECG-gating ± gradient-echo TEE Descending aortic dissection following surgery for type A dissection Masani et al. (24) 14 0.5 ECG and respiratory-gated T1 echo images TEE Descending aortic dissection following surgery for type A dissection Yamada et al. (29) 7 1.5 Spin-echo IVUS Chronic aortic dissection Deutsch et al. (25) 25 1.5 ECG-gated spin-echo or gradient-echo TEE Chronic aortic dissection Williams et al. (26) 27 1.5 T1 spin-echo IVUS and TEE Aortic dissection Rofsky et al. (19) 24 0.5 ECG-gated spin-echo ± gradient-echo FAME CT Descending aortic dissection following surgery for type A dissection Grenier et al. (20) 17 0.5 Spin-echo ± ECG-gating CT Medically managed type B dissection Pernes et al. (21) 29 0.5 Spin-echo ± ECG-gating CT and aortography Chronic aortic dissection MRI, magnetic resonance imaging; ECG, electrocardiography; 3D, three-dimensional; SSFP, steady state free precession; MRA, magnetic resonance angiography; FAME, fast acquisition with multiple excitation; CT, computed tomography; TEE, trans-esophageal echocardiography; IVUS, intravascular ultrasound. Table 1 \| Patient and MRI data from the included studies. MRI, magnetic resonance imaging; ECG, electrocardiography; 3D, three-dimensional; SSFP, steady state free precession; MRA, magnetic resonance angiography; FAME, fast acquisition with multiple excitation; CT, computed tomography; TEE, trans-esophageal echocardiography; IVUS, intravascular ultrasound. MRI, magnetic resonance imaging; ECG, electrocardiography; 3D, three-dimensional; SSFP, steady state free precession; MR FAME, fast acquisition with multiple excitation; CT, computed tomography; TEE, trans-esophageal echocardiography; IVUS, intr aspects of blood-ﬂow and the aortic wall environment. CURRENT MRI TECHNOLOGY This key potential advantage that MRI affords over other imaging modal- ities offers the prospect of developing new prognostic indicators that move beyond the simple measurement of vessel dimensions alone (32). that the initial dimension of the descending thoracic aorta and the non-invasive pulse pressure to be independent predictors of late progression to aneurysm (37). Computational 3D models of descending thoracic aortic dissection have previously demon- strated a positive correlation between wall shear stress and disease progression (38). A typical MRI protocol for imaging of the aorta will include GRE and black blood-weighted images for anatomic deﬁnition (Figure 1B). The addition of steady state-free precession cine imaging (true FISP) affords a high blood/tissue contrast with- out the need for intravenous contrast administration (Figure 1C). Gadolinium-contrast-enhanced magnetic resonance angiography (MRA) (Figure 1A) provides high resolution three-dimensional (3D) data, similar to CT. However uncommon, gadolinium toxic- ity can nonetheless occur when such intravenous contrast is used, especially in those with pre-existing renal impairment (33). Frontiers in Cardiovascular Medicine \| Cardiovascular Imaging ECHOCARDIOGRAPHY ECHOCARDIOGRAPHY Echocardiography is generally tolerated well by patients and does not require use of contrast or ionizing radiation. It is also widely available and may be performed at the bedside. It can assess FL ﬂow, FL thrombosis, and FL communication with the true lumen (TL);allsigniﬁcantprognosticmarkers(26,28,34,43).Assessment of ﬂow using echocardiography is optimal owing to high tempo- ral resolution and is able to quantitate maximum ﬂow velocity. However, the quantitative assessment of ﬂow is limited due to the angular dependence of Doppler measurements, which makes this a highly operator-dependent modality. The recent addition of a 3D component to TEE has helped better quantify entry tear site and size (44). Early reports comparing it with MRI in the setting of chronic aortic dissection showed both modalities to be useful in follow-up, even when limited by inferior technology compared with more modern techniques (24, 25). In several studies exam- ining patients following surgery for acute type A dissection, no difference between MRI and TEE was shown regarding the assess- ment of persistence and extent of aortic dissection (23). FL ﬂow was better assessed with TEE in some cases, however, slow FL ﬂow was unreliably detected by MRI sequences at this stage of technol- ogy development (23). A major limitation of echocardiography is the difﬁculty in viewing all sections of the thoracic aorta (23–25). MRI, as a cross-sectional modality, gives excellent coverage of the aorta throughout its course. Furthermore, MRI has shown lower inter-observer variability (compared with echocardiography) for aortic diameter measurement, a critical prognostic indicator (45). In a study in 2000 by Di Cesare et al., 29 patients who had undergone surgery for type A dissection all underwent follow- up imaging with TEE, conventional MRI, and contrast-enhanced 3D breath-hold MRA (22). Imaging follow-up time ranged from 1 to 110 months post-operatively. A high correlation co-efﬁcient was observed for diameter of the descending aorta in all three imaging types, however, TEE showed greater inter-observer vari- ability of measurements made at the distal surgical anastomosis (22). Contrast-enhanced MRA was the most reliable in detection of FL ﬂow; MRI was considered the modality of choice for the follow-up of surgically treated patients with persisting distal aortic dissection (22). Echocardiography is generally tolerated well by patients and does not require use of contrast or ionizing radiation. It is also widely available and may be performed at the bedside. MRI AS A PROBLEM-SOLVING TOOL IN CHRONIC AORTIC DISSECTION MRI AS A PROBLEM-SOLVING TOOL IN CHRONIC AORTIC DISSECTION An important application of MRI is in the identiﬁcation of a num- ber of important post-operative conditions. For example, the use of contrast-enhanced 3D MRA can distinguish aortic pseudoa- neurysm from periprosthetic hematoma via identiﬁed areas of high signal intensity that are indicative of peri-graft ﬂow (39). Fur- thermore, contrast-enhanced“breath-hold”MRA has been shown to be superior to “black blood” MRI for the assessment of intimal ﬂaps and in assessing aortic branch vessel involvement (40). This is attributed to both the higher spatial resolution and the delineation of a hypointense intimal ﬂap surrounded by contrast-enhanced bright blood (40). This beneﬁt becomes particularly useful when classifying the dissection according to intimal tear location (41). A major advantage of MRI is the existence of multiple process- ing techniques that all provide speciﬁc and unique information. This permits a“problem-based”approach, where a number of dif- ferent MRI sequences are used to characterize the features of the dissected aorta (17–21, 40). The ability to assess and quantify blood-ﬂow movement within the aorta is gained when PC imaging is utilized (Figure 1D). The ability to more reliably assess FL thrombosis (compared with CT) is an advantage of MRI in this context as FL thrombosis has been shown to be associated with reduced aortic expansion rates (7, 10, 34). Amano et al. reported their series of 16 chronic thoracic aortic dissection patients who underwent MRI at 1.5 T utilizing cardiac- gating, respiratory compensation, and fat suppression acquisition techniques together with 3D PC imaging of ﬂow patterns (9, 35). They demonstrated that time-resolved 3D MRI may be used to assess the presence of blood-ﬂow within the FL which has previ- ously been shown to be prognostically signiﬁcant (35, 36). In a group of 70 patients undergoing MRI (1.5 T) following surgically repaired type A aortic dissection, Almeida et al. demonstrated Blood-ﬂow quantiﬁcation using PC or velocity mapping can demonstrate bidirectional ﬂow within a FL; such turbulent ﬂow may induce aortic wall shear stress which may be associated with elevated risk of aneurysmal dilatation or tear (22–26, 42). Frontiers in Cardiovascular Medicine \| Cardiovascular Imaging February 2015 \| Volume 2 \| Article 5 \| 4 MRI in chronic aortic dissection Sherrah et al. secondary to the reduced associated morbidity, lack of invasive- ness,and lower cost associated with this modality. ECHOCARDIOGRAPHY It can assess FL ﬂow, FL thrombosis, and FL communication with the true lumen (TL);allsigniﬁcantprognosticmarkers(26,28,34,43).Assessment of ﬂow using echocardiography is optimal owing to high tempo- ral resolution and is able to quantitate maximum ﬂow velocity. However, the quantitative assessment of ﬂow is limited due to the angular dependence of Doppler measurements, which makes this a highly operator-dependent modality. The recent addition of a 3D component to TEE has helped better quantify entry tear site and size (44). Early reports comparing it with MRI in the setting of chronic aortic dissection showed both modalities to be useful in follow-up, even when limited by inferior technology compared with more modern techniques (24, 25). In several studies exam- ining patients following surgery for acute type A dissection, no difference between MRI and TEE was shown regarding the assess- ment of persistence and extent of aortic dissection (23). FL ﬂow was better assessed with TEE in some cases, however, slow FL ﬂow was unreliably detected by MRI sequences at this stage of technol- ogy development (23). A major limitation of echocardiography is the difﬁculty in viewing all sections of the thoracic aorta (23–25). MRI, as a cross-sectional modality, gives excellent coverage of the aorta throughout its course. Furthermore, MRI has shown lower inter-observer variability (compared with echocardiography) for aortic diameter measurement, a critical prognostic indicator (45). The time to complete a CTA examination is relatively short (4– 20 s) and the use of electrocardiographic (ECG) gating techniques allows reliable artifact-free imaging of the aortic root and coronary arteries when necessary (5, 50, 51). CTA can show superior visu- alization of vessel calciﬁcation (compared with echocardiography or MRI where it is often observed as artifact or “signal drop-out”) and extremely precise aortic lumen diameter measurements. In the context of endovascular aortic stent-grafts and some mechan- ical heart valves, CT is deemed as the imaging modality of choice (4). MRI may become feasible in this subset of patients, however, inadequate visualization of stent struts and incompatibility with stainless steel implants remains at present (52). The presence of stainless steel wires used for operative sternal closure additionally inhibits image acquisition due to artifact. g q Despite improvements in modern multi-detector CT technol- ogy, limited temporal resolution can prevent reliable resolution of rapidly moving structures such as an intimal ﬂap or native valve leaﬂets (53). Ganten et al. OTHER IMAGING MODALITIES As the traditional gold standard (2), aortography has been super- seded by the less invasive approaches. Its high speciﬁcity and sensitivity are countered by risk of further iatrogenic aortic dissec- tion and need for intravenous iodinated contrast (27, 55). Its use in the chronic dissection setting is difﬁcult to justify given other available modalities. IVUS has similarly been reported to have ECHOCARDIOGRAPHY have shown the potential for ECG-gated CTA imaging in chronic aortic dissection patients, particularly regarding determination of vessel distensibility (51). Thirty-two patients with conservatively treated type B dissection showed a reduction in aortic distensibility as measured by CTA (versus healthy age-matched controls), which may be a predisposing fac- tor for dissection or a part of the vascular remodeling process following dissection (51). Such information may prove prognosti- cally useful regarding the progression of aneurysmal disease. The feasibility of other novel CTA measures may also have potential as prognostic markers in chronic aortic dissection, for example, aortic displacement (a potential contributor to vessel wall shear stress) during the cardiac cycle (54), or four-dimensional (4D) CTA to assess aortic pulsatility (50). In a study in 2000 by Di Cesare et al., 29 patients who had undergone surgery for type A dissection all underwent follow- up imaging with TEE, conventional MRI, and contrast-enhanced 3D breath-hold MRA (22). Imaging follow-up time ranged from 1 to 110 months post-operatively. A high correlation co-efﬁcient was observed for diameter of the descending aorta in all three imaging types, however, TEE showed greater inter-observer vari- ability of measurements made at the distal surgical anastomosis (22). Contrast-enhanced MRA was the most reliable in detection of FL ﬂow; MRI was considered the modality of choice for the follow-up of surgically treated patients with persisting distal aortic dissection (22). Although TEE can provide useful prognostic information in the acute setting, current recommendations suggest MRI or CT to be more useful for long-term follow-up (5, 43). Given its com- paratively low cost, favorable temporal resolution, and bedside utility, previous recommendations have included TEE as a ﬁrst line follow-up imaging modality in chronic dissection (23), how- ever, this may not be entirely appropriate when measurement of absolute aortic dimensions are of such high importance. ALTERNATIVE IMAGING TECHNIQUES ECHOCARDIOGRAPHY ALTERNATIVE IMAGING TECHNIQUES ECHOCARDIOGRAPHY MRI AS A PROBLEM-SOLVING TOOL IN CHRONIC AORTIC DISSECTION CTA is addition- ally readily available in the vast majority of clinical centers,and has a high reproducibility and low intra- and inter-operator variabil- ity (46). Its drawbacks include the need for iodinated contrast and the exposure to radiation. For patients greater than 60 years of age with normal renal function, the potential negative effects of such radiation exposure may be negligible when compared with the risks of their aortic disease (2), however the increasing recognition of multiple types of genetically deﬁned aortopathies means that for younger patients requiring long-term monitoring, radiation dose may be an important consideration. New advances in recon- struction algorithms, prospective gating, and detector efﬁciency have permitted large reductions in radiation dose. Although not speciﬁc to chronic aortic dissection, this has been demonstrated in several recent reports, where “low radiation” CT assessment of aortic coarctation (47), endoleak (48), and the ascending aorta (49) has been achieved. Pressure-dependent movement of a dissection membrane and consequent branch obstruction also indicates that morphologic assessment alone may be insufﬁcient (21, 27, 42). Additionally, MRI affords the ability to accurately assess blood-ﬂow at low velocities and hence differentiate this from FL thrombosis (17). ALTERNATIVE IMAGING TECHNIQUES COMPUTATIONAL FLUID DYNAMICS The integration of computational ﬂuid dynamics (CFD) as an adjunctive assessor of prognosis in chronic aortic dissection has been explored in several studies (56–58). Such approaches uti- lize computer-based algorithms involving Newtonian ﬂuid ﬂow and complement MRI to provide speciﬁc hemodynamic parame- ters. A case study from Karmonik et al. of a 45-year-old male with chronic aortic dissection where dual phase MRA and two- dimensional (2D) PC results were compared with CFD studies demonstrated that such simulation could quantify changes in both total pressure and wall shear stress during follow-up using patient- derived data (56). Karmonik’s group have additionally compared such changes with that which occurs in the healthy aorta, albeit with a small case series (n = 2) (58). When using MRI at 1.5 T and CFD, both the ascending aorta and TL diameter increased by a factor of 1.36 times in the chronic dissection patient compared with the patient with a healthy aorta. Abnormal wall shear stress values (considerably lower in the healthy aorta) were attributed to aneurysmal dilatation, rather than simply an increase in FL pres- sure or increase in the intra-arterial pressure gradient (58). As is noted in these studies, however, CFD simulations describe ﬁxed mechanical forces and effects and do not take into account the multiple biological factors that exist in a native vessel. FIGURE 3 \| 3D velocity-encoded (4D) view with velocity streamlines of the thoracic aorta of the patient presented in Figure 1. Blood-ﬂow vectors passing through the true lumen (solid white arrow) and the false lumen (dotted white arrow) have been isolated; the reconstructed outline of the entire thoracic aorta is shown. Notably, ﬂow acceleration is observed within the true lumen at the aortic arch (black arrow). Courtesy by Dr. F. Callaghan, Sydney Translational Imaging Laboratory, The Charles Perkins Centre, and The University of Sydney, Sydney, Australia. of 2D PC MRI) (17). In their series of 12 patients,the utilization of a 4D PC sequence demonstrated a correlation between the rate of rotation of helical blood-ﬂow and the rate of aortic expansion in theFL(17).Francoisetal.havesimilarlydemonstratedthefeasibil- ity of integration of 4D ﬂow techniques using MRI at 3 T,where the entire thoracic aorta can be imaged in a single acquisition without signiﬁcant additional scan time (32). COMPUTED TOMOGRAPHY Computed tomography angiography has essentially replaced conventional angiography in the assessment of aortic disease February 2015 \| Volume 2 \| Article 5 \| 5 www.frontiersin.org www.frontiersin.org MRI in chronic aortic dissection Sherrah et al. both high sensitivity and speciﬁcity, however, is similarly compro- mised by its invasiveness (2, 29). The use of positron emission tomography (PET) has also been reported; in the acute setting areas of elevated metabolic activity at freshly disrupted segments of aortic wall show increased uptake of radionuclide tracer (12). In asymptomatic patients with chronic aortic dissection, however, no noticeable uptake is detected, hindering its use as a prognostic indicator in this setting (12). Furthermore, PET has limited spatial resolution, where precise delineation of ﬁne structural detail is not always possible. FIGURE 3 \| 3D velocity-encoded (4D) view with velocity streamlines of the thoracic aorta of the patient presented in Figure 1. Blood-ﬂow vectors passing through the true lumen (solid white arrow) and the false lumen (dotted white arrow) have been isolated; the reconstructed outline of the entire thoracic aorta is shown. Notably, ﬂow acceleration is observed within the true lumen at the aortic arch (black arrow). Courtesy by Dr. F. Callaghan, Sydney Translational Imaging Laboratory, The Charles Perkins Centre, and The University of Sydney, Sydney, Australia. COMPUTATIONAL FLUID DYNAMICS Their computation of blood- ﬂow streamlines has allowed the observation of such helicity and vortical blood-ﬂow as well as signiﬁcantly more retrograde ﬂow in the FL compared with the TL (32). Such approaches raise the potential to derive secondary biomarkers or wall shear stress forces from acquired 4D ﬁelds as clinical prognosticators (32, 61). The signiﬁcance of such observations upon aortic disease progression, risk of future acute events, and ultimately patient mortality and morbidity is yet to be determined. 4D FLOW MRI Hemodynamic quantiﬁcation appears to be the major advantage of MRI over other imaging modalities in chronic aortic dissec- tion. Encoding of all three spatial directions of a volumetric data set utilizing 3D velocity-encoded cine MRI relative to the cardiac cycle, is commonly referred to as 4D ﬂow MRI (59). The velocity and direction of aortic blood-ﬂow can be represented as a“stream- line” image (Figure 3). A reported MRI at 1.5 T of a single patient with chronic dissection from Müller-Eschner et al. with the use of velocity-colored “streamlines” of blood-ﬂow showed acceleration of ﬂow entering the FL through the primary entry tear (59). This evident vortical ﬂow in the FL may be a further contributing factor to progressive expansion of the FL and aneurysmal dilatation. Maj et al. have similarly published hemodynamic information acquired usingtime-resolved,contrast-enhancedMRA(60).Theyshowthat with a sufﬁcient blood velocity difference between the FL and TL, it is possible to achieve separate contrast enhancement of the dissected luminal channels (60). REFERENCES 20. Grenier P, Pernes JM, Desbleds M-T, DeBrux J-L. Magnetic resonance imag- ing of aneurysms and chronic dissections of the thoracic aorta. Ann Vasc Surg (1987) 1:534–41. doi:10.1016/S0890-5096(06)61436-2 1. Tsai TT, Trimarchi S, Nienaber CA. Acute aortic dissection: perspectives from the international registry of acute aortic dissection (IRAD). Eur J Vasc Endovasc Surg (2009) 37(2):149–59. doi:10.1016/j.ejvs.2008.11.032 21. Pernes JM,Grenier P,Desbleds M-T,de Brux JL. MR evaluation of chronic aortic dissection. J ComputAssistTomogr (1987) 11(6):975–81. doi:10.1097/00004728- 198711000-00009 2. Erbel R, Alfonso F, Boileau C, Dirsch O, Eber B, Haverich A, et al. 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J Vasc Surg (2012) 55(4):914–23. doi:10.1016/j.jvs. 2011.11.005 CONCLUSION h f Inthesettingof chronicaorticdissection,theidealimaging modal- ity for use in follow-up has high sensitivity and speciﬁcity, is non-invasive, and can accurately identify not only aortic dimen- sions but also progressive changes in relative ﬂow between true and FLs. MRI can achieve these objectives, as well as the assessment of signiﬁcant prognostic indicators such as FL thrombosis. CT and echocardiography remain the two most widely used alternatives, given their easy access, rapid acquisition, and non-invasiveness. Clough et al. have shown excellent accuracy of 4D PC MRI in velocity assessment (when compared with the MRI gold standard February 2015 \| Volume 2 \| Article 5 \| 6 Frontiers in Cardiovascular Medicine \| Cardiovascular Imaging MRI in chronic aortic dissection Sherrah et al. There is a paucity in the current literature comparing these imag- ing modalities in the context of chronic aortic disease. In patients requiring multiple studies, the risks of repeated contrast/radiation exposure (especially in the young) and inter-observer variability in assessed aortic dimensions remain a concern. New techniques in MRI include more accurate hemodynamic assessment and the use of 4D imaging to demonstrate blood-ﬂow and potential wall shear stress. However, the relevance of such techniques to long- term prognosis warrants further prospective investigation. Given its advantages, and when available, MRI is suggested as a suitable imaging modality in the follow-up of chronic aortic dissection. 12. Reeps C, Pelisek J, Bundschuh RA, Gurdan M, Zimmermann A, Ockert S, et al. Imaging of acute and chronic aortic dissection by 18F-FDG PET/CT. J Nucl Med (2010) 51(5):686–91. doi:10.2967/jnumed.109.072298 13. Sherrah AG, Vallely MP, Grieve SM, Jeremy RW, Hendel PN, Puranik R. Clinical utility of magnetic resonance imaging in the follow-up of chronic aortic type B dissection. 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Accuracy, image quality, and radi- ation dose of prospectively ECG-triggered high-pitch dual-source CT angiogra- phy in infants and children with complex coarctation of the aorta. Acad Radiol (2014) 21(10):1248–54. doi:10.1016/j.acra.2014.04.019 Copyright © 2015 Sherrah, Grieve, Jeremy, Bannon, Vallely and Puranik. 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 publica- tion 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. Received: 26 September 2014; paper pending published: 21 October 2014; accepted: 05 February 2015; published online: 19 February 2015. Citation: Sherrah AG, Grieve SM, Jeremy RW, Bannon PG, Vallely MP and Puranik R (2015) MRI in chronic aortic dissection: a systematic review and future directions. Front. Cardiovasc. Med. 2:5. doi: 10.3389/fcvm.2015.00005 This article was submitted to Cardiovascular Imaging, a section of the journal Frontiers in Cardiovascular Medicine. Copyright © 2015 Sherrah, Grieve, Jeremy, Bannon, Vallely and Puranik. 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 publica- tion 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. REFERENCES Copyright © 2015 Sherrah, Grieve, Jeremy, Bannon, Vallely and Puranik. 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 publica- tion 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. 48. Koike Y, Ishida K, Hase S, Kobayashi Y, Nishimura J,Yamasaki M, et al. Dynamic volumetric CT angiography for the detection and classiﬁcation of endoleaks: application of cine imaging using a 320-row CT scanner with 16-cm detectors. J Vasc Interv Radiol (2014) 25(8):1172–80. doi:10.1016/j.jvir.2014.03.019 February 2015 \| Volume 2 \| Article 5 \| 8 Frontiers in Cardiovascular Medicine \| Cardiovascular Imaging

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